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K. A. Mears, L. A. Levin; In vivo Imaging of Reactive Oxygen Species in Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5384. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Reactive oxygen species not only are generated as a result of cellular injury, but also serve as signaling molecules for a variety of critical processes, including mitosis and cell death. We previously reported that injury to retinal ganglion cell axons induces a burst of superoxide within the cell body, probably of mitochondrial origin (IOVS 47:1477, 2006). We now describe a method for imaging of reactive oxygen species within the retina of the living animal using a confocal scanning laser ophthalmoscope equipped with dual lasers.
Hydroethidium (HEt), HEt incubated with superoxide generated by xanthine/xanthine oxidase (oxy-Et), and oxy-Et incubated with salmon sperm DNA (oxy-Et-DNA) were imaged using a Heidelberg Retina Angiograph 2 (HRA2) with fluorescein filters. The resultant images were quantified using ImageJ. The left or right optic nerve of Long-Evans rats was crushed intraorbitally, sparing the retinal circulation. In some rats with the superior colliculi of Long-Evans rats had been previously exposed via craniotomy and overlaid with Gelfoam saturated with indocyanine green (ICG). At varying time points the animals were injected intravenously or intravitreally with HEt or 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and imaged with fluorescein and/or ICG filters.
Although not optimized for oxy-Et or oxy-Et-DNA, the HRA2 detected both species using fluorescein filters. There was virtually no fluorescence with the non-oxidized HEt control, nor when HEt was incubated with hydrogen peroxide. These results indicate that HEt can be used to detect the presence of superoxide with the HRA2. The ICG filters clearly demonstrated multiple bright foci of fluorescence in the ganglion cell layer, indicating that ICG was a retrogradely transported dye that could be detected with the HRA2. There was no cross-talk between the fluorescein and ICG channels when detecting ICG or oxy-Et-DNA, respectively.
Retinal ganglion cells can be simultaneously identified and levels of reactive oxygen species measured using a dual frequency confocal scanning laser ophthalmoscope.
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