Morphological and Immunohistochemical Patterns of the Intrinsic Ganglionated Nerve Plexus in the Mouse Heart ; Pelės širdies vidusieninio nervinio rezginio morfologija ir imunohistochemija
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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES MEDICAL ACADEMY Kristina Rysevaitė MORPHOLOGICAL AND IMMUNOHISTOCHEMICAL PATTERNS OF THE INTRINSIC GANGLIONATED NERVE PLEXUS IN THE MOUSE HEART Doctoral Dissertation Biomedical Sciences, Biology (01 B) Kaunas, 2011 The dissertation has been prepared during the period of 2007–2011 at the Institute of Anatomy, Medical Academy, Lithuanian University of Health Sciences Scientific Supervisor: Prof. Dr. Neringa Paužienė (Medical Academy, Lithuanian University of Health Sciences, Biomedical Sciences, Biology – 01 B) 2 CONTENTS LIST OF FREQUENTLY USED ABBREVIATIONS............................. 5 INTRODUCTION........................................................................................ 7 Actuality of the Study .............................................................................. 7 Aim and Objectives.................................................................................. 8 Originality and Implications .................................................................... 8 1. REVIEW OF LITERATURE................................................................. 9 1.1. Innervation of the Heart .................................................................... 9 1.2. Neurotransmiters in Cardiac Innervation........................................ 11 1.3. Physiology of Intrinsic Cardiac Neurons........................................
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Publié par
Publié le
01 janvier 2011
Nombre de lectures
61
Langue
English
Poids de l'ouvrage
16 Mo
LITHUANIAN UNIVERSITY OF HEALTH SCIENCES MEDICAL ACADEMY Kristina Rysevait MORPHOLOGICAL AND IMMUNOHISTOCHEMICAL PATTERNS OF THE INTRINSIC GANGLIONATED NERVE PLEXUS IN THE MOUSE HEART Doctoral Dissertation Biomedical Sciences, Biology (01 B) Kaunas, 2011
The dissertation has been prepared during the period of 2007– 2011 at the Institute of Anatomy, Medical Academy, Lithuanian University of Health Sciences Scientific Supervisor: Prof. Dr. Neringa Paužien (Medical Academy, Lithuanian University of Health Sciences, Biomedical Sciences, Biology – 01 B)
CONTENTS LIST OF FREQUENTLY USED ABBREVIATIONS ............................. 5 INTRODUCTION........................................................................................ 7 Actuality of the Study .............................................................................. 7 Aim and Objectives.................................................................................. 8 Originality and Implications .................................................................... 8 1. REVIEW OF LITERATURE ................................................................. 9 1.1. Innervation of the Heart .................................................................... 9 1.2. Neurotransmiters in Cardiac Innervation ........................................ 11 1.3. Physiology of Intrinsic Cardiac Neurons ........................................ 14 1.4. Intrinsic Cardiac Nerve Plexus ....................................................... 16 1.5. Immunohistochemical Characterization of Intrinsic Cardiac Neurons .................................................................................................. 23 1.6. Immunohistochemistry of Nerves and Nerve Fibers in Intrinsic Cardiac Nervous System ........................................................................ 25 1.7. Autonomic Control and Innervation of Mammalian Cardiac Conduction System ................................................................................ 27 1.8. Studies on Intrinsic Cardiac Nervous System by Kaunas Anatomists ............................................................................................. 33 2. MATERIALS AND METHODS .......................................................... 34 2.1. Material ........................................................................................... 34 2.2. Methods........................................................................................... 34 2.2.1. Total heart preparations .................................................... 34 2.2.2. Thorax-dissected preparations .......................................... 34 2.2.3. Whole-mount preparations................................................ 35 2.2.4. Immunohistochemistry...................................................... 35 2.3. Microscopic Examinations and Measurements............................... 37 2.4. Statistical Analysis .......................................................................... 38 3. RESULTS ............................................................................................... 39 3.1. Distribution of TH-IR and ChAT-IR Nerve Fibers in the Intrinsic Cardiac Nerves......................................................................... 39 3.2. Access of Mediastinal Nerves into the Mouse Heart ...................... 39 3.3. Architecture and Topography of the Itrinsic Cardiac Nerve Plexus .................................................................................................. 39 3.4. Morphology of Mouse Cardiac Ganglia ......................................... 40 3.5. Distribution of Immunochemically Distinct Intrinsic Cardiac Ganglia and Neurons.............................................................................. 41 3.6. ChAT-IR Neurons........................................................................... 41 3.7. TH-IR Neurons and Small Intensively Fluorescent (SIF) Cells ..... 42
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3.8. Distribution of ChAT-IR and TH-IR Nerve Fibers......................... 3.9. Distribution of ChAT-IR and TH-IR Nerve Fibers in the Sinuatrial and Atrioventricular regions .................................................. 3.10. Distribution of SP-IR and CGRP-IR Nerve Fibers ....................... 4. DISCUSSION ......................................................................................... CONCLUSIONS ........................................................................................ PUBLICATIONS ....................................................................................... RERERENCES ..........................................................................................
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LIST OF FREQUENTLY USED ABBREVIATIONS ACh – acetylcholine AChE – acetylcholinesterase AV – atrioventricular AVN atrioventricular node – cChAT – conventional form of ChAT CGRP – calcitonin gene related peptide ChAT – choline acetyltransferase CHT – high-affinity choline transporter DBH – dopamine beta hydrohylase DRA – dorsal right arial subplexus DVM – dorsal motor nucleus of the vagus ENP – epicardiac neural plexus GNPHH – ganglionated nerve plexus of the heart hilum HCN4 – hyperpolarization activated cyclic nucleotide-gated potassium channel 4 HH – hilum of the heart ICG – intrinsic cardiac ganglia ICNS – intrinsic cardiac nervous system ICNs – intrinsic cardiac neurons INP – intrinsic neural plexus IR – immunoreactive IVC – inferior vena cava LA – left atria LAu – left auricle LC left coronary subplexus – LD – left dorsal subplexus LOM – ligament of Marshall or neural fold of the left atrium LPV – left pulmonary vein MD – middle dorsal subplexus MPV – middle pulmonary vein NA – nucleus ambiguus NOS – nitric oxide synthase NE – norepinephrine NPY – neuropeptide Y pChAT – choline acetyltransferase of a peripheral type PGP 9.5 – protein gene product 9.5 PVs – pulmonary veins RA – right atria
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RAu – right auricle RPV – right pulmonary vein RV – right ventral subplexus SA – sinoatrial SAN – sinoatrial node SIF – small intensively fluorescent cell SP – substance P TH – tyrosine hydroxylase VAChT – vesicular acetylcholine transporter VC – vena cava VLA – ventral left atrial subplexus VRA – ventral right arial subplexus
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INTRODUCTION Actuality of the Study The intrinsic cardiac nervous system plays a crucial role in the regu-lation of heart rate, atrioventricular nodal conduction, and inotropism of atria and ventricles (Baumgart and Heusch, 1995; Cifelli et al., 2008; Feigl, 1998; Gorman et al., 2000; Randall et al., 2003; Tsuboi et al., 2000). Intrinsic ganglionated cardiac plexus integrates input from multiple sources including vagal efferent and afferent neurons, extrinsic sympathetic and spinal sensory neurons. The balance between the stimulatory sympathetic and inhibitory parasympathetic inputs are important for control of cardiac function (Ardell et al., 1991; Pauza et al., 1997b; Smith, 1999). It is widely recognized that autonomic nervous system modulates the cardiac electro-physiology of the heart and influences the genesis of cardiac arrhythmias or sudden cardiac death (Racker and Kadish, 2000). The recent elucidation of the complete mouse genome in combination with transgenesis and gene targeting in embryonic stem cells have opened excellent opportunities to breed numerous genetically modified mouse lines for experimental modeling and investigation of molecular mechanisms of the role of sympathetic-parasympathetic imbalance in cardiac arrhythmia predisposition (Kanazawa et al., 2010; Koentgen et al., 2010). The mouse, as an animal model, is widely used in cardiovascular researches. It have been developed transgenic mouse models, in which specific genes involved in cardiac development, are modified (Feintuch et al., 2007). It is reported that neuronal populations within the intrinsic cardiac neurons of adult mice and human are competently comparable as they exhibit a similar neuroche-mical phenotype manifested predominantly by choline acetyl-transferase and tyrosine hydroxylase (Mabe et al., 2006). Morphologically, the intrinsic cardiac nervous system corresponds to the neural ganglionated plexus, which is frequently subdivided according to layers of heart wall into epi-cardial, myocardial and endocardial (Marron et al., 1995). Cardiac neuro-anatomical investigations have demonstrated that intrinsic cardiac neural plexus may be considered as a complex of distinct ganglionated subplexuses (Pauza et al., 2000). Intrinsic ganglia related to particular subplexuses are distributed at specific atrial or ventricular regions around the sinuatrial node, the roots of caval and pulmonary veins, and near the atrioventricular node (Arora et al., 2003; Batulevicius et al., 2008; Pauza et al., 2002b; Pauza et al., 2000). In contrast to human and very few species of laboratory animal hearts, neuroanatomy of the mouse heart was rather poorly examined for a
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long while. In spite of very few investigations to this date (Ai et al., 2007; Hoard et al., 2008; Hoard et al., 2007; Mabe et al., 2006; Maifrino et al., 2006), both the distribution of sympathetic, vagal and sensory nerve fibers within the mouse intrinsic cardiac nerve plexus was almost unknown.
Aim and Objectives The aim of the present studywas to determine the structural organiza-tion of the intrinsic neural plexus in the total, non–sectioned mouse heart, identifying the immunohistochemistry of nerve fibers and neurons located within this intrinsic neural plexus. The objectives of the study: 1. To seek out the neural sources and neural pathways, by which mediastinal nerves supply the mouse heart. 2. To ascertain the structural organization of the mouse cardiac neural plexus. 3. the number of ganglionic cells in theTo assess the ganglion size and mouse hearts in order to compare this animal model with others. 4. To identify the distribution of cholinergic, adrenergic and peptidergic neural structures in the whole-mount mouse heart preparations using double immunohistochemical labeling. Originality and Implications This is the first detailed anatomical investigation of intrinsic gangliona-ted nerve plexus in the total (i.e., non-parceled and non-sectioned) mouse heart. This study demonstrates for the first the distribution of cholinergic, adrenergic and peptidergic nerve fibers and neurons in the whole-mount preparation of the mouse heart. The technique of the whole-mount prepa-ration allows precise identifying and mapping of the all intrinsic cardiac ganglia. We also identified their immunohistochemical properties and inter-connections of intrinsic cardiac neurons within the atria, interatrial and interventricular septa. This study mapped in detail the distribution of the mouse intrinsic cardiac nerves and ganglia and this may help in attempts to stimulate and/or to ablate selectively the functionally distinct intrinsic neural pathways for investigations of arrhythmic heart. The neuroanatomy of the mouse heart demonstrated in this study facilitates further investigations with this animal model and, thereby, it should increase our knowledge of physiologic roles of distinct intrinsic nerves and individual intrinsic ganglia.
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1. REVIEW OF LITERATURE 1.1. Innervation of the Heart Parasympathetic innervation of the heart is carried by the Xth cranial (vagus) nerve, which originates in the medulla oblongata. In the human, it is the superior, inferior, and thoracic branches of the vagus nerve that innervate the heart (Kawashima, 2005). The nerves supplying the heart join in the cardiac plexus, an accumulation of mixed neurons located cranial and dorsal to the heart. In the human heart, left and right-sided cardiac plexuses surround the brachiocephalic trunk and the aortic arch, respectively, and form part of a larger cardiac plexus that lies between the aorta and pulmo-nary trunk (Kawashima, 2005; Pauza et al., 1997a; Pauza et al., 2000). Nuclei in the ventral lateral medulla project via the vagus nerve to postganglionic neurons in the cardiac nerve plexus. Physiological, viral-tracing, and degeneration studies showed that neurons of the dorsal motor nucleus of the vagus (DVM) and of the nucleus ambiguus (NA) innervate the heart (Standish et al., 1994). Furthermore, it has been shown that the ganglion cluster located at the left atrium adjacent to the inferior vena cava receives input from both the DVM and NA, (Massari et al., 1995; Standish et al., 1995) whereas, the ganglion cluster located at the right pulmonary vein-left atrial junction receives fibers from the NA only (Massari et al., 1994). A submacroscopic anatomical investigations of the human extrinsic cardiac nervous system showed that (1) the superior, and middle cervical, and the cervicothoracic (stellate) ganglia, composed of the inferior cervical and 1st thoracic ganglia, were mostly consistent sources of symphatetic imputs to the heart; (2) the superior, middle, and inferior cardiac nerves innervated the heart by simple following the descent great arteries; (3) the thoracic cardiac nerve in the posterior mediastinum followed a complex course because of the long distance to the middle mediastinum; (4) the cranial cardiac nerve and branch tended to distribute into the heart medially, and the caudal cardiac nerve and branch tended to distribute into the heart laterally; (5) the mixing positions (cardiac plexus) of the sympathetic cardiac nerve and the vagal cardiac branch, as well as the definitive morphology of brachial arteries with the recurrent laryngeal nerves, tended to differ on both sides (Kawashima, 2005). The dorsal right atrial, right ventral and middle dorsal subplexuses of the sheep intrinsic cardiac nervous system (ICNS) receive the main extrinsic neural input from the right cervi-cothoracic and right thoracic sympathetic T2and T3ganglia as well as from the right vagal nerve. The left dorsal subplexus is supplied by sizeable
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extrinsic nerves from the left thoracic T4–T6sympathetic ganglia and the left vagal nerve (Saburkina et al., 2010). Convergence of inputs from extrinsic cardiac (vagal and cardiopulmonary (CPN)) nerves on intrinsic cardiac neurons was investigated in the pig (Smith, 1999). There is a degree of asymmetry in the distribution of preganglionic symphatetic neurons in the cat medulla (Massari et al., 1995; Massari et al., 1994). Injection of a retrograde tracer into the atrioventricular (AV) ganglion of the cat results in the labeling of twice as many cells on the left side of the medulla as on the right side (Massari et al., 1995), whereas injection of a retrograde tracer into the sinoatrial (SA) ganglion showed asymmetrical distribution of labeled preganglionic neurons (Massari et al., 1994). Preganglionic neurons form synapses on postganglionic neurons in autonomic ganglia, and there is a substantial degree of "convergence" in the parasympathetic nervous system. However, there is considerable diversity in the degree of convergence and divergence among species, also among different autonomic ganglia and individuals of a single species (Wang et al., 1995). The cardiac ganglion of the cat appears to be a degree of divergence rather than convergence: one preganglionic neuron projects on average to 13 or 32 postganglionic neurons (Wang et al., 1995). Similarly, studies by Massan and colleagues (Massari et al., 1995) indicate that a small number of neurons in the medulla are capable of controlling significant numbers of postganglionic cardiac neurons. The ultrastructural development of the cardiac ganglia in the chicken can be divided into three phases: (1) migration and aggregation of neuroblasts on days 3.5-5; (2) differentiating ganglia, days 5-10; (3) maturing ganglia, days 11 to hatching. The development of cholinergic me-chanisms precedes that of adrenergic mechanisms. As a consequence the parasympathetic-cholinergic control becomes functional and plays a role in cardiac function earlier than the sympathetic-adrenal neural control (Baptista and Kirby, 1997). In the mouse, nerve cells are first seen in the dorsal mesocardium at 10.5 days after fertilization. Well developed nerve tracts that can be identified using the neurofilament marker NF160D, extend through this region and reach the heart by 12.5 days after fertilization (Hildreth et al., 2008). Innervation of the outflow tract also occurs later in the mouse, with the first neural elements first seen between the separating aorta and pulmonary trunk. Staining against tyrosine hydroxylase (TH) and the parasympathetic neuron marker vasoactive cholinesterase transporter protein (VAChT) reveals the presence of sympathetic and parasympathetic neurons within the main nerves innervating the arterial and venous poles of the heart at 11.5
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days after fertilization and 12.5 days after fertilization (Hildreth et al., 2008). Sympathetic innervation has been shown to occur at later stages compared with parasympathetic innervation in both avian and mammalian species (Kirby et al., 1980; Shoba and Tay, 2000). In the mouse, autonomic efferent innervation precedes sensory innervation, as shown by the later appearance of calcitonin gene related peptide (CGRP) positive nerve fibers in comparison to neuropeptide Y (NPY) immunoactivity in the rat (Shoba and Tay, 2000). It has been proposed that cardiac parasympathetic neurons from the DVM and the NA project their axons to the intrinsic cardiac neurons and that the neurons from the DVM regulate cardiac inotropism, while those in the NA are related to heart rate control (Armour, 2008; Gatti et al., 1995). Some studies have shown the importance of the intrinsic cardiac ganglia in modulating relay between extrinsic autonomic nerves and the nodal tissues of the cardiac conduction system. Specifically, autonomic inputs from the left and right vagal branches are regulated to varying degrees by different cardiac ganglia, revealing complex interlinking pathways in the control of heart rate. These pathways have been revealed in studies of atrial fibrillation, which can be induced through electrical stimulation of auto-nomic nerves or cardiac ganglia (Patterson et al., 2005; Scherlag et al., 2005a; Scherlag et al., 2005b). A study in the dog (Hou et al., 2007) revealed that the right and left vagal trunks exert sympathetic control over heart rate, but that both inputs are modulated through specific, interlinking pathways, with these pathways differing between each node. There is, however, a degree of variation in these pathways between different individuals. Failure completely to attenuate the autonomic responses following ablation of these ganglia suggested that the modulation involves other ganglia within the cardiac network (Hou et al., 2007). 1.2. Neurotransmiters in Cardiac Innervation Regulation of cardiac function by the autonomic nervous system plays
crucial roles in the response of the organism to external stimulation. The sympathetic nervous system increases heart rate and the force of contraction via effects on the function of the sarcoplasmic reticulum within the cardio-myocytes and on ion channel activity. Preganglionic sympathetic axons from neurons in the T1-T5 spinal segments project to secondary sympa-thetic neurons that are located in the sympathetic chain, as well as the me-diastinal and intrinsic cardiac ganglia (Armour, 2008; Horackova et al., 1999; Kawashima, 2005; Richardson et al., 2006). Most of these sympa-thetic neurons contain TH, which is required for the synthesis of norepi-
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