title: Dynamic optical imaging of metabolic and NADPH oxidase-derived superoxide in live mouse brain using fluorescence lifetime unmixing.
abstract: Superoxide is the single-electron reduction product of molecular oxygen generated by mitochondria and the innate immune enzyme complex, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox), and its isoforms. Initially identified as critical to the host defense against infection, superoxide has recently emerged as an important signaling molecule and as a proposed mediator of central nervous system injury in stroke, neurodegenerative conditions, and aging itself. Complete understanding of superoxide in central nervous system disease has been hampered by lack of noninvasive imaging techniques to evaluate this highly reactive, short-lived molecule in vivo. Here we describe a novel optical imaging technique to monitor superoxide real time in intact animals using a fluorescent probe compound and fluorescence lifetime contrast-based unmixing. Specificity for superoxide was confirmed using validated mouse models with enhanced or attenuated brain superoxide production. Application of fluorescence lifetime unmixing removed autofluorescence, further enhanced sensitivity and specificity of the technique, permitted visualization of physiologically relevant levels of superoxide, and allowed superoxide in specific brain regions (e.g., hippocampus) to be mapped. Lifetime contrast-based unmixing permitted disease model-specific and brain region-specific differences in superoxide levels to be observed, suggesting this approach may provide valuable information on the role of mitochondrial and Nox-derived superoxide in both normal function and pathologic conditions in the central nervous system.
authors.affiliation: Department of Radiology, University of California, San Diego, San Diego, California 92093-0746, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Hall
authors.forename: Sung-Ho
authors.initials: SH
authors.lastname: Han
authors.forename: Andre
authors.initials: A
authors.lastname: Chepetan
authors.forename: Edny G
authors.initials: EG
authors.lastname: Inui
authors.forename: Mike
authors.initials: M
authors.lastname: Rogers
authors.forename: Laura L
authors.initials: LL
authors.lastname: Dugan
mesh_terms: D000375:Aging / Q000378:metabolism / Q000473:pathology; D000818:Animals; D001921:Brain / Q000378:metabolism* / Q000473:pathology / Q000502:physiology; D001931:Brain Mapping / Q000295:instrumentation / Q000379:methods; D004996:Ethidium / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000008:administration & dosage* / Q000493:pharmacokinetics; D007091:Image Processing, Computer-Assisted; D066298:In Vitro Techniques; D007274:Injections, Intraperitoneal; D051379:Mice; D008815:Mice, Inbred Strains; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism*; D055096:Optical Devices; D010084:Oxidation-Reduction; D013481:Superoxides / Q000378:metabolism*; D014018:Tissue Distribution
chemical_list: D005456:Fluorescent Dyes; C067883:dihydroethidium; D013481:Superoxides; D019255:NADPH Oxidases; D004996:Ethidium