"En Face" detection of nitric oxide and superoxide in endothelial layer of intact arteries

Yu, Yi Cardiovascular and Aging Research, Department of Medicine, Division of Physiology, University of Fribourg, Switzerland
Xiong, Yuyan Cardiovascular and Aging Research, Department of Medicine, Division of Physiology, University of Fribourg, Switzerland
Montani, Jean-Pierre System Physiology, Department of Medicine, Division of Physiology, Faculty of Science, University of Fribourg, Switzerland
Yang, Zhihong Cardiovascular and Aging Research, Department of Medicine, Division of Physiology, University of Fribourg, Switzerland
Ming, Xiu-Fen Cardiovascular and Aging Research, Department of Medicine, Division of Physiology, University of Fribourg, Switzerland

25.02.2016

Published in:

Journal of Visualized Experiments. - 2016, no. 108, p. e53718

English 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.

Faculty

Faculté des sciences et de médecine

Department

Département de Médecine

Language