Sympathetic efferent axons regulate cardiac functions. However, the topographical distribution and morphology of cardiac sympathetic efferent axons remain insufficiently characterized due to the technical challenges involved in immunohistochemical labeling of the thick walls of the whole heart. In this study, flat-mounts of the left and right atria and ventricles of FVB mice were immunolabeled for tyrosine hydroxylase (TH), a marker of sympathetic nerves. Atrial and ventricular flat-mounts were scanned using a confocal microscope to construct montages. We found (1) In the atria: A few large TH-immunoreactive (IR) axon bundles entered both atria, branched into small bundles and then single axons that eventually formed very dense terminal networks in the epicardium, myocardium and inlet regions of great vessels to the atria. Varicose TH-IR axons formed close contact with cardiomyocytes, vessels, and adipocytes.Multiple intrinsic cardiac ganglia (ICG) were identified in the epicardium of both atria, and a subpopulation of the neurons in the ICG were TH-IR. Most TH-IR axons in bundles traveled through ICG before forming dense varicose terminal networks in cardiomyocytes. We did not observe varicose TH-IR terminals encircling ICG neurons. (2) In the left and right ventricles and interventricular septum: TH-IR axons formed dense terminal networks in the epicardium, myocardium, and vasculature. Collectively, TH labeling is achievable in flat-mounts of thick cardiac walls, enabling detailed mapping of catecholaminergic axons and terminal structures in the whole heart at single-cell/axon/varicosity scale. This approach provides a foundation for future quantification of the topographical organization of the cardiac sympathetic innervation in different pathological conditions.
The sympathetic nervous system is crucial for controlling multiple cardiac functions. However, a comprehensive, detailed neuroanatomical map of the sympathetic innervation of the heart is unavailable. Here, we used a combination of state-of-the-art techniques, including flat-mount tissue processing, immunohistochemistry for tyrosine hydroxylase (TH, a sympathetic marker), confocal microscopy and Neurolucida 360 software to trace, digitize, and quantitatively map the topographical distribution of the sympathetic postganglionic innervation in whole atria of C57Bl/6 J mice. We found that (1) 4–5 major extrinsic TH-IR nerve bundles entered the atria at the superior vena cava, right atrium (RA), left precaval vein and the root of the pulmonary veins (PVs) in the left atrium (LA). Although these bundles projected to different areas of the atria, their projection fields partially overlapped. (2) TH-IR axon and terminal density varied considerably between different sites of the atria with the greatest density of innervation near the sinoatrial node region (P < 0.05, n = 6). (3) TH-IR axons also innervated blood vessels and adipocytes. (4) Many principal neurons in intrinsic cardiac ganglia and small intensely fluorescent cells were also strongly TH-IR. Our work provides a comprehensive topographical map of the catecholaminergic efferent axon morphology, innervation, and distribution in the whole atria at single cell/axon/varicosity scale that may be used in future studies to create a cardiac sympathetic-brain atlas.
This protocol describes the process of mapping the topographical organization of tyrosine hydroxylase immune reactive sympathetic postganglionic axons and terminals in the mouse heart. Hearts were removed and separated as whole mounts, then scanned using confocal or zeiss microscopy
Neurofibromatosis type 1 (NF-1) is known to be associated with increased risk of malignancy by at least fourfold. Malignant lymphomas are rare in adults with NF-1. Hereby, we present a 75-year-old male with NF-1 complaining of weakness, nausea, and vomiting associated with abdominal pain. Three months prior to presentation, he had suffered a motor vehicle accident (MVA) resulting in multiple rib fractures that was seen in chest X-ray. For the following three months, he had intermittent chest pain, but it was attributed to the recent rib fracture. During this admission, the severity of chest pain worsened and the associated vomiting inclined further investigation; including CT imaging and bone biopsy, it was revealed to be a rare case of diffuse B cell lymphoma in a patient with NF-1. However, we believe the recent MVA caused an anchoring bias in making a prompt diagnosis. In addition, the appearance of the neurofibroma, resulted in suboptimal physical examination, and hence, there was a delay in reaching the diagnosis. We will discuss here the presentation of this case, to highlight the rare association and to increase awareness of when encountering a challenging diagnosis.
The dorsal root ganglia (DRG) project spinal afferent axons to the stomach. However, the distribution and morphology of spinal afferent axons in the stomach have not been well characterized. In this study, we used a combination of state-of-the-art techniques, including anterograde tracer injection into the left DRG T7-T11, avidin-biotin and Cuprolinic Blue labeling, Zeiss M2 Imager, and Neurolucida to characterize spinal afferent axons in the flat-mounts of the whole rat stomach muscular wall. We found that spinal afferent axons innervated all regions with a variety of distinct terminal structures innervating different gastric targets: 1) The ganglionic type: some axons formed varicose contacts with individual neurons within myenteric ganglia. 2) The muscle type: most axons ran in parallel with the longitudinal and circular muscles and expressed spherical varicosities. Complex terminal structures were observed within the circular muscle layer. 3) The ganglia-muscle mixed type: some individual varicose axons innervated both myenteric ganglia and circular muscles, exhibiting polymorphic terminal structures. 4) The vascular type: individual varicose axons ran along the blood vessels and occasionally traversed the vessel wall. This work provides a foundation for future topographical anatomical and functional mapping of spinal afferent axon innervation of the stomach under normal and pathophysiological conditions.
To understand and treat cardiac pain, it is important to have an understanding of the topographical distribution of nociceptive axons in the heart. While previous studies have detected nociceptive axons in the sectioned heart, their full distribution has not yet been determined. In this study, we used a nociceptive marker calcitonin gene‐related peptide (CGRP) to determine the distribution of nociceptive afferent axons in the whole heart. We first removed the hearts of male Sprague‐Dawley rats (n=6), and C57BL/6 mice (n=6). The hearts were dissected into left and right atria and 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 Fluro 488 secondary antibody for fluorescence. The flat‐mounts were imaged with a Zeiss M2 Imager (automatic fluorescence microscope) using a 488 nm excitation wavelength. In both rats and mice, we found that CGRP‐IR nerve bundles entered the heart and innervated the left and right atria and ventricles. For the right/left atria, CGRP‐IR axons entered as 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 which distributed throughout the tissue, including cardiac ganglia, the SA/AV nodes, auricles, and the blood vessels. In the right/left ventricles and interventricular septum, CGRP‐IR axons entered as large bundles through the base before bifurcating into small branches towards the apex Ultimately, single varicose axons and terminals innervated myocardium and blood vessels. Our data shows the distribution of CGRP‐IR axons in the whole heart at single cell/axon resolution. With this information, we will gain insight into the function of these axons and the pathways that they follow, paving the way for the development of better treatments for cardiac pain and disease. The first two authors contributed equally to this work.
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