Abstract 16458: Tissue Clearance Technique Enables to Investigate Three-Dimensional Distribution of Cardiac Sympathetic Nervous System
Introduction: Sympathetic nervous system plays an important role in cardiac function and occurrence of cardiac arrhythmias. Visualization of sympathetic nervous network gives us useful information about its anatomical status such as denervation or hyperinnervation, however, only limited information was obtained with conventional method regarding its three-dimensional (3D) distribution. Recently, in neuroscientific field, tissue clarifying techniques were developed to attain 3D imaging of central nervous system, whether the cardiac nervous system can be visualized has remained to be examined.
Hypothesis: We assessed the hypothesis that tissue clearance technique could be applied to heart tissue and that cardiac sympathetic nerves (SNs) could be visualized in 3D manner.
Methods: Hearts isolated from C57BL/6J mice of 8 weeks old were optically cleared using ‘CUBIC’ protocol as previously reported. Briefly, hearts were fixed with 4%PFA and processed for two weeks with CUBIC-1 reagent containing 25% urea, 25% N,N,N’,N’-tetrakis(2-hydropropyl)ethylenedyamine and 15% Triton X-100. After clarification, SNs were immunofluorescently labeled with anti-tyrosine hydroxylase antibody and then images were acquired using light-sheet fluorescence microscope. In addition, we evaluated 3D structure of cardiac SNs in myocardial infarction (MI) model.
Results: Hearts were sufficiently clarified using CUBIC method and distribution of cardiac SNs could be visualized in 3D manner through immune-fluorescent labeling (upper panel). In MI model mice, we could see denervation or abrupt extinction of SNs (lower panel) and hyperinnervation at ischemic border zone, as is called nerve sprouting, was also observed.
Conclusions: In the transparent heart, the 3D structures of fluorescently-labeled cardiac SNs have been visualized. The technique would enable us to investigate the role of sympathetic nervous system in the pathophysiological condition of the heart.
Author Disclosures: T. Yokoyama: None. J. Lee: Research Grant; Modest; Astellas Pharma Inc., Takeda Pharmaceutical Inc., Daiichi Sankyo Inc.. Research Grant; Significant; Shionogi Pharmaceutical Inc.. K. Miwa: None. S. Tomoyama: None. H. Nakanishi: None. A. Yoshida: None. S. Miyagawa: None. Y. Sawa: None. Y. Sakata: None. I. Komuro: None.
- © 2016 by American Heart Association, Inc.