Quantification of sympathetic hyperinnervation and denervation after myocardial infarction by three-dimensional assessment of the cardiac sympathetic network in cleared transparent murine hearts

• Published in: PloS one 2017-07, Vol.12 (7), p.e0182072-e0182072
• Main Authors: Yokoyama, Teruki, Lee, Jong-Kook, Miwa, Keiko, Opthof, Tobias, Tomoyama, Satoki, Nakanishi, Hiroyuki, Yoshida, Akira, Yasui, Haruyo, Iida, Tadatsune, Miyagawa, Shigeru, Okabe, Shigeo, Sawa, Yoshiki, Sakata, Yasushi, Komuro, Issei
• Format: Article
• Language: English
• Subjects: Animals; Arrhythmias, Cardiac - metabolism; Arrhythmias, Cardiac - pathology; Biology and Life Sciences; Blood vessels; Cardiology; Cardiomyopathy; Care and treatment; Coronary artery disease; Coronary Vessels - metabolism; Coronary Vessels - pathology; Denervation; Engineering and Technology; Fluorescent antibody technique; Heart; Heart - innervation; Heart attack; Heart attacks; Heart diseases; Heart surgery; Image acquisition; Infarction; Ischemia; Laboratory animals; Male; Medicine; Medicine and Health Sciences; Methods; Mice; Myocardial infarction; Myocardial Infarction - metabolism; Myocardial Infarction - pathology; Myocardium; Myocardium - metabolism; Myocardium - pathology; Nerves; Nervous system; Neurobiology; Neurosciences; Ostomy; Perfusion; Research and Analysis Methods; Spatial distribution; Substrates; Sympathetic nerves; Sympathetic nervous system; Sympathetic Nervous System - metabolism; Sympathetic Nervous System - pathology; Three dimensional imaging; University graduates; Ventilators
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0182072

The sympathetic nervous system is critical in maintaining the normal physiological function of the heart. Its dysfunction in pathological states may exacerbate the substrate for arrhythmias. Obviously, knowledge of its three-dimensional (3D) structure is important, however, it has been revealed by conventional methods only to a limited extent. In this study, a new method of tissue clearance in combination with immunostaining unravels the 3D structure of the sympathetic cardiac network as well as its changes after myocardial infarction. Hearts isolated from adult male mice were optically cleared using the CUBIC-perfusion protocol. After making the hearts transparent, sympathetic nerves and coronary vessels were immunofluorescently labeled, and then images were acquired. The spatial distribution of sympathetic nerves was visualized not only along the epicardial surface, but also transmurally. They were distributed over the epicardial surface and penetrated into the myocardium to twist around coronary vessels, but also independent from the coronary vasculature. At 2 weeks after myocardial infarction, we were able to quantify both denervation distal from the site of infarction and nerve sprouting (hyperinnervation) at the ischemic border zone of the hearts in a 3D manner. The nerve density at the ischemic border zone was more than doubled in hearts with myocardial infarction compared to intact mice hearts (3D analyses; n = 5, p