Topographical Distribution and Morphology of Calcitonin Gene-Related Peptide (CGRP)-IR Nociceptive Afferent Axons and Terminals in Flat-Mounts of the Heart of Rats: Tracing, Digitation, and 3D Scaffold
Authors: Chen Jin , Bendowski Kohlton , Bizanti Ariege , Zhang Yuanyuan , Heal Maci , Christie Richard , Hunter Peter , Cheng Zixi 
Published: May 2023

To treat cardiac pain, it is important to elucidate the topographical distribution of nociceptive axons in the heart. Previous studies have detected nociceptive axons in the sectioned heart. However, the topographical distribution of cardiac nociceptive afferent innervation has not been determined. In this study, we used calcitonin gene-related peptide (CGRP), a nociceptive marker, to determine the topographical distribution of nociceptive afferent axons in the whole heart. The hearts of Sprague-Dawley rats (male, n=6) were removed, dissected into left and right atria, left and right ventricles, and interventricular septum, and prepared as flat-mounts. These samples were then processed with immunohistochemical labeling using a primary antibody which binds to CGRP and an Alexa Fluor 488 secondary antibody for fluorescence. The flat mounts were imaged with a Leica confocal microscope (40X oil lens) for regions of interest and a Zeiss M2 Imager (20X oil lens) for imaging the flat mounts of the whole atria, ventricles and interventricular septum to wholistically examine the heart at high resolution. To increase antibody penetration into the thick tissues (right ventricle: 1 mm; left ventricle: 3 mm; interventricular septum: 2.5 mm, approximately), we modified the tissue clearing steps of an iDISCO/CLARITY protocol combined with our established immunohistochemical procedures. We found that: 1) For the right/left atria, CGRP-immunoreactive (CGRP-IR) axons entered as several large bundles near the superior/inferior vena cava, left pre-caval vein and the pulmonary veins before bifurcating into small branches, and finally formed single varicose axons and terminals to innervate the cardiac ganglia, sinoatrial and atrioventricular nodes, auricles, and blood vessels. 2) In the right/left ventricles and interventricular septum, CGRP-IR axons entered as several large bundles from the base. The CGRP-IR axon network covered the whole ventricles from the base towards the apex densely. Ultimately, single varicose axons and terminals innervated the epicardium, myocardium and blood vessels. 3) To establish a 3D innervation map, the distribution of CGRP-IR axons in flat-mounts was traced, digitized, and integrated into a heart scaffold using the novel Neurolucida 360 software and the organ mapping tool provided by MBF Bioscience and the NIH SPARC Map-Core. For the first time, we showed the topographical distribution of CGRP-IR nociceptive axons in the whole heart at single cell/axon/varicosity resolution in rats. Our work will provide foundation of the functional mapping of nocice This study was supported by NIH HEAL/SPARC U01 NS113867-01 (ZJC), NIH R15HL137143-01A1 (ZJC), NIH SPARC OT3OD025349-01 (PH). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.