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1.
J Mol Cell Cardiol ; 75: 188-97, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25101952

RESUMO

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.


Assuntos
Miócitos Cardíacos/citologia , Miócitos Cardíacos/ultraestrutura , Nó Sinoatrial/citologia , Nó Sinoatrial/inervação , Animais , Colina O-Acetiltransferase/metabolismo , Imunofluorescência , Átrios do Coração/ultraestrutura , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Miócitos Cardíacos/enzimologia , Fibras Nervosas/metabolismo , Fibras Nervosas/ultraestrutura
2.
Auton Neurosci ; 176(1-2): 32-47, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23403121

RESUMO

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.


Assuntos
Sistema Nervoso Autônomo/anatomia & histologia , Sistema de Condução Cardíaco/anatomia & histologia , Sistema de Condução Cardíaco/química , Nó Sinoatrial/anatomia & histologia , Animais , Sistema Nervoso Autônomo/química , Feminino , Coração/anatomia & histologia , Coração/inervação , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Imagem Óptica , Técnicas de Cultura de Órgãos , Nó Sinoatrial/química , Nó Sinoatrial/inervação
3.
Auton Neurosci ; 167(1-2): 61-5, 2012 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-22281046

RESUMO

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.


Assuntos
Axônios/patologia , Ablação por Cateter/efeitos adversos , Degeneração Neural/patologia , Pericárdio/inervação , Veias Pulmonares/patologia , Veias Pulmonares/cirurgia , Acetilcolinesterase/metabolismo , Animais , Axônios/ultraestrutura , Colina O-Acetiltransferase/metabolismo , Feminino , Imuno-Histoquímica , Masculino , Microscopia Eletrônica de Transmissão , Fibras Nervosas/patologia , Fibras Nervosas/ultraestrutura , Fibras Nervosas Mielinizadas/patologia , Veias Pulmonares/inervação , Células de Schwann/patologia , Células de Schwann/ultraestrutura , Ovinos , Toracotomia , Tirosina 3-Mono-Oxigenase/metabolismo
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