LITHUANIAN UNIVERSITY OF HEALTH SCIENCES

(1)

1

LITHUANIAN UNIVERSITY OF HEALTH SCIENCES

MEDICAL ACADEMY, FACULTY OF MEDICINE, INSTITUTE OF ANATOMY

On Itai Solomon

Comparative morphological study of epicardial nerve plexus

of ventricles in the human and experimental animals

MEDICAL INTEGRATED MASTER'S STUDY PROGRAMME

Thesis Supervisor

Dr. Inga Saburkina

(2)

2

Acknowledgments

• The authors sincerely thank Nida Rutkauskiene for technical assistance throughout the study. This study was fully supported by the Grant MIP-13037 from the Research Council of Lithuania.

• I would like to thank my supervisor Dr. Inga Saburkina, for her expert advice and encouragement throughout the master thesis process.

(5)

5

Abbreviation list

AV node - atrioventricular node SA node – sinoatrial node

SIF cells - small intensely fluorescent cells INP - intrinsic neural plexus

ENP - epicardiac neural plexus

B-MHC – Major Histocompatibility Complex Class B RV - right ventral ganglionated nerve subplexus LD - left dorsal ganglionated nerve subplexus MD - middle dorsal ganglionated nerve subplexus LC – left coronary subplexus

RC – right coronary subplexus

Pre-CA – region laying in front of the conus arteriosus DLV - dorsal left ventricular region

PBS - phosphate-buffered saline AChE - acetylcholinesterase PFA - paraformaldehyde solution SDs - standard deviations

Ao – aorta

CG - coronary groove CV - caudal vein

IVC - inferior vena cava LV - left ventricle PT - pulmonary trunk RV - right ventricle PT - pulmonary trunk

(6)

6 CV - caudal vein

LAV - left azygos vein LPV - left pulmonary vein MPV - middle pulmonary vein RPV - right pulmonary vein CdV - caudal vein

SVC - superior vena cava

RSPV - right superior pulmonary vein RIPV - right inferior pulmonary vein LSPV - left superior pulmonary vein LIPV - left inferior pulmonary vein Post-Ao – post-aortic region

Pre-Ao – pre-aortic region

VLCS – the region of the ventral left coronary sulcus VLV – ventral left ventricular region

VRCS – the region of the ventral right coronary sulcus VRV – ventral right ventricular region

DLCS – the region of the dorsal left coronary sulcus DRCS – the region of the dorsal right coronary sulcus

(7)

7

Thesis summary

➢ Title – Comparative morphological study of epicardial nerve plexus of ventricles in the human and experimental animals (sheep).

➢ Introduction – the use of animal models demonstrated a better understanding of the field of cardiovascular diseases and pathophysiology. The models continue to be an essential tool in the forecasting and prevention of those cardiac diseases. Sheep, as a big animal model with its cardiac resemblance to the human heart, is approved to serve as an appropriate pre-clinical animal model for cardiovascular studies. Regarding cardiac kinetics and performance, similar aspects in the sheep heart comparable to the human heart regarding the contractility and relaxation of the cardiac cardiomyocytes were noticed. It has been shown that human and sheep hearts share the same values ranges and frequencies in resting, systolic and diastolic actions of the heart. The main aspects of this comparative study are to process and demonstrate the entrance points of the nerves in the ventricles of human and ovine, to show the distribution pathways of the nerves through the ventricles and specifically through the epicardium, to demonstrate a topographic distribution of the ganglia and to perform its quantitative evaluation.

➢ The aim of the thesis- to conduct a comparative morphological study of the epicardial nerve plexus of ventricles in humans and sheep.

➢ Objectives of the study-

• To determine the entrance points of the nerves into the cardiac ventricles.

• To study the distribution pathways of the nerves through the epicardium of ventricles. • To estimate the distribution of the ventricular epicardiac ganglia (topography).

• To evaluate the total nerve width (sum) quantitatively across different epicardial subplexuses • To evaluate the total ganglion areas (sum) quantitatively across different ventricular

homological regions. ➢ The methodology-

• Five newborn German black-colored-faced lambs of both sexes were used. The hearts were perfused with phosphate-buffered saline (PBS) through a syringe needle inserted in the left ventricle.

• Five human hearts were taken from autopsy cases without cardiac problems or complications no more than 12 to 24 hours after death at the morgues of Hospital of Lithuanian University of Health Sciences. The hearts were obtained from fetuses of both sexes at the gestation age of 23 -40 weeks.

(8)

8 • Stain in Histochemically manner by acetylcholinesterase of pressure-inflated hearts on both

species.

• Statistical analysis was performed using R v4.0.2. Categorical data are presented as counts and percentages.

➢ General conclusions –

• Mediastinal nerves accessed the human and sheep ventricles similarly, but some topographical and structural interspecific differences exist.

• The main aspects of the distribution pathways of the nerves through the human and ovine ventricles do not fully differ in both species, neither structurally nor topographically.

• The distribution pattern of ventricular epicardial ganglia differs in humans and sheep.

• The distribution trend and the order of magnitude of the total nerve width across different epicardial subplexuses of both species are different and not comparable.

• The distribution trend and the order of magnitude of the ganglion sum areas across different ventricular areas of both species are different and not comparable.

Santrauka

➢ Pavadinimas – Žmogaus ir avies skilvelių epikardinio nervinio rezginio lyginamasis tyrimas. ➢ Įvadas – Eksperimentinių gyvūnų naudojimas tyrimuose leidžia geriau suprasti širdies ir

kraujagyslių ligas ir jų patofiziologiją. Avis, kurios širdis atitinka dydžiu žmogaus, yra populiarus fundamentinių širdies ir kraujagyslių sistemos tyrimų modelis. Avies širdis panaši į žmogaus savo fiziologija, taip par yra pastebėta analogija tarp žmogaus ir avies miokardo susitraukimo ir atsipalaidavimo procesų. Yra nustatyta, kad žmogaus ir avies širdžių atsipalaidavimas, sistolė ir diastolė yra panašios. Šis lyginamosios anatomijos tyrimas atskleidžia ir palygina nervų patekimo į žmogaus ir avies skilvelius bei jų plitimo skilvelių epikardu kelius, taip pat parodo nervinių mazgų pasklidimo ypatumus bei pateikia nervinių struktūrų kiekybinę analizę.

➢ Tikslas- palyginti žmogaus ir avies skilvelio epikardinį nervinį rezginį. ➢ Uždaviniai-

• Nustatyti nervų patekimo į skilvelius vietas.

• Ištirti epikardinių nervų išplitimo po skilvelius kelius.

• Atskleisti epikardinių nervinių mazgų pasklidimą (topografiją) skilvelių epikarde. • Kiekybiškai įvertinti suminį nervų plotį skirtinguose nerviniuose subrezginiuose.

(9)

9 • Kiekybiškai įvertinti suminį nervinių mazgų plotą skilvelių homologinėse zonose.

➢ Metodika-

• Tyrime panaudotos intaktinės (nesupjaustytos) penkios žmogaus naujagimių širdys ir penkios vokiečių juodagalvių veislės abiejų lyčių naujagimių ėriukų širdys, kuriose nervinės struktūros buvo išryškintos histocheminiu acetilcholinesterazės būdu.

• Morfometrinė nervų ir nervinių mazgų analizė buvo atlikta, naudojant stereoskopinį mikroskopą ir Axiovision 4.8.2 programinį paketą (Zeiss, Jena, Vokietija).

➢ Išvados –

• Tarpuplaučio nervai pasiekė žmogaus ir avies skilvelius panašiai, nors tam tikri tarprūšiniai topografiniai ir struktūriniai ypatumai egzistuoja.

• Pagrindiniai nervų plitimo žmogaus ir avies skilvelių epikardu keliai iš esmės buvo panašūs, nors egzistuoja tam tikri rūšiniai ypatumai.

• Nervinių mazgų pasklidimas skiriasi žmogaus ir avies skilvelių epikarde.

• Suminio nervo pločio pasiskirstymo tendencijos skirtinguose subrezginiuose ir ganglijų suminiai plotai homologinėse zonose yra skirtingi žmogaus ir avies skilveliuose ir jų negalima palyginti.

(10)

10

Introduction

Heart failure among cardiovascular diseases is the prominent reason for mortality and morbidity in the developed world (1). Both activation of the sympathetic nervous system and partial reduction in the activeness of vagal stimulation led to the development of heart failure (2) (3) (4). The previous reports demonstrate that increased parasympathetic tone on the heart directly relates to the decrease in myocardial contractility, refractory periods prolongation, and normalized cardiac rhythm, making chronic vagal stimulation of therapeutic importance value (3).

Additionally, chronic stimulation of the thoracic spinal cord induces significant remodeling of cardiac sympathetic innervation after myocardial infarction and improves left ventricular function together with degradation in spillover of myocardial norepinephrine (5).

The use of animal models has shown better understanding and comprehension in the field of cardiovascular diseases and especially in the field of cardiac pathophysiology (6). The models remain an important tool in the prediction and prevention of those cardiac diseases (7).

Sheep, as a large animal model with similarities to the human heart, is approved to serve as a good pre-clinical animal model for cardiovascular research (6) (8). In terms of cardiac functionality and kinetics, the contractility and relaxation of the sheep cardiac cardiomyocytes have high similarity lines comparable to the human heart, and both human and sheep hearts have a positive force-frequency relationship (6)_{. It has been demonstrated that human and sheep hearts share the same values ranges and}

frequencies in resting, systolic and diastolic actions of the heart (6)_.

Although the mammalian heart has species-dependent neuroanatomical differences (9) (10) (11) (12)

(13) (14) (15) (16)_{, recent neuroanatomic investigations have demonstrated that the intrinsic cardiac neural}

plexus with intrinsic cardiac ganglia are distributed at specific atrial or ventricular regions around the sinoatrial node, the roots of caval and pulmonary veins, and near the atrioventricular node (9) (10) (11) (12)

(13) (14) (15)_{. Unfortunately, the regional distribution of the ovine ventricular nerves and ganglia has still}

not been examined in detail.

Likewise, several authors have described intrinsic neuron distribution within the walls of ovine cardiac ventricles (17) (14), but the accurate number of those cells still remains unknown. And therefore, the aim of this comparative study is to observe and demonstrate the entrance points of the nerves in the ventricles of human and ovine, to show the distribution pathways of the nerves through the ventricles and specifically through the epicardium, demonstration of topographic distribution of the ganglia and its quantitative evaluation.

(11)

11

Aim and objectives of the thesis

➢ The aim of the thesis- to conduct a comparative morphological study of the epicardial nerve plexus of ventricles in humans and sheep.

➢ Objectives of the study-

• To determine the entrance points of the nerves into the cardiac ventricles.

• To study the distribution pathways of the nerves through the epicardium of ventricles. • To estimate the distribution of the ventricular epicardiac ganglia (topography).

• To evaluate the total nerve width (sum) quantitatively across different epicardial subplexuses • To evaluate the total ganglion areas (sum) quantitatively across different ventricular

(12)

12

1. Role of the Autonomic Nervous System in Cardiovascular Diseases

Autonomic nervous system activity exerts a strong influence in the cardiovascular field. According to many types of cardiovascular diseases, evidence has been approved to show a neurogenic factor, which may be acute, long-term catalysts, or both. Most eminent conditions which also has neural component and exacerbating cardiovascular dysfunction are coronary artery disease, arrhythmias, heart failure and hypertension (18)_{. The elemental mechanisms are complex and composed of many factors.}

There are prominent causes and factors which could be direct and intermediary and responsible for the adverse influence of unbalanced autonomic nervous system function, which influences healthy cardiovascular activity. Those factors variant in hemodynamic function, particularly elevated arterial blood pressure and neurotransmitters, direct effect on the vascular endothelium and myocardium.

Activation of the sympathetic nerve has been strongly implicated in provoking arrhythmias which can be life-threatening both in humans and animals (19). Infusion of catecholamines, stimulation of the peripheral and central adrenergic structures, and application of behavioral stress result in increased cardiac vulnerability in normal heart and in ischemic one (20). Those pro-fibrillatory influences are markedly reduced by beta-adrenergic receptor blockade.

Diversion in supraventricular arrhythmias can advance by activation of the autonomic nervous system. A striking surge in the activity of the sympathetic nerve appears within minutes of myocardial ischemia by experimental left anterior descending coronary artery occlusion, has been recorded and documented by direct nerve recording measurements and recently by complex demodulation of heart rate variability. The increase in sympathetic nerve activeness is associated with increased vulnerability to ventricular fibrillation, has been witnessed by the spontaneous appearing of arrhythmia, decreasing ventricular fibrillation threshold, and a t-wave alternans magnitude increasing (21). in time of reperfusion, the second peak in vulnerability occurs, which is probably due to the washout of ischemic byproducts from the myocardium.

Stellectomy blunting the increasing vulnerability to ventricular fibrillation during occlusion but enlarges during reperfusion. These observations coincide with the comprehension that adrenergic factors are mandatory in arrhythmogenesis in time of ischemia and that stellectomy enhances reactive hyperemia during reperfusion which results in a higher release of pro-fibrillatory ischemic byproducts. The mechanism in which sympathetic nerve activity increases the cardiac vulnerability in a healthy heart and ischemic one is highly complex. The leading indirect effects involved afflicted oxygen supply-demand ratio due to elevated cardiac metabolic activity and coronary vasoconstriction, especially in vessels with endothelium been injured and in the presence of altered preload and afterload. The

(13)

13 influences which are direct pro-fibrillatory and affect cardiac electrophysiological function are contributors to the order in impulse development, conduction, or both.

Increasing catecholamines levels stimulate beta-adrenergic receptors, which eventually modify adenylate cyclase activity and the flux of intracellular calcium. Those actions are probably conducted and mediated by the cyclic nucleotide and the protein kinase regulatory cascade, which is able to disturb the spatial heterogenicity of calcium transients and eventually cause an elevation in the scattering of repolarization. The influence effect is consequently an increasing vulnerability to ventricular fibrillation.

Contrariwise, stellectomy procedure has been proven to be anti-fibrillatory as this technique reduces cardiac sympathetic drive. Activation of the Vagus nerve decreases the occurrence of ischemia-induced spontaneous ventricular fibrillation. Extensive experimental research demonstrated the direct effect of cardio-protection in the manner of vagus nerve activation. This includes stimulation of the vagus nerve, vago-mimetic agents administration, and vagotomy, which has been recently reviewed (22). The main result of the study can be summarized as follows. Vagotomy is pro-fibrillatory in the time of acute myocardial ischemia and reperfusion.

In an animal that is conscious, induction of stress decreases in cardiac electrical stability is significantly lowered when a blockage is conducted to vagus nerve activity by atropine. Electrical stimuli of the vagus nerves reduce the occurrence of fibrillation which linked with exercise superimposed on acute ischemia in animals that had a prior infarction. The prominent component of the anti-fibrillatory action of the vagus nerve basically results from antagonism of the destructive effects of sympathetic nerve action (23). The molecular and cellular base for this underlined antagonism appears to be presynaptic norepinephrine inhibition and activation of the muscarinic receptors, which cause inhibition of secondary messenger formation by catecholamines. Vagus nerve action is not effective in preventing reperfusion which induces ventricular fibrillation during fixed rate pacing. However, in time of spontaneous rhythm, the rate-reducing effect of the vagus nerve excitation reduces susceptibility to fibrillation by elevation of diastolic perfusion time and decreasing oxygen deficit of the myocardium, a process that reduces the number of ischemic byproducts in humans. There is no clear evidence for an antiarrhythmic effect of vagus nerve activation at the ventricular level.

In any case, circumstantial data suggest the part of the mechanisms described in experimental animals may operate in the clinical setting. Disturbance of or a reduction in either vagal tone has been assessed by heart rate variability, or during reflex activation of the nerve, as assessed by sensitiveness of baroreceptor in respond to phenylephrine infusion, are both connected with increase mortality and incidence of sudden death among patient which is post-myocardial infarction (24). The risk potential in blocking tonic vagus nerve activity during the acute phase of myocardial ischemia has been reported and

(14)

14 observed in the past (25) (26) and deal with atropine administration which triggered ventricular fibrillation.

As a summarization, activation of the vagus nerve is able to exert a significant effect on heart rhythm. Stimulation of the vagus nerve can provide an anti-fibrillatory influence by norepinephrine presynaptic inhibition, which gets released from adrenergic nerve endings and by direct muscarinic antagonism of second messenger formation. Additionally, Cardiac vagal tone protecting the heart against ischemia which induces susceptibility to ventricular fibrillation; it happens by reducing heart rate and attendant cardiac metabolic demands.

Electrophysiology direct effects of sympathetic nervous system stimulation modify pacemaker direction from sinus node to junctional region, shorten P-R interval, changing P-wave morphology, increase automaticity of Purkinje fiber, increases soon after depolarization, cause prolongation of Qt-interval on the body surface, increases TQ-depression and enhances reentry in time of acute myocardial infarction, it reduces the threshold of ventricular fibrillation, induces alternans of T-wave in the long QT-syndrome and in time of myocardial infarction (27).

Vagal tonicity manifests a rate-independent elevation in myocardial electrical stability, which leads to a reduction in vulnerability to ventricular fibrillation at the time of myocardial ischemia. Vagal nerve action causes a decrease in heart rate, an action that plays an essential role during myocardial ischemia and in time of reperfusion because it elevates diastolic perfusion time and decreases cardiac metabolic demand. Vagal activity enhancement has an extra anti-fibrillatory effect, and that is due to the antagonism of adrenergic influences. The basic principles for sympathetic–parasympathetic synergy is norepinephrine release inhibition from nerve endings and attrition in response to catecholamines at receptor sites. Essential effects in the activity of the vagus nerve may be corrupted if extensive bradycardia and hypotension ensue.

Myocardial infarction condition may modify the influences which take part by the autonomic nervous system and cause damage to the neural fibers (28). The main subject that has been reviewed is in order to discuss the aspects and impacts which autonomic nervous system activity exerts on cardiovascular health. The potentially destructive effect can manifest in many of the prominent forms of heart disease, including heart failure, hypertension, disease of the coronary artery, and susceptibility to life-threatening cardiac arrhythmias. In general, hyperadrenergic activity is harmful, and parasympathetic neural effects are cardioprotective because they act as antagonists to the destructive actions of excessive release of catecholamines (29).

Cell bodies or somata of the intrinsic cardiac neurons get perfused by a coronary artery which taking part in an obstructive process and may go through a pathological changing process over time in such a way that their activity becomes malfunction. Additionally, locally ventricular ischemia may

(15)

15 induce regional nerve sprouting or pathology; the latter is result in local sensory and motor neurite function. Nevertheless, the existence of nerves passing through transmural ventricular infarction has remained unimpaired by that state as the prominent intrinsic cardiac nerves are joined by their own rich blood supply arising from extracardiac sources.

Some chemicals such as peptides, purinergic agents, and hydroxyl radicals get released in high amounts from the ischemic myocardium, and that alter regional cardiac sensory neurite activity. A lot of cardiac afferent neurons are extensively affected on restoration in the manner of the arterial blood supply to their sensory neurites in time of reperfusion in its early stage. Apparently, it when the highest concentration of regionally accumulated metabolites becomes available to affect their sensory neurites. The various situations of ischemia-induced reflex modulation affect not only the cardio dynamics (30) but also on local flow of coronary arterial blood.

The current existing information shows that the symptoms that accompanied myocardial ischemia depend to a considerable extensive manner on the capacity of afferent neuron P1-purinoceptors to cause such an event, That their capacity to transduce myocardial ischemia becomes blunted in the presence of adenosine receptor blockadeaid to support to that allegation. Peptides that get released from the ischemic myocardium take a prominent supporting role in the appearance of such symptoms. Peripheral and central processing of cardiovascular sensory goes through a remodeling process during the development of heart failure. Heart failure is highly associated with elevated circulation release of catecholamines, and that is because of the global enhancement of sympathetic efferent neuronal tonicity.

It is known that chronic activity of adrenergic efferent neurons attending heart failure may cause downregulation of cardiac myocyte B-adrenoceptor activity in such a way that the response to adrenergic agonist becomes week. And that why extensive heart failure therapy, which involves blockage of the adrenergic receptor, has been considered in situations of cardiomyocyte B-adrenoceptor modulation and afterload reduction. It has been proved that B-adrenergic or angiotensin receptor blockade also pointed in select populations of intrathoracic cardiac neurons. However, the reduction process of ventricular norepinephrine content in canine models of heart failure, adrenergic efferent neurons during activation release sufficiently high quantities of catecholamines into the myocardial interstate to exerts ventricular contractility. And this is because, partly, retention of the cardiomyocytes cell surface B-adrenoceptor function in such a state.

Cardiomyocytes are not the only ones that possess B-adrenoceptors; in fact, intrathoracic neurons are also possessed it, while the latter is also sensitive to angiotensin II. Those B-adrenoceptor or angiotensin II receptor blockade has an impact on the neurons which reside the intrathoracic nervous system and make an indication, that those therapies share a common aim. For instance, the block of

(16)

16 angiotensin II receptor narrows the sympathetic efferent neuronal inputs to the SA node and to the ventricles, accounting partly for the concomitant reduction in heart rate and blood pressure attending such therapy. Those therapies lower the remodeling process such that the cardiac neuronal hierarchy undergoes during the evolution of heart failure as well (31).

2. Structure of the intrinsic cardiac nervous system

The main function of innervating the tissue of the heart by motor neurons is dependent to a significant extent on the capacity of afferent neurons, which is located in intrathoracic, nodose (32),_and

dorsal root ganglia to transduce the cardiovascular milieu. The Association of unipolar neurons with cardiac sensory neurites has been identified by anatomical means with both the nodose ganglia and the dorsal root ganglia (33), which from the C7 to T4 segment of the spinal cord (34). It also has been visualized in intrathoracic, extracardiac (35) (36), and intrinsic cardiac ganglia (36) (37). Such kinds of neurons exhibit various chemical markers.

When cardiac motor neurons are activated, an influence of heart rate and atrioventricular nodal conduction occurs, as well as atrial and ventricular inotropism (38). Cardiac parasympathetic efferent preganglionic neuronal somata identified in the past by both functional and anatomical means, primarily located (not exclusively) in the ventral lateral region of the nucleus ambiguous. A lesser amount is found in the dorsal motor nucleus and in the intermediate zone, which is located in between these two medullary nuclei. It has been suggested that cardiac motor neurons, which are located in the dorsal motor nucleus, may be mostly occupied with the regulation of cardiac inotropism, while those located in the nucleus ambiguous may be mainly occupied with regulating cardiac heart rate (39). These somata are projecting axons to parasympathetic efferent postganglionic neurons, which are found throughout the entire atrial or ventricular ganglionated plexus (40).

Sympathetic efferent preganglionic neurons of the spinal cord project axons through the T1-T5 rami to synapse with sympathetic efferent preganglionic cardiac neurons (41),_{which are found in both the}

superior and middle cervical ganglia, and additionally in the cranial poles of stellate ganglia. They are also found in mediastinal ganglia that lie alongside the heart, as well as each prominent intrinsic cardiac ganglionated plexus.

Postganglionic sympathetic somata in each intrinsic cardiac ganglionated plexus spread axons to various regions of the heart (42). Some specific adrenergic somata even project two axons through different cardiopulmonary nerves in order to innervate variant cardiac regions. This type of anatomical

(17)

17 order probably ensures that the somata of sympathetic efferent postganglionic neurons, which are located on the particularly intrathoracic ganglionic locus, will have a wide effect on various cardiac regions.

The density of the adrenergic efferent neurites is linked with these somata varies considerably throughout the ventricles. Their local anatomical density does not reflect the capacity of local neurons to cause regional inotropic. For example, the capacity of adrenergic postganglionic somata to increase regionally inotropic is markedly greater in the ventricular outflow tracts and pupillary muscles with no regard to the fact that the density of their adrenergic efferent postganglionic neurites is akin to that which located in other ventricular areas (38). This manner presents a problem if one is trying to equate the local tissue density of those neurites to the capacity of their associated somata in order to cause an influence of regional cardiac contractility function. Previous studies demonstrate that a lot of neuronal somata in individual intrathoracic ganglia, including those on the heart, spread axons exclusively to other neurons within the same ganglion. Other somata spread axons to neurons in other intrathoracic ganglia, while others spread axons (43) to central neurons.

A lot of intrathoracic local circuit neurons receive inputs through extra thoracic sources as well. Some of those intrathoracic, large-diameter somata (i.e., ∼30 μm) form rosettes within their respective ganglion (44) (37), together with their dendrites, which adjoining within the ganglionic center and projects axons to neurons in different intrathoracic ganglia, while others spread axons to central neurons. Additionally, A lot of intrathoracic regional circuit neurons get inputs from extrathoracic sources as well.

Intrinsic cardiac cholinergic efferent postganglionic neurons get direct synaptic inputs from medullary preganglionic neurons that are found specifically in the nucleus ambiguous, with lesser amounts being found in the dorsal motor nucleus and medullary regions in the middle of these two nuclei

(45) (8)_{. The cardiac cholinergic postganglionic neuronal population is, in fact, quite limited with}

consideration to all the neurons within the intrinsic cardiac nervous system. Sympathetic efferent postganglionic neurons participate in cardiac regulation, get inputs from caudal cervical and cranial thoracic spinal cord preganglionic neurons (41)_{. The former is found in stellate, superior cervical, middle}

cervical, mediastinal ganglia, and also in intrinsic cardiac ganglia. When activated to the maximum, those sympathetic efferent neurons elevate the cardiac chronotropism, dromotropism, and inotropism while at the same time reduce the left ventricular chamber end-diastolic volume (46).

Parasympathetic efferent postganglionic neurons, during activation, do the opposite action, including the suppression of ventricular inotropism. Additionally, studies conducted recently shows that a lot of the neurons in intrathoracic ganglia, in addition to those on the heart (47), constantly communicates with one another in such that reciprocal antagonism between adrenergic and cardiac cholinergic postganglionic neurons may be considered as the exception instead of the constant rule (48).

(18)

18 So, the wide population of intrathoracic neurons permanently interacting with each other and with the central neurons. Create temporarily dependent reflexes that regulate, and control extensionally determined overlapping cardiac regions.

Fig.1 - Model for the cardiac neuronal hierarchy, which demonstrates intrathoracic components (49)_.

The locally cardiac mechanosensory and chemosensory milieu is transduced by afferent neuronal somata found not only in nodose and dorsal root ganglia but additionally in intrathoracic intrinsic and extrinsic cardiac ganglia. This information engenders intrathoracic, as well as central (medullary and spinal cord) reflexes. The lower right-hand box indicates that circulating catecholamines influencing cardiomyocytes not only directly but also indirectly via intrinsic cardiac adrenergic neurons.

Recent studies show that the intrinsic cardiac afferent neurons transduce the local mechanical and chemical milieu of the heart, and additionally those major intrathoracic vessels (37) (43) to other neurons in their own ganglion, as well as to those in different intrinsic cardiac and intrathoracic extracardiac ganglia. Recent research indicates that the synaptic interactions that happen among intrinsic and intrathoracic extracardiac neurons utilize a variety of neurochemicals. The peripheral neurons communicate with the central neurons and, because of that, involve the complete cardiac neuronal hierarchy (19). Basically, the sensory neurons located in each prominent intrinsic cardiac ganglionated plexus transduce majorly, but not exclusively, the chemical milieu of regions throughout the heart (51)

(50)_{. It has been suggested that such sensory input signals are considered for the generally stochastic}

behavior occurring by many atrial and ventricular neurons (52).

It has been suggested that short-latency reflexes involved in modulating cardiac indices involve neuronal stomata, which are located near the cardiomyocytes, which lie in the intrinsic cardiac and mediastinal ganglia. Such kind of short-latency of intrathoracic reflexes apparently affects cardiac indices throughout every cardiac cycle.

(19)

19 In the manner of longer-latency intrathoracic reflexes involving intrathoracic neurons regulate cardiac efferent neurons for a few cardiac cycles, and it occurs after the starting event. Those various intrathoracic autonomic reflexes involve regional neuronal circuitry that generally displays memory (53) such that cardiac sensory information processed in time of one cardiac cycle regulates cardiac efferent neurons not only during that cardiac cycle but also in subsequent ones. Maybe such polysynaptic reflex loops allow amplification of the efferent neuronal inputs to the heart, additionally with the time-dependent effects described above relating to several cardiac cycles.

Certain intrinsic cardiac and intrathoracic extracardiac neurons are influenced by the tonicity of the spinal cord neurons (54). Other intrinsic cardiac neurons are under a tonicity influence of medullary neurons. Moreover, there is an intrinsic cardiac local circuit that obviously receives input signals from both of those central neuronal populations (53). It seems that the hierarchy of cardiac neuronal structure is organized to supply the flexibility necessary for beat-to-beat coordination of local cardiac indices through short-latency (the intrinsic cardiac ganglia), medium-latency (intrathoracic), and relatively long-latency feedback (spinal cord and brain) (55) (56)_{. The somewhat long-latency reflexes involving central}

neurons set the tonic statues of the peripheral nervous system.

The descending inputs to the intrinsic cardiac neurons derive from the preganglionic neurons of the medullary and spinal cord, and it directly influences a limited amount of intrinsic cardiac motor neurons, nevertheless at the same moment influencing many local circuit neurons in an indirect process

(53)_{. Additionally, the Spinal cord and medullary neurons interact with each other, and it is under the}

influence of neurons in the level of the insular cortex. The afferent neurons, which are located in the dorsal root cardiovascular ganglion, initiate spinal cord reflexes while the neurons of the nodose ganglion (56) are initiating the brainstem-derived reflexes. This kind of information exchange among neurons can also be indirectly influenced by sensorial inputs from receptors found in different body regions.

Latencies that are the shortest and are such centrally derived cardio-cardiac reflexes range from 125 – 350 ms (57), mainly affecting a change over more extended periods of time. The importance of these tonic central neuronal inputs to the intrathoracic nervous system is shown by the idea that a lot of intrathoracic neurons, additionally to those on the heart, no longer perform a spontaneous activity in the physiologic state when their connections to the central neurons are severed. Nevertheless, there are some intrathoracic sympathetic reflexes that receive tonic suppressor inputs from the spinal cord motor neurons in a way that they appear more prominent when they get disconnected from their central inputs.

When excitation occurs in the intrathoracic cardiac neurons that express catecholaminergic phenotypic properties (58)_{, it led to increase heart rate, Dromotropism, and force of contraction. It is}

(20)

20 occurring when groups of intrinsic cardiac neurons are exposed to locally applied adrenergic agonists or to nicotine. Intrinsic cardiac neurons, which express acetylcholinesterase or butyrylcholinesterase-like actions at the moment of activation, reduces those cardiac indexes (59). It has been thought by some researchers that neurons found in one cardiac ganglionated plexus yield motor control solely over adjacent cardiac regions. In that kind of scenario, neurons found in the right atrial ganglionated plexus solely modulate adjacent SA nodal tissue, despite the ones in the inferior vena cava, which inferior atrial ganglionated plexus is controlling the atrioventricular node (39) (40).

Neurons found in the ventral ventricular ganglionated plexus would selectively way reduce ventricular contractility. Neurons found in each prominent ganglionated plexus generate control over electrical and mechanical events in all cardiac chambers. That accompanied by the fact that neurons in every intrinsic cardiac ganglionated plexus are in continuous contact with each other (47).

Cholinergic neurons found in the right atrial ganglionated plexus, when chemically activated, yield control over the SA node, left atrium, Av node, and distant ventricular tissues (60). The sensory data are arising from the heart (61)_{and the major blood vessels initiating the peripheral and central reflexes,}

which monitoring cardiac motor neurons. This kind of control can be divided into two basic sections, first is the way in which afferent neurons transduce the cardiovascular milieu in a direct or indirect manner to the cardiac motor neurons. Second, the type and time scale of information spreads to such neurons. As part of the intrathoracic neurons are sensitive to excitatory or inhibitory amino acids (62), those reflexes take part in excitatory and inhibitory synapses. Their diverse latencies depend on the distance of afferent somata from where the target organ is found (distance of the control center from the heart) and the number of synapses they are using.

Direct transduction of cardiac mechanical events to cardiac motor neurons may show an unstable control state (63). The somewhat slow transduction of the cardiac chemical milieu via regional circuit neurons to cardiac motor neurons over many cardiac cycles impart longer-term and stable control. Short-term scaling properties of fast responding cardiac mechanosensory neurons yield reflexes which are short-latency that yield rapid control over a selected group of cardiac motor neurons. Their short distance to the first synapse applies differential activation of the cardiac motor neurons in time of specific phases of the cardiac cycle to create beat-to-beat coordination of heart rate and regional heart contractility (64). Those reflexes based on the short-loop mechanosensory process are under central neuronal control.

Arterial-cardiac reflexes are starting by mechanosensory neurites found in the arteries of the carotid and the intrathoracic aorta. The mechanosensory neurites associated with individual afferent neurons are found concentrated in the adventitia of the carotid bulbs and also in the inner arch of the thoracic aorta. They are sending wrong information of the arterial wall, which there they are located with

(21)

21 considerable fidelity through their nodose ganglion somata to neurons found on the nucleus of the solitary tract, causing initiation of short-latency medulla-based activation of the cardiac parasympathetic efferent preganglionic neurons (65). They apparently account for the idea that a lot of cardiac vagal efferent neurons show phase-related activity, which reflecting contemplate arterial events (55), thereby causing an influence on the parasympathetic efferent neuronal control of the sinoatrial node differentially through every cardiac cycle in order to initiate short-term heart rate variability (66). Part of the intrathoracic sympathetic efferent neurons also show phase-related action reflective of reflexes and initiated by carotid artery, aortic, or cardiac mechanosensory neurons.

Vena cava mechanosensory afferent neurons additionally regulate the cardiac efferent neurons through intrathoracic reflexes (67). Groups of afferent neurons spread the cardiac and arterial blood chemical milieu to intrathoracic and also to a higher central neuron; by doing it for a longstanding period, it reflects the normal slow-changing local chemical milieu (68). Exposing the sensory neurites of individual cardiac chemosensory neurons to elevated numbers of a chemical can reset their action for a certain period of time after the discharge of that chemical stimulus, apparently a memory indication. Due to their cause of relative numbers, reflexes that are chemosensory-based can affect a high amount of cardiac motor neurons over multiple cardiac cycles.

For those reasons, it seems that intrathoracic sympathetic efferent postganglionic neurons exhibit somewhat phase-related or stochastic behavior-based majorly on whether it transduces mechanosensory-vs chemosensory-based inputs, respectively. It is now understood that remodeling of the cardiac neuronal structure may either initiate or increase the severity of the cardiac disease. Specific intrinsic cardiac neuronal components remodel in time of evolution of heart failure (69) or after long-term dismissal of their central neuronal inputs. This is in consent with the comprehension that arterial baroreceptor transduction resets in the evolution of hypertension or in time of heart failure.

The high complex cardiac nervous system seems to withstand some linkage malfunction, such as happens when the arterial blood supply to part of its neurons becomes compromised. It seems that this process occurs because of the filtering capacity of its relatively large intrathoracic local circuit neuronal population acting to balance the cardiac efferent neuronal function in the presence of extrasensory inputs which emerge from an ischemic myocardium (70).

Despite irreversible cardiomyocyte or intrinsic cardiac neuronal damage, which are secondary to any narrowing in the supply of the arterial blood, this nervous system remodeling occurs in lack of cellular damage and, as such, represents state change that eventually may be retrieved. When myocardial ischemia is transduced to the second-order neurons through the cardiac neuronal hierarchy (2), certain

(22)

22 neurons may become sufficiently activated to affect suprabulbar neurons like the limbic system, hypothalamus, insular cortex, etc., and initiate symptoms.

The cardiac sympathetic and parasympathetic efferent preganglionic neurons get activated when a sufficient amount of centrally projecting cardiac afferent neurons transmits myocardial ischemia to central neurons. Myocardial ischemia starts cardiac depressor or augmentor reflexes, depends on the way alternations in the regional cardiac milieu are transmitted throughout the neuroaxis (71). The ischemic process, which occurs either in the anterior or posterior wall of the left ventricle, is transduced by both dorsal root and nodose ganglion afferent neurons. Bradycardia appears when a certain amount of nodose ganglion cardiac afferent neurons transducing such an event which activates parasympathetic motor neurons (56). Dorsal root ganglia afferent neuron excitation, which is induced by ischemia in a reflexive action, activated sympathetic efferent neurons which innervating the heart and the systemic vasculature (72) to initiate arterial hypertension. Additionally, Regional myocardial ischemia may induce influence on the ascending spinal cord pathways to affect medullary cardio motor neurons, still, another cardio-cardiac reflex.

By observation of the multiplicity of those reflexes, it is hard to prove the thesis that selected hurt rate changes can be associated with reflexes that started from a particular left ventricular area. Rather, functional and anatomical (63)_{data indicate that central reflexes initiated by local ventricular}

ischemia induce regionally specific or global (cardiac and systemic vasculature) effects depending on how that event is spread throughout the whole cardiac neuroaxis (71). In which way those reflexes acting to stabilize or destabilize the cardiac control in the presence of modified baroreceptor sensitivity (73) is still remains undiscovered.

Compromised locally coronary arterial flow may induce an effect on intrinsic cardiac neuronal function and initiate intrathoracic reflexes. The somata of neurons that are perfused by a coronary artery that taking part in an occlusive process may go through pathological change over time such that their function becomes compromised. Local ventricular ischemia may, additionally, induce local nerve terminal sprouting (74) or pathology, which results in the loss of local sensory and neurite's motor function. Nevertheless, the nerve viability, which coursing over a transmural ventricular infarction, stays unimpaired by that state as prominent cardiac nerves are accompanied by their own rich blood flow, which is coming from extracardiac roots. Some chemicals such as peptides, purinergic agents (75), and hydroxyl radicals (76) are getting released in increasing amounts from the ischemic myocardium, which modifies the activity of cardiac sensory neurite (77).

A lot of cardiac afferent neurons are progressively affected on the restoration of the arterial blood flow to their sensory neurites during the initial point of reperfusion, and it seems that it is when the

(23)

23 highest concentration of regionally accumulated metabolites becomes available to influence their sensory neurites. Peripheral and central processing of cardiovascular sensory inputs go through modification in the evolution of heart failure (78); additionally, The arterial baroreflexes are getting blunted.

There is an association between heart failure and increasing circulating amounts of catecholamines, and that is due to the global enhancement of the sympathetic efferent neuronal tone. It has been suggested that chronic activity of adrenergic efferent neurons attending heart failure downregulates cardiac myocyte B-adrenoceptor function in a way that their response to adrenergic agonist is getting obtunded. So, that why heart failure therapy which involves adrenergic receptor blockade, has been counted in terms of cardiomyocyte B-adrenoceptor modification and afterload reduction (79). Now, it is a fact that B-adrenergic or angiotensin receptor blockade is also targeting a certain population of cardiac intrathoracic neurons.

The reduction of ventricular norepinephrine content in canine models of heart failure and adrenergic efferent neurons in the time when it gets activated, it is releasing high amount of catecholamines into the interstitium of the myocardium (69)_{, and that, in order to enhance ventricular}

contractility, and this is partly due to the retention of cardiomyocyte cell surface B-adrenoceptor function in that kind of state. Cardiomyocytes are not the only ones that possess B-adrenoceptors; in fact, intrathoracic neurons also have it and also sensitive to angiotensin II.

A blockade of B-adrenoceptors and angiotensin II receptors which affects neurons inside the intrathoracic nervous system, suggests that those therapies share a common aim. For example, angiotensin II receptor blockade decreases sympathetic efferent neuronal inputs to the SA node and ventricles, which Accounting in part for the associated decrease in heart rate and blood pressure attending such therapy.

Those therapies minimize the remodeling that the cardiac neuronal structure undergoes during the evolution of heart failure as well. Neurons that are at the level of the insular cortex to the intrinsic cardiac nervous system may take part in the progression of cardiac arrhythmias (80) (81). A certain amount of extracardiac parasympathetic and sympathetic efferent neurons, when maximally activated, initiate atrial (82) or ventricular arrhythmias.

In Association with the previous information, ventricular fibrillation may occur when a sufficient amount of intrinsic cardiac neurons is activated by, for example, locally applied neurochemicals such as adrenoceptor agonists, endothelin I (83), or angiotensin II. From a therapeutic view, it may be essential that the enhancement of intrinsic cardiac neuronal activity secondary to their transduction of an ischemia myocardium can be overcome by elevating their spinal cord neuronal inputs (84)_{. Thus, the harmful}

(24)

24 results that activate a high amount of intrinsic cardiac neurons consequent to their transducing regional ventricular ischemia, which can be compliant to therapy.

Activation of a certain amount of intrinsic cardiac cholinergic efferent neurons innervating either the SA or AV node can suppress heart rate or AV nodal transmission, respectively. Although it has been pointed that neurons, which in the right atrial ganglionated plexus project entirely to the adjacent SA node, whereas the ones in the inferior vena cava - inferior atrial ganglionated plexus, a project entirely to the adjacent AV node (43), a cholinergic efferent neuron that controls SA nodal or AV nodal function are located throughout the intrinsic cardiac nervous system. Thus, AV nodal conduction postponed can occur to balance ventricular rate in the presence of atrial tachydysrhythmias by activating not only neurons in the right atrial ganglionated plexus but also the ones found in others as well.

Focal electrical stimuli, which are transduced to loci within an intrinsic cardiac ganglionated plexus, activate associated somata and also efferent and afferent axons of passage, whereas regionally applied chemicals affect adjacent somata. That, together with the occurrence that local circuit neurons throughout the intrinsic cardiac nervous system are in continuous contact, apparently accounts for the varied cardiac responses elicited between subjects by such interventions.

Atrial ventricular arrhythmias can be aggravated when a certain amount of intrinsic cardiac neurons become highly over-activated (85)_{; depending on the population of neurons that participated,}

these can degenerate into fibrillation. Thus, the removal of certain neuronal elements responsible for such events may be deliberated for rhythm control by eroding somata rather than axons of passage; long-term consequences may be expected as axons promptly reinnervate distal cardiac tissues after their sectioning (86). The functional interconnectivity showed among atrial and ventricular neurons makes it likely that ablating or stimulating a locus within one intrinsic cardiac ganglionated plexus or, in the same manner, an entire intrinsic cardiac ganglionated plexus, will lead to variable and even unexpected results among individuals (87). In that regard, it should be mentioned again that activating certain intrathoracic neuronal elements can also arouse ventricular or atrial arrhythmias. By targeting somata at prominent centrifugal and centripetal convergence points, nexus points within this structure may be more predictable, and long-term results will get progress (87) (62).

3. Topography of the cardiac nerve plexus

A particular presentation of the distribution, location and projections of the intracardiac ganglia has been given for the heart in couple species of mammals (37) (89) (12) (90) (91) (92) (36) (93) (94) (95) (96) (97) (98) (99)

(25)

25 myocardium and endocardium (97) (106) (18) (102) (99), which most of the ganglia are epicardial type while the septal ganglia are found on the inner surface part of the atria.

The location of the cardiac ganglionic cells in the right atrium collaborates with sinoatrial node control and neurons found in the region of the inferior vena cava, which modify AV conduction (108). The specific intracardiac ganglia anatomical distribution varies among different species (102). Additionally, Cardiac ganglia are spreads in different areas of the atria in a couple of mammalian species, surrounding the SA node, around the vena cava roots, pulmonary veins, interatrial septum, and in close proximity to the AV node. The typically cardiac ganglion composes of neurons, small intensely fluorescent cells (SIF cells), and satellite cells. The cardiac ganglia of mammals contain unipolar, bipolar, and multipolar neurons with various dimensions and shape patterns.

The satellite cells and neurons of the cardiac ganglia are primitively developed from neural crest cells that get migrated to the heart. In the time it is arriving at the outflow tract, the cells are divided into parasympathetic neurons and supporting cells in order to form cardiac ganglia (109).

The human intrinsic cardiac nerve plexus is divided extensively; a prominent part of its ganglia is being located on the posterior surfaces of the atria and the superior part of the ventricles. The atrial ganglia at the human heart were characterized on the superior surface area of the right atrium, on the upper surface of the left atrium, in the posterior surface area of the right atrium, in the posterior medial surface of the left atrium (the latter two fuse medially where they extend anteriorly into the interatrial septum) and to the lower and lateral part of the posterior - left atrium.

The ventricular ganglionated plexuses are located in fat which are surrounded the 18 aortic roots, at the origins of the left and right coronary arteries (the latter extending to the origins of the left anterior descending and circumflex coronary arteries), at the origin of the posterior descending coronary artery, near the origin of the right, acute marginal coronary artery and at the origins of the left obtuse marginal coronary artery (37) (94) which concluded that the human heart is innervated by seven sub-plexuses.

The right atrium was originally innervated by two sub-plexuses which are the left atrium by three, the right ventricle by one, and the left ventricle by three sub-plexuses. The maximum density of epicardiac ganglia was identified adjacent to the heart hilum, especially on the dorsal and dorsolateral surfaces of the left atrium, where up to 50% of all cardiac ganglia are found. The studies of epicardiac ganglia of the human fetuses demonstrate the topography and structural pattern of epicardiac neural plexus were typical for hearts of adult humans. The highest ganglion number comprising 77% of all counted ganglia was recognized on the dorsal atrial surface. The fetal epicardiac plexus in gestation term of 15-40 weeks compose 929±62 ganglia (104).

(26)

26 A three-dimensional presentation of the distribution and structure of the canine intrinsic cardiac nervous system was developed to characterize its full extent physiologically. Assemble of ganglia which associated with nerves and ganglionated plexuses has identified in certain points in epicardial fat and cardiac tissue. Distinct epicardial ganglionated plexus were constantly watched in four atrial and three ventricular regions, with sometimes, a neuron being found throughout atrial and ventricular tissues. Certain ganglionated plexus extended from the ventral to dorsal surfaces of the right atrium. Another ganglionated plexus, composed of three components, were identified in fat on the left atrial ventral surface. A ganglionated plexus was found on the mid-dorsal surface of the two atria, spreading ventrally in the interatrial septum. The fourth atrial ganglionated plexus was found at the origin of the inferior vena cava extending to the dorsal, caudal surface area of the two atria. On the cranial surface area of the ventricles, a ganglionated plexus that are surrounding the aortic root was recognized. This plexus spreads to the right and left main coronary arteries and the origins of the ventral descending and circumflex coronary arteries. The two other ventricular ganglionated plexuses were recognized near the origins of the right and left marginal coronary arteries (94)_.

Later, a study was performed on whole canine hearts to mark the differences of intrinsic neural plexus (INP) in human and dog (96)_{. There have been 13 locations identified between the canine}

ascending aorta and pulmonary trunk, which are around the pulmonary veins, and on every side of the superior vena cava, through which mediastinal cardiac nerves accessed the canine heart—the intrinsic nerves from nineteens locations spread within the canine epicardium by seven neuronal sub-plexuses.

The intrinsic nerves and ganglia were set to be vastly distributed in topographically consistent atrial and ventricular regions. The right atrium of the canine, which includes the sinoatrial node, was innervated by two sub-plexuses, the left atrium wall by three, and the right and left ventricles by two sub plexuses. Counting on the age of the animal, the number of intrinsic ganglia per one canine heart might range from 400 and up to 1500. By considering the ganglion size and the approximate potential number of neurons found inside a ganglion of a specific size, it was evaluated that, on average, about 80,000 intrinsic neurons are in Association with the canine heart.

A comparative analysis of the morphological structures of the canine and human intrinsic cardiac neural plexus demonstrated that the topography of these plexuses might be considered as kind of similar, but the structural and quantitative differences of the intrinsic cardiac neural sub plexuses between dogs and humans are significant (95) (96) (100) (94).

Sheep are constantly used in experimental cardiac electrophysiology and surgery, but it is mentioned that a possible distinct neural control of the ventricles in the human and sheep hearts (24). The intrinsic cardiac nerves extend from the venous part of the ovine heart hilum through the roots of the

(27)

27 cranial (superior) caval and the left azygos veins to the atria and ventricles through five epicardial routes, first, the dorsal right atrial, middle dorsal, left dorsal, right ventral, and ventral left atrial nerve sub-plexuses. The intrinsic nerves proceed from the arterial section of the heart hilum through the roots of the aorta and pulmonary trunk, which extend entirely into the ventricles as the right and left coronary sub plexuses. The sheep hearts comprise an average of 769±52 epicardial ganglia. Cumulative regions of epicardial ganglia in the root of the cranial vena cava and also on the wall of the coronary sinus were the biggest of all areas (99). However, substantial interindividual various variability in the morphology of the ovine epicardiac neural plexus (ENP), right-sided epicardial neural sub-plexuses which supplying the sinoatrial and atrioventricular nodes are mostly organized at a fat pad in between the right pulmonary veins and the cranial vena cava. This specific finding is in severe contrast with a solely left lateral neural input to the atrioventricular node of human, which is spread prominently from the left dorsal and middle dorsal sub-plexuses (99) (18).

The nerves were entering the epicardium of the porcine epicardium at five sites: ventromedially to the origin of the superior vena cava, dorsally to the origin of the superior vena cava, amongst the pulmonary veins, dorsomedial to the origin of the left azygos vein, and ventrally to the left pulmonary vein (98)_{. Certain ganglia and 20 interconnecting nerves were found to be concentrated in epicardial fat}

in five atrial and six ventricular regions of the porcine heart (89)_{. The five atrial ganglionic fields have}

been identified in the ventral right atrial, the right vena cava-right atrial, the dorsal atrial, the interatrial septal, and the left superior vena cava-left atrial ones. Six ventricular ganglionic fields were recognized in close distance to the roots of the aorta and pulmonary artery (craniomedial), extending proximity to the main coronary artery to the ventral interventricular and circumflex coronary arteries. Some ganglionic fields were also recognized around the origin of the dorsal interventricular coronary artery as well as the right man and right marginal coronary arteries; additionally, There was a scattering of neurons throughout the cranial interventricular septum (89).

Certain investigators split the porcine ENP according to the neural pathways (sub-plexuses): the left atrium received nerves by four sub-plexuses, left ventricle by three sub-plexuses, right atrium, and right ventricle, each by two sub plexuses (106). By evaluation, the total number of intrinsic ganglia per porcine heart was 362±52. Around 36% of the ganglia in the porcine heart were accumulated on the right atrium, while 55% of the ganglia were on the left atrium and the percentage of the ganglia within the porcine ventricular and paraaortic regions was 7.6% and 1.6%, respectively (98).

The nerves which entered the guinea pig heart were located both in the venous and arterial sections of the heart hilum (106), while the nerves which are from the arterial part of the heart hilum

(28)

28 proceeded into the ventricles, but the nerves from the venous part of the hilum formed a nerve plexus of the cardiac hilum located on the heart base.

Inside the guinea pig epicardium, the intrinsic nerves divided into six routes and proceeded to splits atrial, septal, and ventricular regions (106). The intracardiac neurons from adult guinea pigs were accumulated within 329±15 ganglia. The hearts of young guinea pig contained extremely fewer ganglia, only 211±27 (106). Between all recognized neurons in the guinea pig heart, 85-90% are found in ganglia (ganglionic neurons), the rest being isolated (individual neurons) (36). A remarkable similarity was found in the structure of the intracardiac nerve plexuses between a guinea pig and rat (97) (106) (110).

The extrinsic nerves entered the rat heart in both the arterial and venous parts of the cardiac hilum. Extracardiac nerves entering the rat heart were located amid the 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 specifically to the ventricles, but the nerves from the venous part of the hilum interconnected between themselves generate a nerve plexus of the cardiac hilum on the heart base. Inside the rat epicardium, intrinsic nerves clustered into six routes by which they selectively projected to different rat heart regions. The ventral wall of the ventricles was supplied by three neural sub-plexus, dorsal ventricular wall by one sub-plexus; each atrium received nerves from two distinct sub-plexuses (110)_.

Additionally, cardiac ganglia and vagal efferent projections distributions to cardiac ganglia in rats and mice were somewhat similar both qualitatively and quantitatively (107).

By using a tracer, cardiac ganglia of various sizes and shapes were marked in the sinoatrial node, atrioventricular node, and lower pulmonary vein regions on the dorsal surface of the atria. In every region, several certain ganglia formed a ganglionated plexus and plexuses, which located in different locations were interconnected by nerves.

Vagal efferent fibers branched within the cardiac ganglia, creating a complex network of axons and innervated cardiac ganglia with very dense basket endings around individual cardiac principal neurons (107).

Intrinsic cardiac ganglia of human can range in size from those containing a few neurons to a high number of ganglia measuring up to 0.5 X 1mm (37). The human heart was estimated to have more than 14,000 neurons, and the neural somata are different in size and shape. A lot of axon terminals in intrinsic cardiac ganglia comprise high numbers of clear, small circumscribed vesicles that create asymmetrical axodendritic synapses, whereas a few axons contained large, dense-core vesicles (37). Asymmetrical axodendritic synapses were common.

The canine intrinsic cardiac nervous system comprises a variety of neurons interconnected and communicates through plexuses of nerves. Intrinsic cardiac ganglia range in size from ones comprising

(29)

29 one or a few neurons along the course of a nerve to ones as big as 1 X 3 mm, estimated to comprise just a few hundred neurons. Intrinsic cardiac neuronal somata vary in shape and size, up to 36% containing multiple nucleoli.

Examination by electron microscope is demonstrating typical autonomic neurons and satellite cells in intrinsic cardiac ganglia (94). Many of their axon profiles contained high numbers of round, clear, and dense-core vesicles; Asymmetrical axodendritic synapses were very common (94). Almost 3,000 neuronal somata were estimated to comprise the intrinsic cardiac nervous system (ICNS) of the pig (89), certain ganglia comprise more than 100 neurons. Neuronal somata had dimensions of barely 33.1 (short axis) by 46.3 (long axis) microns (89). Calculations of different authors demonstrate that, on average, the heart of the porcine comprises around 12,000 intrinsic neurons (98).

An estimated amount of neuronal somata in guinea pig individual ganglia was 100-300 (36). The total amount of the intracardiac neurons, which been estimated per atria was 1,510±251 (111), in 22 examinations of the entire heart, a counting of 2,321±215 neurons were marked, and there was no significant change between young and adult animals.

In adult guinea pigs, almost 60% of the intracardiac neurons were distributed inside ganglia of not more than 20 neurons, but the ganglia of such size are accumulating only 45% of the neurons in young animals (106)_{. Ganglia in the guinea pig heart comprised three sub-populations of neurons which}

almost 80% of the ganglionic neurons was large as 15–40-micron diameters, around 20% had smaller diameter – less than 15 microns, and 5% of ganglionic neurons were very small – less than 20 microns

(91)_.

The total amount of intrinsic cardiac neurons in old rats was 6,576±317. Nevertheless, the juvenile animals contained significantly fewer neurons, only around 5,009±332, and around 70% of the entire intracardiac neurons were assembled within the heart hilum, while 30% of the neurons were distributed epicardially. Inside the interatrial septum, only 11±11 neurons were identified in the juvenile and 6±4 neurons in old rats (97).

4. Animal models in the experimental cardiology

A major cause of morbidity and mortality worldwide is known to be a result of cardiovascular disease. A significant achievement has been made in the last several years in the management of cardiovascular disease and in the use of experimental animal models in this field.

The animal models were categorized by small animals (e.g., mice or rats) and larger animal models (e.g., dogs and sheep) (112) (113) (114)_{. In the research process, when the practical clinical approach}

(30)

30 is attending. The initial results that have been tested on small rodents must be confirmed in a larger animal model that closely resembles human with the medical approach of high percentage in genetic conservation (115) (116) (117).

The most frequently used animal model species were canines and swine, while primates usually been excluded due to high cost. A vigorous debate has been processed over the question of which animal species is most closely resembles human. Before using dogs and pigs as experimental animals, important anatomical and functional differences and changes must be considered. For example, the circulation of the coronary arteries in pigs absent anastomoses between vascular branches while the dog's coronary circulation is highly collateralized (118). It has been described that when a young human heart gets evaluate in anatomic manners, it is more "pig-like," whereas when an older human heart with ischemic heart disease is described, it is more as "dog-like."

An ideal model of the human cardiovascular system with precise anatomical and functional accuracy does not exist, and that why researches and studies should not be based on a single particular animal model in addressing all related questions. Nevertheless, the differences between experimental laboratory models and human are progressively decreased as a result of weight matching between the heart/body of animal models and human (8)_.

The human heart and canine heart demonstrate similar features on both organ and cellular levels. The heart rate, body weight, and heart weight in canines are anatomical and physiological features that more resembles the human heart characteristics than the same features in mice, rats, and rabbits. Due to the size of the canine heart, almost the entire "in vivo" techniques that are used to assess the contractility of the human heart can be applied to those animal model's heart. When considering long-term studies and heart disease exploration using canines, financial aspects must be an integral part of consideration because using canines as animal models is drastically more expensive than using small rodents or cats. Furthermore, a prominent disadvantage of using canines as animal models is the complicated bureaucratic process of obtaining the necessary approvals for performing experimental procedures in those animals (8).

Sheep is considered as a large animal model that its heart has high similarities characteristics to the human heart and is fully approved to serve as a reliable pre-clinical animal in the research of the cardiovascular field. In the matter of molecular and cellular levels, myosin heavy chain isoform in the sheep heart is predominate (as 100%), which comparably match to the slow B-MHC in the human heart at very high rates (95%). In functionality terms, the kinetics of contractile and relaxation in cardiomyocytes of sheep heart are also similar to the cardiomyocytes of the human heart, and both hearts have a positive force-frequency relationship.

LITHUANIAN UNIVERSITY OF HEALTH SCIENCES