Distribution and Morphology of Catecholaminergic Axons and Terminals in Flat-Mounts of the Whole Left Ventricles of Rats: Tracing, Digitization, and 3D Scaffold

Cardiac sympathetic innervation plays a vital role in regulating various heart functions, and its remodeling has been linked to a range of cardiovascular diseases. To understand cardiac sympathetic innervation and remodeling, it is important to elucidate the topographical distribution and morphology of sympathetic axons in the whole left ventricle. However, immunohistochemistry (IHC) on whole heart tissue presents challenges due to its thickness, as conventional methods label only small sections, disrupting tissue integrity and neural connections, and thus complicating the comprehensive analysis of sympathetic innervation. To overcome this, we previously developed a whole flat-mount technique for processing the mouse heart (ventricle thickness: 0.5-1 mm). Building on this success, we have adapted this technique to the rat heart (ventricle thickness: 3 mm). The left ventricles of Sprague-Dawley rats (adult male, n=6) were removed and prepared as flat-mounts. These samples were then processed with immunohistochemical labeling of Tyrosine Hydroxylase (TH, a sympathetic marker). Flat mounts of whole left ventricles were imaged using a Zeiss M2 Imager equipped with a 20X oil immersion lens, producing montages composed of several hundred all-in-focus projection images. For high-resolution imaging of regions of interest, a Leica confocal microscope with a 63X oil immersion lens was used. We found that: 1) TH-IR (immunoreactive) axons entered as several large bundles from the base of the left ventricles. The TH-IR axon network densely covered the whole ventricle from the base towards the apex. Specifically, individual varicose axons and terminals were found in the epicardium and myocardium, and ran along blood vessels. 2) To establish a 3D anatomical map of innervation, TH-IR axons in flat-mounts was traced, digitized, and integrated into a 3D 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 distribution of TH-IR sympathetic axons in the whole left ventricle of rats at single cell/axon/varicosity resolution. Our results enabled us to integrate the sympathetic innervation of the entire rat ventricle onto a 3D heart scaffold at an unprecedented resolution to visualize precise anatomical distribution. This 3D model will provide the foundation for functional mapping and for understanding the remodeling of sympathetic innervation of the heart in pathological conditions, such as advanced heart failure. Supported by NIH HEAL/SPARC U01 NS113867-01 (ZJC), NIH SPARC OT3OD025349-01 (PH) and NIH 2R01HL137832 (YL Li). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.