title: En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries.
abstract: Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2(.-)) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2(.-) levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2(.-), it is difficult to measure NO and O2(.-) directly in a blood vessel. Numerous methods have been developed to measure NO and O2(.-) production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2(.-) using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2(.-) levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2(.-) in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.
authors.affiliation: Cardiovascular and Aging Research, Department of Medicine, Division of Physiology, Faculty of Science, University of Fribourg; Kidney Control of Homeostasis, Swiss National Centre of Competence in Research.
authors.forename: Yi
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Cardiovascular and Aging Research, Department of Medicine, Division of Physiology, Faculty of Science, University of Fribourg; Kidney Control of Homeostasis, Swiss National Centre of Competence in Research.
authors.forename: Yuyan
authors.initials: Y
authors.lastname: Xiong
authors.affiliation: Kidney Control of Homeostasis, Swiss National Centre of Competence in Research; System Physiology, Department of Medicine, Division of Physiology, Faculty of Science, University of Fribourg.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Montani
authors.affiliation: Cardiovascular and Aging Research, Department of Medicine, Division of Physiology, Faculty of Science, University of Fribourg; Kidney Control of Homeostasis, Swiss National Centre of Competence in Research; zhihong.yang@unifr.ch.
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Cardiovascular and Aging Research, Department of Medicine, Division of Physiology, Faculty of Science, University of Fribourg; Kidney Control of Homeostasis, Swiss National Centre of Competence in Research.
authors.forename: Xiu-Fen
authors.initials: XF
authors.lastname: Ming
mesh_terms: D000818:Animals; D001011:Aorta / Q000737:chemistry*; D059305:Diet, High-Fat / Q000009:adverse effects; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism; D004996:Ethidium / Q000031:analogs & derivatives; D019793:Fluorescein; D007202:Indicators and Reagents; D008297:Male; D008810:Mice, Inbred C57BL; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D009765:Obesity / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*
chemical_list: C414689:4,5-diaminofluorescein diacetate; D007202:Indicators and Reagents; C067883:dihydroethidium; D013481:Superoxides; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D004996:Ethidium; D019793:Fluorescein