Neringa Pauziene

Morphology, distribution, and variability of the epicardiac neural ganglionated subplexuses in the human heart

Abstract

Morphology of the Intrinsic Cardiac Nervous System in the Dog: A Whole-Mount Study Employing Histochemical Staining with Acetylcholinesterase

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.

Nerve Supply of the Human Pulmonary Veins: An Anatomical Study

BACKGROUND Atrial ectopic discharges originating in the pulmonary veins (PVs) are known to initiate atrial fibrillation (AF), which may be terminated by catheter-based PV isolation. Because a functional relationship exists between cardiac autonomic effects and PVs in arrhythmogenesis, it has been suggested that discharges of the nerves that proceed to the PVs and interconnect with intrinsic ganglionated nerve plexuses are potential triggers of AF in man. OBJECTIVE This study sought to determine the characteristics and distribution of neural routes by which autonomic nerves supply the human PVs. METHODS We examined the intrinsic neural structures of 35 intact (nonsectioned) left atrial (LA)-PV complexes stained transmurally for acetylcholinesterase using a stereomicroscope. RESULTS The epicardial ganglionated nerves pass onto the extrapulmonary segments of the human PVs from the middle, left dorsal, and dorsal right atrial subplexuses. The left and right inferior PVs involved a lesser number of ganglia than the left and right superior PVs. Abundant extensions of epicardial nerves penetrate transmurally the PV walls and form a patchy neural network beneath the endothelium of PVs. The subendothelial neural meshwork with numerous free nerve endings, which appeared to be typical sensory compact nerve endings, was mostly situated at the roots of the 4 PVs. No ganglia were identified beneath the endothelium of the human PVs. CONCLUSION The richest areas containing epicardial ganglia, from which intrinsic nerves extend to the human PVs, are concentrated at the inferior surface of both the inferior and left superior PVs. Therefore, these locations might be considered as potential targets for focal pulmonary vein ablation in catheter-based therapy of AF.

Immunohistochemical characterization of the intrinsic cardiac neural plexus in whole-mount mouse heart preparations

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.

Comparative quantitative study of the intrinsic cardiac ganglia and neurons in the rat, guinea pig, dog and human as revealed by histochemical staining for acetylcholinesterase

This study was conducted to determine the overall number of intrinsic neurons distributed through-out the entire heart, in which most neurons are located inside of intramural ganglia and are hidden to observers. For this reason, we attempted to ascertain: (1) how the number of neurons located inside of intrinsic cardiac ganglion is related to its area, and (2) whether this relationship is dependent on age and species of animals. Hearts of rats, guinea pigs, dogs and humans were used to examine intramural ganglia stained histochemically for acetylcholinesterase (AChE). The number and parameters of neurons located inside of 104 ganglia were estimated in serial sections. Although the revealed intrinsic cardiac ganglia varied extremely in shape and size, two different types were identified: the globular and plain ones. In the plain ganglia, perikarya of side by side situated neurons were always intensely stained for AChE and, being clearly discernible, they could be reliably counted in any plain ganglia on total heart preparations using a contact microscope. Contrarily, neuron somata in the globular ganglia were densely packed above one another and their perikarya were almost indiscernible for the observer. Counting of neurons located inside of globular ganglia was possible in serial sections only. The largest cardiac ganglia were revealed in dogs, in which some globular ganglia containing up to 2000 neurons occupied more than 1 mm2. In spite of evident species-dependent differences with respect to frequency of large ganglia, the majority of intrinsic cardiac ganglia both in humans and animals were comparatively small, involved approximately 100-200 nerve cells and occupied an area ranging from 0.01 to 0.17 mm2. Overall, the number of neurons located inside of globular ganglion was related to its area (correlation coefficient = 0.82). However, the correlation coefficients between the globular ganglion area and its neuron number were unequal in different species (0.92 in guinea pig; 0.80 in dog; 0.72 in human; and 0.44 in rat) as well as dependent on (1) ganglion size (0.8 for ganglia equal to or larger than 0.17 mm2 and 0.6 for ganglia smaller than 0.17 mm2) and (2) age of specimens (respectively, 0.98 for juvenile and 0.87 for adult dogs; 0.71 for infants and 0.54 for aged human). In all examined animals and humans, the mean measurements of neuron perikarya were similar (on average, 23 microm in width, 32 microm in length, and 615 microm2 in area) and differences between them were statistically insignificant. However, neuron perikarya of adult dogs and aged humans were significantly larger than those revealed in the juvenile dogs and infants, respectively. Based on the data of this study, we concluded that the number of intrinsic cardiac neurons may be approximated in the total heart preparation via counting and measuring of intramural ganglia, contours of which are well-discernible following a histochemical reaction for AChE.

Epicardial neural ganglionated plexus of ovine heart: Anatomic basis for experimental cardiac electrophysiology and nerve protective cardiac surgery

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.

Anatomical study of the neural ganglionated plexus in the canine right atrium: implications for selective denervation and electrophysiology of the sinoatrial node in dog.

The aim of the present study was to elucidate the topography and architecture of the intrinsic neural plexus (INP) in the canine right atrium because of its importance for selective denervation of the sinoatrial node (SAN). The morphology of the intrinsic INP was revealed by a histochemical method for acetylcholinesterase in whole hearts of 36 mongrel dogs and examined by stereoscopic, contact, and electron microscopes. At the hilum of the heart, nerves forming a right atrial INP were detected in five sites adjacent to the right superior pulmonary veins and superior vena cava (SVC). Nerves entered the epicardium and formed a INP, the ganglia of which, as a wide ganglionated field, were continuously distributed on the sides of the root of the SVC (RSVC). The epicardiac ganglia located on the RSVC were differentially involved in the innervation of the sinoatrial node, as revealed by epicardiac nerves emanating from its lower ganglia that proceed also into the atrial walls and right auricle. The INP on the RSVC (INP‐RSVC) varied from animal to animal and in relation to the age of the animal. The INP‐RSVC of juvenile dogs contained more small ganglia than that of adult animals. Generally, the canine INP‐RSVC included 434 ± 29 small, 17 ± 4 medium‐sized, and 3 ± 1 large epicardiac ganglia that contained an estimated 44,700, 6,400, and 2,800 neurons, respectively. Therefore, the canine right atrium, including the SAN, may be innervated by more than 54,000 intracardiac neurons residing mostly in the INP‐RSVC. In conclusion, the present study indicates that epicardiac ganglia that project to the SA‐node are distributed more widely and are more abundant than was previously thought. Therefore, both selective and total denervation of the canine SAN should involve the whole region of the RSVC containing the INP‐RSVC. Anat Rec 255:271–294, 1999.

Topographic morphology and age-related analysis of the neuronal number of the rat intracardiac nerve plexus

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.

Neuroanatomy of the murine cardiac conduction system: a combined stereomicroscopic and fluorescence immunohistochemical study.

The mouse heart is a popular model to study the function and autonomic control of the specialized cardiac conduction system (CCS). However, the precise identity and anatomical distribution of the intrinsic cardiac nerves that modulate the function of the mouse CCS have not been adequately studied. We aimed at determining the organization and distribution of the intrinsic cardiac nerves that supply the CCS of the mouse. In whole mouse heart preparations, intrinsic neural structures were revealed by histochemical staining for acetylcholinesterase (AChE). Adrenergic, cholinergic and peptidergic neural components were identified, respectively, by immunohistochemical labeling for tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), calcitonin gene related peptide (CGRP), substance P (SP), and protein gene product 9.5 (PGP 9.5). Myocytes of the CCS were identified by immunolabeling of hyperpolarization activated cyclic nucleotide-gated potassium channel 4 (HCN4). In addition, the presence of CCS myocytes in atypical locations was verified using fluorescent immunohistochemistry performed on routine paraffin sections. The results demonstrate that four microscopic epicardial nerves orientated toward the sinuatrial nodal (SAN) region derive from both the dorsal right atrial and right ventral nerve subplexuses. The atrioventricular nodal (AVN) region is typically supplied by a single intrinsic nerve derived from the left dorsal nerve subplexus at the posterior interatrial groove. SAN myocytes positive for HCN4 were widely distributed both on the medial, anterior, lateral and even posterior sides of the root of the right cranial (superior caval) vein. The distribution of HCN4-positive myocytes in the AVN region was also wider than previously considered. HCN4-positive cells and thin slivers of the AVN extended to the roots of the ascending aorta, posteriorly to the orifice of the coronary sinus, and even along both atrioventricular rings. Notwithstanding the fact that cholinergic nerve fibers and axons clearly predominate in the mouse CCS, adrenergic nerve fibers and axons are abundant therein as well. Altogether, these results provide new insight into the anatomical basis of the neural control of the mouse CCS.

Morphologic pattern of the intrinsic ganglionated nerve plexus in mouse heart

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.