Search

Neuronal nitric oxide synthase (nNOS)-derived nitric oxide (NO) plays a major role in the neural control of circulation and in many cardiovascular diseases. However, the exact mechanism of how NO regulates these processes is still not fully understood. This study was designed to determine the possible sources of nitrergic nerve fibres supplying the heart attempting to imply their role in the cardiac neural control. Sections of medulla oblongata, vagal nerve, its rootlets and nodose ganglia, vagal cardiac branches, Th 1 -Th 5 spinal cord segments, dorsal root ganglia of C 8 -Th 5 spinal nerves, and stellate ganglia from 28 Wistar rats were examined applying double immunohistochemical staining for nNOS combined with choline acetyltransferase (ChAT), peripherin, substance P, calcitonin gene-related peptide, tyrosine hydroxylase or myelin basic protein. Our findings show that the most abundant population of purely nNOS-immunoreactive (IR) neuronal somata (NS) was observed in the nodose ganglia (37.4 ± 1.3%). A high number of nitrergic NFs spread along the vagal nerve and entered its cardiac branches. All nitrergic neuronal somata (NS) in the nucleus ambiguus were simultaneously immunoreactive (IR) to ChAT and composed only a small subset of neurons (6%). In the dorsal nucleus of vagal nerve, biphenotypic nNOS-IR/ChAT-IR neurons composed 7.0 ± 1.0%, while small purely nNOS-IR neurons were scarce. Nitrergic NS were plentifully distributed within the nuclei of solitary tract. In the examined dorsal root and stellate ganglia, a few nitrergic NS were sporadically present. The majority of sympathetic NS in the intermediolateral nucleus were simultaneously immunoreactive for nNOS and ChAT. In conclusion, an abundant population of nitrergic NS in the nodose ganglion implies that neuronal NO is involved in afferent cardiac innervation. Nevertheless, nNOS-IR neurons identified within vagal nuclei may play a role in the transmission of preganglionic parasympathetic nerve impulses., (© 2020 Anatomical Society.)

A significant challenge when investigating autonomic neuroanatomy is being able to reliably obtain tissue that contains neuronal structures of interest. Currently, histochemical staining for acetylcholinesterase (AChE) remains the most feasible and reliable method to visualize intrinsic nerves and ganglia in whole organs. In order to precisely visualize and sample intrinsic cardiac nerves and ganglia for subsequent immunofluorescent labeling, we developed a modified histochemical AChE method using material from pig and sheep hearts. The method involves: (1) chemical prefixation of the whole heart, (2) short-term and weak histochemical staining for AChE in situ, (3) visual examination and extirpation of the stained neural structures from the whole heart, (4) freezing, embedding and cryostat sectioning of the tissue of interest, and (5) immunofluorescent labeling and microscopic analysis of neural structures. Firstly, our data demonstrate that this modified AChE protocol labeled intrinsic cardiac nerves as convincingly as our previously published data. Secondly, there was the added advantage that adrenergic, cholinergic and peptidergic neuropeptides, namely protein gene product 9.5 (PGP 9.5), neurofilament (NF), tyrosine hydroxylase (TH), vesicular monoamine transporter (VMAT2), neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), calcitonin gene related peptide (CGRP), and substance P may be identified. Our method allows the precise sampling of neural structures including autonomic ganglia, intrinsic nerves and bundles of nerve fibers and even single neurons from the whole heart. This method saves time, effort and a substantial amount of antisera. Nonetheless, the proof of specific staining for many other autonomic neuronal markers has to be provided in subsequent studies.

Although the rabbit is routinely used as the animal model of choice to investigate cardiac electrophysiology, the neuroanatomy of the rabbit heart is not well documented. The aim of this study was to examine the topography of the intrinsic nerve plexus located on the rabbit heart surface and interatrial septum stained histochemically for acetylcholinesterase using pressure-distended whole hearts and whole-mount preparations from 33 Californian rabbits. Mediastinal cardiac nerves entered the venous part of the heart along the root of the right cranial vein (superior caval vein) and at the bifurcation of the pulmonary trunk. The accessing nerves of the venous part of the heart passed into the nerve plexus of heart hilum at the heart base. Nerves approaching the heart extended epicardially and innervated the atria, interatrial septum and ventricles by five nerve subplexuses, i.e. left and middle dorsal, dorsal right atrial, ventral right and left atrial subplexuses. Numerous nerves accessed the arterial part of the arterial part of the heart hilum between the aorta and pulmonary trunk, and distributed onto ventricles by the left and right coronary subplexuses. Clusters of intrinsic cardiac neurons were concentrated at the heart base at the roots of pulmonary veins with some positioned on the infundibulum. The mean number of intrinsic neurons in the rabbit heart is not significantly affected by aging: 2200 ± 262 (range 1517-2788; aged) vs. 2118 ± 108 (range 1513-2822; juvenile). In conclusion, despite anatomic differences in the distribution of intrinsic cardiac neurons and the presence of well-developed nerve plexus within the heart hilum, the topography of all seven subplexuses of the intrinsic nerve plexus in rabbit heart corresponds rather well to other mammalian species, including humans., (© 2014 Anatomical Society.)

Background: The intrinsic neural plexus of the mouse heart has not been adequately investigated despite the extensive use of this species in experimental cardiology., Objective: The purpose of this study was to determine the distribution of cholinergic, adrenergic, and sensory neural components in whole-mount mouse heart preparations using double immunohistochemical labeling., Methods/results: Intrinsic neurons were concentrated within 19 ± 3 ganglia (n = 20 mice) of varying size, scattered on the medial side of the inferior caval (caudal) vein on the right atrium and close to the pulmonary veins on the left atrium. Of a total of 1,082 ± 160 neurons, most somata (83%) were choline acetyltransferase (ChAT) immunoreactive, whereas 4% were tyrosine hydroxylase (TH) immunoreactive; 14% of ganglionic cells were biphenotypic for ChAT and TH. The most intense ChAT staining was observed in axonal varicosities. ChAT was evident in nerve fibers interconnecting intrinsic ganglia. Both ChAT and TH immunoreactivity were abundant within the nerves accessing the heart. However, epicardial TH-immunoreactive nerve fibers were predominant on the dorsal and ventral left atrium, whereas most ChAT-positive axons proceeded on the heart base toward the large intrinsic ganglia and on the epicardium of the root of the right cranial vein. Substance P-positive and calcitonin gene-related peptide-immunoreactive nerve fibers were abundant on the epicardium and within ganglia adjacent to the heart hilum. Small intensely fluorescent cells were grouped into clusters of 3 to 8 and were dispersed within large ganglia or separately on the atrial and ventricular walls., Conclusion: Although some nerves and neuronal bundles of the mouse epicardial plexus are mixed, most express either adrenergic or cholinergic markers. Therefore, selective stimulation and/or ablation of the functionally distinct intrinsic neural pathways should allow the study of specific effects on cardiac function., (Copyright © 2011 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Background: Both normal and genetically modified mice are excellent models for investigating molecular mechanisms of arrhythmogenic cardiac diseases that may be associated with an imbalance between sympathetic and parasympathetic nervous input to the heart., Objective: The purpose of this study was to (1) determine the structural organization of the mouse cardiac neural plexus, (2) identify extrinsic neural sources and their relationship with the cardiac plexus, and (3) reveal any anatomic differences in the cardiac plexus between mouse and other species., Methods: Cardiac nerve structures were visualized using histochemical staining for acetylcholinesterase (AChE) on whole heart and thorax-dissected preparations derived from 25 mice. To confirm the reliability of staining parasympathetic and sympathetic neural components in the mouse heart, we applied a histochemical method for AChE and immunohistochemistry for tyrosine hydroxylase (TH) and/or choline acetyltransferase (ChAT) on whole mounts preparations from six mice., Results: Double immunohistochemical labeling of TH and ChAT on AChE-positive neural elements in mouse whole mounts demonstrated equal staining of nerves and ganglia for AChE that were positive for both TH and ChAT. The extrinsic cardiac nerves access the mouse heart at the right and left cranial veins and interblend within the ganglionated nerve plexus of the heart hilum that is persistently localized on the heart base. Nerves and bundles of nerve fibers extend epicardially from this plexus to atria and ventricles by left dorsal, dorsal right atrial, right ventral, and ventral left atrial routes or subplexuses. The right cranial vein receives extrinsic nerves that mainly originate from the right cervicothoracic ganglion and a branch of the right vagus nerve, whereas the left cranial vein is supplied by extrinsic nerves from the left cervicothoracic ganglion and the left vagus nerve. The majority of intrinsic cardiac ganglia are localized on the heart base at the roots of the pulmonary veins. These ganglia are interlinked by interganglionic nerves into the above mentioned nerve plexus of the heart hilum. In general, the examined hearts contained 19 ± 3 ganglia, giving a cumulative ganglion area of 0.4 ± 0.1 mm(2)., Conclusion: Despite substantial anatomic differences in ganglion number and distribution, the structural organization of the intrinsic ganglionated plexus in the mouse heart corresponds in general to that of other mammalian species, including human., (Copyright © 2011 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Background: Sheep are routinely used in experimental cardiac electrophysiology and surgery., Objective: The purpose of this study was to (1) ascertain the topography and architecture of the ovine epicardial neural plexus (ENP), (2) determine the relationships of ENP with vagal and sympathetic cardiac nerves and ganglia, and (3) evaluate gross anatomic differences and similarities of ENP in humans, sheep, and other species., Methods: Ovine ENP and extrinsic sympathetic and vagal nerves were stained histochemically for acetylcholinesterase in whole heart and/or thorax-dissected preparations from 23 newborn lambs, with subsequent examination by stereomicroscope., Results: Intrinsic cardiac nerves extend from the venous part of the ovine heart hilum along the roots of the cranial (superior) caval and left azygos veins to both atria and ventricles via five epicardial routes: dorsal right atrial, middle dorsal, left dorsal, right ventral, and ventral left atrial nerve subplexuses. Intrinsic nerves proceeding from the arterial part of the heart hilum along the roots of the aorta and pulmonary trunk extend exclusively into the ventricles as the right and left coronary subplexuses. The dorsal right atrial, right ventral, and middle dorsal subplexuses receive the main extrinsic neural input from the right cervicothoracic and right thoracic sympathetic T(2) and T(3) ganglia as well as from the right vagal nerve. The left dorsal is supplied by sizeable extrinsic nerves from the left thoracic T(4)-T(6) sympathetic ganglia and the left vagal nerve. Sheep hearts contained an average of 769 +/- 52 epicardial ganglia. Cumulative areas of epicardial ganglia on the root of the cranial vena cava and on the wall of the coronary sinus were the largest of all regions (P <.05)., Conclusion: Despite substantial interindividual variability in the morphology of ovine ENP, right-sided epicardial neural subplexuses supplying the sinoatrial and atrioventricular nodes are mostly concentrated at a fat pad between the right pulmonary veins and the cranial vena cava. This finding is in sharp contrast with a solely left lateral neural input to the human atrioventricular node, which extends mainly from the left dorsal and middle dorsal subplexuses. The abundance of epicardial ganglia distributed widely along the ovine ventricular nerves over respectable distances below the coronary groove implies a distinctive neural control of the ventricles in human and sheep hearts., (Copyright 2010 Heart Rhythm Society. All rights reserved.)

The aim of the present study was to map the topography of the porcine epicardiac nerve plexus (ENP) and to re-examine the total number and distribution of the porcine intracardiac ganglia and neurons. Eleven juvenile pigs (Sus scrofa domestica, 3-4 weeks of age) were examined employing histochemistry for acetylcholinesterase to reveal the ENP on total hearts. The nerves entered porcine epicardium at five sites: (1) ventro-medially to the origin of the superior vena cava, (2) dorsally to the origin of the superior vena cava, (3) among the pulmonary veins, (4) dorso-medially to the origin of the left azygos vein, and (5) ventrally to the left pulmonary vein. Within the porcine epicardium, the nerves connected to the groups of the intrinsic ganglia and proceeded into the discrete atrial and ventricular regions via five topographical pathways (subplexuses). In general, the porcine left atrium received nerves by four subplexuses, left ventricle by three subplexuses, right atrium and right ventricle each by two subplexuses. The estimated total number of the intrinsic ganglia per porcine heart was 362+/-52. About 55% of ganglia per porcine heart were accumulated on the left atrium while 36% on the right atrium. The percentage of ganglia within porcine ventricular and para-aortic regions was 7.6% and 1.6%, respectively. On average, porcine heart contained about 12,000 intrinsic neurons. In summary, the results of the present study imply that (1) the porcine epicardiac nerves are grouped into distinct topographical pathways, and (2) the porcine atria contain significantly more intrinsic ganglia and neurons compared to the ventricles.

The aim of the present study was to elucidate both the topography and architecture of the rat intrinsic cardiac nerve plexus, as this species becomes a frequent mammalian model for electrophysiological investigations of the intracardiac nervous system. Fifteen adult rats were examined employing histochemistry for acetylcholinesterase to visualize the intracardiac nerve plexus in hearts. Extracardiac nerves entering the rat heart were found amid aorta and pulmonary trunk as well as along both right and left cranial veins. The nerves from the arterial part of the heart hilum extended directly to the ventricles but the nerves from the venous part of the hilum interconnected among themselves forming a nerve plexus of the cardiac hilum on the heart base. Within the rat epicardium, intrinsic nerves clustered into six routes by which they selectively projected to different rat heart regions. Ventral wall of the ventricles was supplied by three neural subplexuses, dorsal ventricular wall--by one subplexus; each atrium received nerves from two distinct subplexuses. In conclusion, this morphological study demonstrates that rat intracardiac nerve plexus compounds to anatomical scheme of the same plexus in human, therefore rat is a usable model for electrophysiological experiments of the intracardiac nervous system.

The study was designed to determine the three-dimensional organization of the rat intrinsic cardiac neural plexus (ICNP) and to ascertain whether the rat heart undergoes a decrease in neuronal number with aging as has been reported for other mammalian species, including human. Juvenile (3-4 weeks of age, n = 14) and adult (more than 2 months of age, n = 23) animals were examined using enzyme histochemistry for acetylcholinesterase in order to visualize the ICNP in total hearts. The number of intrinsic cardiac neurons was estimated by counting nerve cells in serial sections of the atrial pieces stained with cresyl fast violet. The total number of intrinsic cardiac neurons in old rats was 6576 +/- 317. The juvenile animals contained significantly fewer such neurons, only 5009 +/- 332. Approximately 70% of all intracardiac neurons were amassed within the heart hilum, while 30% of the neurons were distributed epicardially. Within the interatrial septum, only 11 +/- 11 neurons were identified in the juvenile and 6 +/- 4 neurons in old rats. Extrinsic nerves entered the rat heart in both the arterial and venous parts of the cardiac hilum. The nerves from the arterial part of the cardiac hilum extended directly to the ventricles but the nerves from the venous part of the hilum formed a particular nerve plexus of the cardiac hilum on the heart base. Within the rat epicardium, intrinsic nerves clustered into six routes by which they selectively projected to different atrial and/or ventricular regions. In conclusion, this study provides a detailed description of the three-dimensional organization of the rat ICNP and contradicts the decrease in neuronal number with aging in the rat heart.

The intrinsic cardiac nervous system is known to be important both in the prevention and treatment of risky heart diseases. The present study was designed to determine the topography and 3-dimensional architecture of the intrinsic nervous system in the canine heart highlighting the differences of this system in dogs and humans. The morphology of the intrinsic cardiac neural plexus was revealed with a histochemical method using acetylcholinesterase in whole hearts of 18 mongrel dogs and examined with the aid of dissecting stereoscopic and contact microscopes. The present study identified 13 locations between the canine ascending aorta and pulmonary trunk, around the pulmonary veins, and on every side of the superior vena cava, through which mediastinal cardiac nerves accessed the canine heart. Intrinsic nerves from these locations extended within the canine epicardium by seven neuronal subplexuses. Intrinsic nerves and ganglia were found to be widely distributed in topographically consistent atrial and ventricular regions. In general, the canine right atrium, including the sinoatrial node, was innervated by two subplexuses, the wall of the left atrium by three, and the right and left ventricles by two subplexuses. Depending on the age of the animal, the number of intrinsic ganglia per one canine heart might range from 400 up to 1,500. By taking into account the ganglion size and potential approximate number of neurons residing inside a ganglion of a certain size, it was estimated that on average about 80,000 intrinsic neurons are associated with the canine heart. A comparative analysis of the morphological patterns of the canine and human intrinsic cardiac neural plexuses showed that the topographies of these plexuses may be considered as quite similar, but the structural and quantitative differences of the intrinsic cardiac neural subplexuses between dogs and humans are significant., (Copyright 2003 S. Karger AG, Basel)

Since many human heart diseases involve both the intrinsic cardiac neurons and nerves, their detailed normal ultrastructure was examined in material from autopsy cases without cardiac complications obtained no more than 8 h after death. Many intracardiac nerves were covered by epineurium, the thickness of which was related to nerve diameter. The perineurial sheath varied from nerve to nerve and, depending on nerve diameter, contained up to 12 layers of perineurial cells. The sheaths of the intracardiac nerves therefore become progressively attenuated during their course in the heart. The intraneural capillaries of the human heart differ from those in animals in possessing an increased number of endothelial cells. A proportion of the intraneural capillaries were fenestrated. The number of unmyelinated axons within unmyelinated nerve fibres was related to nerve diameter, thin cardiac nerves possessing fewer axons. The most distinctive feature was the presence of stacks of laminated Schwann cell processes unassociated with axons that were more frequent in older subjects. Most unmyelinated and myelinated nerve fibres showed normal ultrastructure, although a number of profiles displayed a variety of different axoplasmic contents. Collectively, the data provide baseline information on the normal structure of intracardiac nerves in healthy humans which may be useful for assessing the degree of nerve damage both in autonomic and sensory neuropathies in the human heart.

Concomitant with the development of surgical treatment of cardiac arrythmias and management of myocardial ischemia, there is renewed interest in morphology of the intrinsic cardiac nervous system. In this study, we analyze the topography and structure of the human epicardiac neural plexus (ENP) as a system of seven ganglionated subplexuses. The morphology of the ENP was revealed by a histochemical method for acetylcholinesterase in whole hearts of 21 humans and examined by stereoscopic, contact, and bright-field microscopy. According to criteria established to distinguish ganglionated subplexuses, they are epicardiac extensions of mediastinal nerves entering the heart through discrete sites of the heart hilum and proceeding separately into regions of innervation by seven pathways, on the courses of which epicardiac ganglia, as wide ganglionated fields, are plentifully located. It was established that topography of epicardiac subplexuses was consistent from heart to heart. In general, the human right atrium was innervated by two subplexuses, the left atrium by three, the right ventricle by one, and the left ventricle by three subplexuses. The highest density of epicardiac ganglia was identified near the heart hilum, especially on the dorsal and dorsolateral surfaces of the left atrium, where up to 50% of all cardiac ganglia were located. The number of epicardiac ganglia identified for the human hearts in this study ranged from 706 up to 1,560 and was not correlated with age in most heart regions. The human heart contained on average 836 +/- 76 epicardiac ganglia. The structural organization of ganglia and nerves within subplexuses was observed to vary considerably from heart to heart and in relation to age. The number of neurons identified for any epicardiac ganglion was significantly fewer in aged human compared with infants. By estimating the number of neurons within epicardiac ganglia and relating this to the number of ganglia in the human epicardium, it was calculated that approximately 43,000 intrinsic neurons might be present in the ENP in adult hearts and 94,000 neurons in young hearts (fetuses, neonates, and children). In conclusion, this study demonstrates the total ENP in humans using staining for acetylcholinesterase, and provides a morphological framework for an understanding of how intrinsic ganglia and nerves are structurally organized within the human heart., (Copyright 2000 Wiley-Liss, Inc.)

Background: Organs with the juxtaposed entry or exit of their communications have the sites which should be termed hila. Despite juxtaposed communications of the heart, the name hilum cordis is absent in the Nomina Anatomica., Results: This paper describes the hilum of the heart as the site bounded by the serous pericardium above heart base, ascending aorta and pulmonary trunk., Conclusion: Because the hilum of the heart is an equivalent to the hila of the other organs enveloped by the splanchnic mesoderm and thus far the term hilum cordis is absent in the Nomina Anatomica, the authors suggest to recognize a novel anatomical term-hilum cordis.

The paper describes the morphological pattern of neurons in the nerve plexus on the heart base of rats and guinea pigs. The nerve plexus, containing the investigated neurons, lies beneath the pulmonary arteries on the myocardium of the left atrium. This plexus is not covered by the epicardium. Therefore, contrary to the subepicardiac nerve plexus the investigated plexus was termed the nerve plexus of the cardiac hilum (NPCH). The morphology of neurons in the NPCH was revealed by ionophoretic injection of Lucifer Yellow via an intracellular microelectrode in vitro. A total of 139 neurons in 31 rats and 15 guinea pigs were labeled with dye and examined without chemical fixation with a fluorescent microscope. In the NPCH of both species, two types of neuron were revealed: unipolar and multipolar. The unipolar predominated (61.2% of the labeled nerve cells), whereas the multipolar were encountered less frequently (38.8% of the sampled neurons). Morphometrically, both types were similar and there was no significant difference in their length or width. The dyed neurons of both types were divided into separate groups according to indentations on the surface of their soma. Most of the unipolar nerve cells were encompassed into a group of "smooth' neurons because the surface of their soma was without noticeable prominences or grooves. The rest of the unipolar neurons were distinguished from the 'smooth' by various types of unevenness of the surface of their body, such as spine-like sprouts and grooves of different depth. The latter were attached to another group, the 'unsmooths', which made up 22.4% of all the labeled cells. The multipolar neurons were subdivided into two groups according to the number of long processes. The first group included neurons with a single long process, whereas the other group encompassed the nerve cells with two or more processes. The latter groups made up 31.6% and 7.2%, respectively, of the total number of labeled nerve cells. The obtained data have shown that the neurons in the NPCH of the rats and guinea pigs are morphologically different, and therefore it is proposed that the function of the neurons in the diverse groups may also be different.