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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. [ABSTRACT FROM AUTHOR]

Background: Ganglionated plexuses (GP) are terminal parts of cardiac autonomous nervous system (ANS). Radiofrequency ablation (RFA) for atrial fibrillation (AF) possibly affects GP. Changes in heart rate variability (HRV) after RFA can reflect ANS modulation., Methods: Epicardial RFA of GP on the left atrium (LA) was performed under the general anesthesia in 15 mature Romanov sheep. HRV was used to assess the alterations in autonomic regulation of the heart. A 24 - hour ECG monitoring was performed before the ablation, 2 days after it and at each of the 12 following months. Ablation sites were evaluated histologically., Results: There was an instant change in HRV parameters after the ablation. A standard deviation of all intervals between normal QRS (SDNN), a square root of the mean of the squared differences between successive normal QRS intervals (RMSSD) along with HRV triangular index (TI), low frequency (LF) power and high frequency (HF) power decreased, while LF/HF ratio increased. Both the SDNN, LF power and the HF power changes persisted throughout the 12 - month follow - up. Significant decrease in RMSSD persisted only for 3 months, HRV TI for 6 months and increase in LF/HF ratio for 7 months of the follow - up. Afterwards these three parameters were not different from the preprocedural values., Conclusions: Epicardial RFA of GP's on the ovine left atrium has lasting effect on the main HRV parameters (SDNN, HF power and LF power). The normalization of RMSSD, HRV TI and LF/HF suggests that HRV after epicardial RFA of GPs on the left atrium might restore over time.

Fluorescent immunohistochemistry on the cardiac conduction system in whole mount mouse heart preparations demonstrates a particularly dense and complex network of nerve fibres and cardiomyocytes which are positive to the hyperpolarization activated cyclic nucleotide-gated potassium channel 4 (HCN4-positive cardiomyocytes) in the sinoatrial node region and adjacent areas around the root of right cranial vein. The present study was designed to investigate the morphologic and histochemical pattern of nerve fibres and HCN4-positive cardiomyocytes using fluorescent techniques and/or electron microscopy. Adrenergic and cholinergic nerve fibres together with HCN4-positive cardiomyocytes were identified using primary antibodies for tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), and the HCN4 channel respectively. Amid HCN4-positive cardiomyocytes, fluorescence and electron microscopy data demonstrated a dense distribution of nerve fibres immunoreactive for ChAT and TH. In addition, novel electron microscopy data revealed that the mouse sinoatrial node contained exclusively unmyelinated nerve fibres, in which the majority of axons possess varicosities with clear mediatory vesicles that can be classified as cholinergic. Synapses occurred without any clear terminal connection with the effector cell, i.e. these synapes were of "en passant" type. In general, the morphologic pattern of innervation of mouse HCN4-positive cardiomyocytes identified using electron microscopy corresponds well to the dense network of nerve fibres demonstrated by fluorescent immunohistochemistry in mouse sinoatrial node and adjacent areas. The complex and extraordinarily dense innervation of HCN4-positive cardiomyocytes in mouse sinoatrial node underpins the importance of neural regulation for the cardiac conduction system. Based on the present observations, it is concluded that the occurrence of numerous nerve fibres nearby atrial cardiomyocytes serves as a novel reliable extracellular criterion for discrimination of SA nodal cardiomyocytes using electron microscopy., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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.)

Aims: Pulmonary vein ganglia (PVG) are targets for atrial fibrillation ablation. However, the functional relevance of PVG to the normal heart rhythm remains unclear. Our aim was to investigate whether PVG can modulate sinoatrial node (SAN) function., Methods and Results: Forty-nine C57BL and seven Connexin40+/EGFP mice were studied. We used tyrosine-hydroxylase (TH) and choline-acetyltransferase immunofluorescence labelling to characterize adrenergic and cholinergic neural elements. PVG projected postganglionic nerves to the SAN, which entered the SAN as an extensive, mesh-like neural network. PVG neurones were adrenergic, cholinergic, and biphenotypic. Histochemical characterization of two human embryonic hearts showed similarities between mouse and human neuroanatomy: direct neural communications between PVG and SAN. In Langendorff perfused mouse hearts, PVG were stimulated using 200-2000 ms trains of pulses (300 μs, 400 µA, 200 Hz). PVG stimulation caused an initial heart rate (HR) slowing (36 ± 9%) followed by acceleration. PVG stimulation in the presence of propranolol caused HR slowing (43 ± 13%) that was sustained over 20 beats. PVG stimulation with atropine progressively increased HR. Time-course effects were enhanced with 1000 and 2000 ms trains (P < 0.05 vs. 200 ms). In optical mapping, PVG stimulation shifted the origin of SAN discharges. In five paroxysmal AF patients undergoing pulmonary vein ablation, application of radiofrequency energy to the PVG area during sinus rhythm produced a decrease in HR similar to that observed in isolated mouse hearts., Conclusion: PVG have functional and anatomical biphenotypic characteristics. They can have significant effects on the electrophysiological control of the SAN.

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: In treatment of atrial fibrillations (AF), radiofrequency ablation (RFA) at the pulmonary vein (PV) roots isolates AF triggers in the myocardial sleeves, but also can destroy PV ganglia and branches of the intrinsic cardiac nerve plexus., Aim: To determine the long-term impact of RFA at the PV roots on the structure of epicardial nerves located distally from the RFA site., Methods: Five black-faced sheep underwent epicardial RFA of the left and middle PV roots. Two to 3 months after RFA, we obtained samples of epicardial nerves from remote locations of the left dorsal (LD) neural subplexus that extends along the roots of the superior PVs toward the coronary sinus (CS) and dorsal left ventricle (LV). Right atrial epicardial nerves from the right ventral (RV) neural subplexus of the ablated animals and epicardial nerves from LD neural subplexus of five additional intact sheep were used as control. Nerve morphology was examined using histochemical, immunohistochemical and transmission electron microscopy., Results: Histochemical acetylcholinesterase staining did not reveal any epicardial nerve alterations. However, tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) staining showed clearly the reduced numbers of TH and ChAT immunoreactive (IR) nerve fibers within epicardial nerves derived from the remote LD subplexus; control samples from all examined animals were full of evenly distributed TH-IR and ChAT-IR nerve fibers. In sharp contrast to control nerves, numerous swollen or disintegrated axons and Schwann cells with pyknotic nuclei inside unmyelinated and myelinated nerve fibers were identified by electron microscopy of ultrathin sections of epicardial nerves from the CS and LV regions in all ablated animals., Conclusions: Degeneration of remote atrial and ventricular epicardial nerves is evident 2-3 months after epicardial RFA at the PV roots. Such nerves are likely to be non-functional. Therefore, long-term autonomic dysfunction is a potential risk of PV isolation by RFA., (Copyright © 2011 Elsevier B.V. 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.)

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.