Lithuanian University Of Health Sciences
Medical Academy
Anatomy Institution
Omar Khalil
THE MORPHOLOGY OF THE EPICARDIAL PLEXUS IN THE
VENTRICLES OF THE PORCINE HEART
Final master’s thesis
Medicine
Supervisor:
Dr. Inga Saburkina
Summary ... 3
Conflict of interest ... 4
Ethics Committee clearance ... 5
Abbreviation list ... 6
Terms ... 7
Introduction ... 8
1. Literature review ... 10
1.1 The morphofunctional description of the autonomic nervous system ... 10
1.2. The anatomy of the intrinsic cardiac nervous system and innervation of the ventricles ... 12
1.3. Porcine heart as a model for experimental neurocardiology... 15
2. Materials and methods ... 18
2.1 Heart preparations ... 18
2.2 staining of the epicardial nerve plexus ... 19
2.3 Microscopy of the epicardiac plexus ... 19
2.4.Statistical analysis of the epicardial nerves and ganglions ... 19
3. Results ... 20
3.1 The penetration points of the cardiac nerves in the epicardium of the ventricles ... 20
3.2 The distribution of the RV and LD subplexal nerves on the surface of the ventricle ... 22
3.2. The morphological differences between the RV and LD subplexuses ... 23
3.3. The morphological differences between ventral and dorsal surfaces of the heart ... 24
3.4 The morphological differences between the surfaces of the right and left ventricles ... 24
3.5. Topography and morphology of the intrinsic cardiac ganglions in porcine ventricles ... 26
4. Discussion ... 27
4.1 The distribution of ganglia and nerves on the ventricles of the porcine heart ... 27
4.2 The morphometry of the epicardial nerves and ganglia on the ventricles of the porcine heart ... 27
4. Conclusions ... 29
Summary
Omar Khalil. Final master thesis “The morphology of the epicardial plexus in the ventricles of the porcine heart”. Supervisor: Dr. Inga Saburkina; Lithuanian university of health sciences, medical
academy, faculty of medicine, anatomy institution.
The morphology of the porcine intrinsic cardiac nervous system in the ventricles of a porcine heart is still poorly understood. This study aims to investigate the morphology of the epicardial nerve plexus of the ventricles of the porcine heart.
The objective of the study are: (i) to study the penetration and distribution of the epicardial nerves and ganglions on the ventricles of a porcine heart, (ii) to perform morphometric analysis, (iii) compare the distribution of the nerves and ganglia on the surfaces of the left and right ventricles.
Material and methods: 5 newborn pigs were dissected, the hearts were prepared and histochemical staining with acetylcholinesterase was performed. The hearts were studied using dissecting stereomicroscope, the intracardiac nerves and ganglia on the ventricles were studied. Morphometric analysis was performed and the distribution of the intracardiac nerves and ganglia along the different surfaces of the ventricles were compared. A comparison was made with the results of previous studies on different mammalian hearts.
Results: The study shows an abundant distribution of intracardiac nerves and scarce distribution of intracardiac ganglia on the ventricles. The ventral surface exhibits a higher distribution of nerves and ganglia than the dorsal side where ganglia were not found. The left ventricle contains thicker and more numerous nerves than the right ventricle.
Conflict of interest
Ethics Committee clearance
Abbreviation list
ACh – acetylcholine.AChE – acetylcholinesterase. AVN – atrioventricular node.
CGRP – calcitonin gene related peptide. DRA – dorsal right arial subplexus.
DVM – dorsal motor nucleus of the vagus. ENP – epicardiac neural plexus
ICG – intrinsic cardiac ganglia.
ICNS – intrinsic cardiac nervous system. ICNs – intrinsic cardiac neurons.
IVC – inferior vena cava. LA – left atrium.
LAV- left azygos vein.
LD – left dorsal subplexus. LPV – left pulmonary vein. MD – middle dorsal subplexus. MPV – middle pulmonary vein. NA – nucleus ambiguus.
NE – norepinephrine. RA – right atria.
Terms
• Intrinsic cardiac nervous system.• Intrinsic cardiac neuron. • Intracardiac ganglion. • Epicardial plexus.
Introduction
The intrinsic cardiac nervous system controls heart rate, contractility, and is involved in various arrhythmic disorders of the heart [1]. It has been shown that in the state of myocardial infarction, the death of the cardiomyocytes release substances that stimulate the endings of the afferent nerves of the vagus (CN X), as well as that of the spinal nerves. The nerve inputs stimulate local intracardiac reflexes , in addition to brainstem and spinal cord reflexes. Myocardial infarction hyperactivates the sympathetic motor neurons, which are the main cause of ventricular arrhythmias and sudden cardiac death, the severity of the complications depends on the location of the infarction [2,3,4,5]. Early studies have shown that the stimulation of the parasympathetic nervous system decreases myocardial contractility, and prolongs the refractory period, hence normalizes the cardiac cycle [6,7,8,9]. On the other hand, the reduction of the sympathetic innervation of the heart through the excision of the inferior part of the stellate ganglion, has been proved to be associated with a decrease in the occurrence of malignant tachyarrythmias, but postoperative complications were noted [2]. The porcine heart has become a common model for studying and improving antiarrhythmic techniques of the epicardial ablation and pulmonary vein isolation [10,11]. Both the endocardium and epicardium of the pig heart unlike the myocardium possess a dense plexus of mixed autonomic and presumptive sensory nerves, with an uneven distribution throughout these cardiac tissues [12]. Moreover, The gross and microscopic anatomy of the porcine intrinsic cardiac nerve plexus have been studied [13], later studies came to map the topography of the epicardiac nerve plexus (ENP) pointing up the disposition of the intracardiac nerves and the connection among the groups of the intrinsic cardiac ganglia [14,15,16].
Aims and objectives
Aim
:
The following study aims to investigate the morphology of the epicardial nerve plexus of the ventricles of the porcine heart.
Objectives:
1. To investigate the penetration points of the cardiac nerves into the epicardium of the ventricles in a pig’s heart.
2. To study the distribution of the epicardial nerves in the ventricular epicardium in a porcine heart. 3. To study the distribution of ganglia in the ventricular epicardium in a porcine heart.
1. Literature review
1.1 The morphofunctional description of the autonomic nervous system
The autonomic nervous system (ANS) is the part of the nervous system that regulates involuntary functions. Examples are the heartbeat, the digestive functions of the intestines, control of respiration, and secretion by glands. The efferent limb is made up of preganglionic and postganglionic fibers and an autonomic ganglion [18]. The efferent limb is further subdivided based on its anatomic and physiological differences into sympathetic and parasympathetic components[18]. The activation of the sympathetic system increases the heart rate, arterial pressure, blood flow to the skeletal muscles, heart, and brain [19].The parasympathetic system depresses the central venous system and increasing the activity of the abdominal viscera. Preganglionic fibers of both the sympathetic and parasympathetic system are myelinated, whereas the post-ganglionic fibers are unmyelinated [18]. Both the divisions of the ANS innervate most of the organs in the body, usually with opposing effects. The effects may also be parallel as seen in the salivary glands [20].
The sympathetic nervous system has two parts the central and the peripheral parts. The central part is located in the lateral horns of the spinal cord between the level of C7 and Th1-L3 segments of the spinal
cord in the intermediolateral nucleus, nucleus intermediolateralis [20]. The peripheral part of the sympathetic nervous system consists of two symmetrical right and left sympathetic trunks, truncus sypatheticus dexter et sinister, stretching on both sides of the spine from the base of the skull to the coccyx where the caudal ends of both trunks meet to form a single common ganglion [18]. Each sympathetic trunk is composed of a series of nerve ganglia called paravertebral ganglia connected by a longitudinal interganglionic branches, rami interganglionares, that consist of nerve fibers. In addition to the ganglia of the sympathetic trunk, ganglia trunci sympathici, prevertebral ganglia lie between the paravertebral ganglia and the target organ (ganglion celiacum, ganglion mesentericum inferior et superior) [21]. The processes of cells located in the lateral horns of the thoracolumbar part of the spinal cord emerge from it through the anterior roots and, on separating from them, pass in the white communicating branches, rami communicantes albicans, to the sympathetic trunk, that is called the preganglionic pathway [18]. From the ganglia of the sympathetic trunk or from the prevertebral ganglia arise non-myelinated nerve fibers of the postganglionic pathways and pass to the blood vessels and viscera [20].
the anterior roots of the spinal cord to the paravertebral ganglia of the sympathetic trunk. Since the centers are situated at the level of the thoracic and upper lumbar segments, the white communicating branches (preganglionic fibers) are only situated between Th1 and L3 segments of the spinal cord [19]. The grey
communicating branches (post-ganglionic fibers) connect the sympathetic trunk with the spinal nerves, they lose the color after exiting the central nervous system and become unmyelinated. The cervical part of the sympathetic trunk is also connected with the cranial nerves. All the plexuses of the somatic nervous system contain therefore fibers of the sympathetic system in their bundles and nerve trunks [21].
The paravertebral sympathetic chain is divided into four parts: (i) a cervical part consisting of three ganglia (superior, middle, and inferior) supplying the head, neck, and thorax. The inferior cervical ganglion fuses with the first thoracic ganglion to form the stellate ganglion, (ii) a thoracic part consisting of series of ganglia from each thoracic segment. Branches from the thoracic ganglia of the sympathetic T1
-T4 chain supply the aortic, cardiac, and pulmonary plexus, (iii) lumbar part: situated in front of the lumbar
vertebral column as the prevertebral ganglia. Branches from the lumbar part form the coeliac plexus, (iv) pelvic part: lies in front of the sacrum and consists of the sacral ganglia [20]. A descending group of nerves stretch to the heart, and a group travel to the organs of the neck almost immediately from the site of origin, the descending group of branches of the cervical sympathetic trunk segment is formed by the cardiac branches of the superior, middle, and inferior cervical ganglia, nervi cardiac cervicales superior, medius, and inferior [20]. They descend into the thoracic cavity and together with the cardiac branches of the sympathetic thoracic ganglia of the sympathetic thoracic ganglia and branches of the vagus nerve contribute to the formation of the cardiac plexuses [21]. The preganglionic fibers originate in the lateral horns of the spinal cord in the upper fourth or fifth thoracic segments, passing the white communicating branches and then through the sympathetic trunk to the five upper thoracic and three lower cervical ganglia [18]. From these ganglia the postganglionic fibers which as the components of the cardiac branches of the superior, middle and inferior cervical and the thoracic ganglion reaching the heart muscle [18].
mid-brain beneath the cerebral aqueduct at the level of the superior colliculus, preganglionic fibers travel through the occulomotor nerve and reach the ciliary ganglion that is located in the orbit lateral to the optic nerve, postganglionic originating from the ciliary ganglion innervate the muscles of the iris, ciliary body, and the pupillary sphincter [18]. The superior salivatory nucleus is located in the pon inside the rhomboid fossa lateral to the motor nucleus of the facial nerve, preganglionic fibers originating from this nucleus travel through the facial nerve to the pterygopalatine ganglion as the major petrosal nerve and to the submandibular ganglion as chorda tympani, the post ganglionic fibers originating from the pterygopalatine ganglion innervate the lacrimal glands and the glands of the nasal cavity and palate, while those originating from the submandibular ganglion innervate the submandibular and sublingual salivary glands [18]. The inferior salivatory nucleus is located in the medulla in the rhomboid fossa and sends preganglionic parasympathetic fibers to the otic ganglion by the glossopharyngeal nerve, the post ganglionic fibers from the otic ganglion innervate the parotid salivary glands [19]. The dorsal motor nucleus in the medulla in the rhomboid fossa send preganglionic fibers through the vagus nerve to the terminal and intramural parasympathetic ganglia, the postganglionic vagal fibers innervate organs of the thorax, head, neck and the gastrointestinal tract [21].
In the pelvic part of the parasympathetic nervous system the preganglionic fibers reach the pelvic organs from the parasympathetic nucleus intermediolateralis that is located in the S2-S4 segments of the
spinal cord [20]. These preganglionic parasympathetic fibers travel through the ventral roots of the spinal cord then by the spinal nerve and ventral rami of the spinal nerve, reaching the pelvic cavity as the pelvic splanchnic nerve [20]. These nerves reach the inferior hypogastric plexus and pelvic ganglia, the post ganglionic fibers innervate the pelvic organs such as the bladder, the intestines and the rectum [21].
nerve terminals makes it possible for transmitters released from the nerve terminals of one division to diffuse readily to terminals of the other division, as well as to cardiac muscle cells. Moreover, the integrative function of the intrinsic cardiac neurons is under the tonic reflex of neuron from the insular cortex, brainstem, and spinal cord [26].
Intrinsic cardiac ganglia contain morphologically different neurons, including unipolar, bipolar, and multipolar types [27]. The extensive network of neuronal cell bodies receiving parasympathetic vagal input and comprising the intrinsic cardiac ganglia of the mammalian heart has long been known [28,29], yet the precise function of the network and the way in which it mediates vagal input are largely unknown. A detailed description of the location, distribution, and projections of the intracardiac ganglia has been provided for the heart in numerous mammalian species [14,15,17,18,31,32,33,34,35,36,37]. Cardiac ganglion neurons located in the right atrium are associated with control of the sinoatrial node and neurons in the region of the inferior vena cava modulate atrioventricular conduction [38]. There appear to be at least 3 different types of neurons in the mammalian cardiac plexus on the bases of morphology, histological studies have shown that a complex network of neuron exists within the mammalian cardiac ganglion. Postganglionic parasympathetic neurons are not the only neurons present and there is evidence suggesting that sensory neurons, interneurons, and efferent neurons are found in intracardiac ganglia.
Neural control of the heart is under the influence of the two major divisions of the autonomic system, the sympathetic and parasympathetic nervous system. The sympathetic and parasympathetic (vagal) nervous systems exert antagonistic effect on the heart and interaction between the two systems is well established [40]. The sympathetic system projects to the heart through the intrathoracic ganglia. Preganglionic sympathetic axons from neurons in the T1-T5 spinal segments project to secondary
sympathetic chain as well as the mediastinal and intrinsic cardiac ganglia [44,41,42,43]. Activation of the sympathetic nervous system causes an increase in heart rate, an increase in conduction velocity, and an increase in ventricular contractile force. The parasympathetic system, in contrast, counteracts the action of the sympathetic nerves, slowing the heart rate [45]. Studies suggest that the cardiac parasympathetic neurons from the dorsal motor nucleus of the vagus (DVM) and the nucleus ambigious (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 [44,46]. The activation of the parasympathetic division of the nervous system, which arises from the neurons in the medulla region of the brain stem, causes a decrease in heart rate, a decrease in conduction velocity through the AV node, and a decrease in the force of atrial and ventricular contraction [46]. Parasympathetic innervation of the heart is carried by the X CN, originating from the medulla oblongata In the human, it is the superior, inferior, and thoracic
vagus (DVM) and the nucleus ambigious (NA) innervate the heart as shown by physiological, viral tracing, and the generation studies [47], furthermore, it has been shown that the ganglion cluster located at the left atrium adjacent to the inferior vena cava (AV ganglion) receives input from both the dorsal motor nucleus of vagus (DVM) and nucleus ambiNA [48,49] whereas, the ganglion cluster located at the right pulmonary vein –left atrial junction-(SA ganglion) receives fibers from the NA only [50]. There is a degree of asymmetry in the distribution of preganglionic neurons in the medulla, the preganglionic neurons form synapses on postganglionic neurons in autonomic ganglia, and there is a substantial degree of "convergence" in the parasympathetic nervous system [42]. 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 [51]. Small intensely fluorescent (SIF) cells, which contain catecholamines, have also been shown to be present within mammalian intracardiac ganglia therefore both the summer and axon terminal and parasympathetic neurons may be under the physiological influence of catecholamines [52,53,54].
The sensory neurons associated with the cardiac function have been identified inside the nodose, C1-T4 dorsal root, mediastinal, and intrinsic cardiac ganglia [41,44,56,55]. Recent findings suggest that intrinsic sensory cardiac neurons are also involved in local neural circuits via their axonal projections to efferent neurons distribute within the same or neighboring intrinsic ganglia [44]. Possibly, this diversity of neurons composes an integrative neuronal network, which modulates extrinsic autonomic projections to the heart and mediates local cardiac reflexes [44,32]. Visceral cardiac afferent neurons exist in cardiac ganglia, which are identified by the presence of neuropeptide substance P (SP) and calcitonin gene-related peptide (CGRP) [57]. SP-containing nerve fibers are found surrounding blood vessels within the myocardium, and within the sinoatrial and atrioventricular nodes [58]. The presence of these afferents in close association with parasympathetic neurons has lead to postulation of the existence of a local reflex circuit within the heart [58].
In addition to parasympathetic efferent and sensory afferent neurons, there also exists a population of interneurons within the cardiac ganglia [62]. The axons of these cells may reside within its particular ganglion or travel out into another ganglion cluster. This arrangement has been found in most species, including the dog [59], guinea pig [60], and rabbit [61]. Inter- neurons within cardiac ganglia mediate lateral interactions between various ganglion neurons and allow a convergence of different inputs. Electrical stimulation of the stellate ganglion or the vagosympathetic trunk produces responses in ganglion cells, with variable latencies indicating polysynaptic connections [62].
endocardial [62]. Recent neuroanatomic investigations have demonstrated that the intrinsic cardiac neural plexus may be considered a complex of distinct ganglionated subplexuses [14]. Each subplexus originates from the specific site in the heart hilum, each subplexus has connectivities to the groups of ganglia located in the discrete epicardial regions, and the nerves of each sub- plexus spread in the distinct epicardial regions [14]. Intrinsic ganglia related to particular subplexuses are distributed at specific atrial or ventricular regions around the sinoatrial node, the roots of caval and pulmonary veins, and near the atrioventricular node. In humans, dogs, sheeps, rats, guinea pigs and mice, the LD subplexus, remarkably extends along the dorsal surface of the ventricles innervating both ventricles [17,37,71]. On contrary to that, the rabbit heart was not shown to have sufficient innervation of the dorsal surface by the LD subplexus as was shown in previous studies [64].
1.3. Porcine heart as a model for experimental neurocardiology
The pig is an important model today in experimental neurocardiology Studies used the porcine model to study the ontogeny of cardiovascular regulation [65], functional innervation of the neonatal heart [12]. The porcine heart has been used to study the functional properties of the intrinsic cardiac neurons [26,66], the effects of surgery on the intrinsic cardiac nervous system [13] and the capacity of the autonomic nerves influence the development of arrhythmias [18]. Recently, the porcine heart became a popular model in research aimed at improving clinical antiarrhythmic techniques of the epicardial ablation and pulmonary vein isolation [67,68]. A histochemical staining using acetylcholinecholinesterase was used by the scientists [17], in addition to that some other scientists used different staining methods like methylene blue staining [13].
Previous studies it was revealed that the porcine intrinsic cardiac neurons are found in different areas of the atrial and the ventricular epicardial tissue, as well as the interartrial septal fat [13].
locations porcine intracardiac nerves spread epicardially by five pathways. The left side of the heart is innervated by four (RV, MD, VLA, and LD) subplexuses, the left atrium is innervated by all four of them and it has the highest density of epicardial neurons, while the left ventricle is innervated by three (RV, LD, MD) subplexuses. On the other hand, both of the structures of the right side of the heart were innervated by two subplexuses each, the right atrium by the RV and DRA subplexuses, the right ventricle by RV and MD subplexuses [17].
Fig. 1. Schematic drawing of the base of the porcine heart to illustrate five neural inputs into epicardium (arrows with numbers) that were regularly found in the hearts examined. Within the epicardium, the nerves proceed by five routes (subplexuses), which are indicated by the abbreviations. Dotted line demarcates the heart hilum. DRA, dorsal right atrial
2. Materials and methods
The study was performed on 5 juvenile pigs of both sexes. The pigs were anaesthetized by a lethal dose of sodium thiopental (100 mg/kg) after an intraperitoneal injection of 1000 units of heparin.
2.1 Heart preparations
Thoracotomy was performed (fig.1), the newborn pigs were infused intracardially as has
been described by previous anatomical studies of the ENP (Pauza el al., 2000, 2002; Batulevicius et al., 2003). The heart was removed and the walls of the atrium were distended by a balloon-tip catheter inserted into atrial chambers throught the superior vena cava and left pulmonary vein. The pericardium, pulmonary arteries and mediastinal fat were removed from the bas of the heart to expose the neural plexus. The preparations were fixed in 4% paraformaldehyde solution 0.1 M phosphate buffer for 30 minutes. After the fixation, the hearts were washed for 1 hour at 4 C in isotonic solution containing hyaluronidase (0.5 mg/100ml, Serva) and tetraisopropylphosphoramide (Iso-OMPA, 0.5 mM, Sigma), and that solution is for inhibiting pseudocholinesterase (butyrylcholinesterase).
2.2 staining of the epicardial nerve plexus
Acetylcholinesterase was used to stain the ENP on total hearts. The hearts were incubated for 30-60 min at 4 C in the medium described by karnovsky and Roots (1964). Afterwards, the preparations were fixed in 4% paraformaldehyde in 0.1M phosphate buffer.
2.3 Microscopy of the epicardiac plexus
Using the software Axiovision rel. 4.7 (ZEISS, Germany) the hearts were studied. Each heart preparation was placed on a stage in distilled water and studied in the light from the fiber optic illuminators using dissecting stereomicroscope (MBS-10, Lomo, Russia) with 1.95-6x magnification (fig. 2). Photos of different views of the ventricles of the heart preparations were taken; anterior, posterior and lateral views of the left and right ventricles of the heart. The intrinsic ventricular ganglia were measured in mm2, and the measurement of the neuronal diameter was recorded in µm.
2.4.Statistical analysis of the epicardial nerves and ganglions
The data are presented as absolute numbers (n), percentages and mean (M) standard deviation (SD). The Kruskal-Willis test was used when comparing non-parametric data of multiple groups. Means of non-parametric data were compared with the Mann-Whitney U-test. Spearman’s correlation coefficient was calculated for the correlation analysis of non-parametric data. A P-value of <0.05 was used as a criterion for statistical significance.
3. Results
3.1 The penetration points of the cardiac nerves in the epicardium of the ventricles
The nerves of the right ventral subplexal nerves enter at the root of the cranial caval vein, then travel down from the ventral walls of both atria towards the interventricular septum where they pass the coronary groove entering the ventricular epicardium of the heart. Spreading in a tree-like pattern along the ventral surface of the heart, the innermost nerves remain adjacent to the interventricular septum travelling down towards the apex of the heart, some of the nerves penetrate deeper into the myocardium and endocardium while other nerves terminate before reaching the apex of the heart. The outermost nerves spread along the ventral and lateral surfaces of both ventricles.
2000 µm 2000 µm
5000 µm
Fig. 4. Macrographs of a porcine heart stained for acetylcholinesterase demonstrating the topography of the nerves of the RV (white arrows) and LD (black arrows) subplexuses. White
arrowheads indicate some intrinsic ganglia. All panels belong to the same
heart. VRV, ventral right ventricle; VLV, ventral left ventricle, DLV, dorsal left ventricle; DRV, dorsal right ventricle, LRV, later right ventricle; LLV, lateral
left ventricle; LAu, left auricle, RAu, right auricle; LAV, left azygos vein. Ventral (a) , dorsal (b), lateral left (c),
Fig.5. Example of the reconstructions of the ventral (left) and dorsal (right) view of the porcine heart stained for acetylcholinesterase to demonstrate the distribution of the RV and LV subplexal neves and their penetration into the ventricular epicardium. The macrographs of all 4 views of the heart are shown in fig. 4. The labellings are the same as fig. 6.
The left dorsal subplexal nerves enter at the root of the left azygos vein, then extend on the dorsal surface of the left atrium along the left azygos vein forming neural ganglionated network on its dorsal wall, from this network the nerves pass the dorsal coronary and travel to 3 different sites. (i) The majority of the nerves innervate the dorsal surface of the left ventricle (fig. 4b, fig. 5b), (ii) a smaller group innervating a small area of the ventral and lateral walls of the left ventricle (fig. 4c), (iii) the smallest group of nerves innervate the dorsal surface of the right ventricle (fig. 4b, fig. 5b).
3.2 The distribution of the RV and LD subplexal nerves on the surface of the ventricle
The RV subplexal nerves innervate mainly the surfaces of the ventral wall of the ventricle in addition to the lateral walls of both ventricles, while the LD subplexal nerves innervate the dorsal surfaces of the heart. However, some of the nerves originating from the LD subplexus tend to spread around the left ventricle supplying the middle, and sometimes the apical part of ventral surface of the left ventricle (fig. 6a). The innervation of the ventral right ventricular surface is restricted to the nerves of the RV subplexus (100%)(fig.2b). Both the LD and RV subplexal nerves innervate the ventral left ventricle. The RV subplexus however, dominates in its innervation (75.8%) over the innervation of the LD suplexus (22.2%)(fig. 6a,b)(fig. 5).
So, the RV subplexus purely innervates the ventral right ventricle. The nerves from the LD subplexus innervate the dorsal surface of the heart. The latter, supplies the dorsal surfaces of both the right and left ventricles. The dorsal surface of the heart is completely innervated by the LD subplexus.
Fig. 5. The percentages of distribution of the RV and LD subplexal nerves onto the ventral surface of the left ventricle.
75.80% 22.20%
Ventral Surface of the Left ventricle
Fig. 6. Drawing of the ventral and dorsal view of the porcine heart illustrating the distribution of the subplexal nerves of the LD(a) and RV(b) suplexuses and there percentages on the different surfaces of the ventricles. RA, right atrium; LA,
left atrium; VRV, ventral right ventricle; VLV, ventral left ventricle; DLV dorsal left ventricle; DRV, dorsal right ventricle; SVP, superior vena cava; IVC, inferior vena cava; Ao, aortic valve; PT, pulmonary valve.
3.2. The morphological differences between the RV and LD subplexuses
The average thickness of the RV subplexal nerves, 1385.9µm, is slightly greater than that of the LD subplexal nerves, 1286.5µm. Additionally, the sum of the width of neurons of the RV subplexus, 2944.422 µm, was also greater than that of the LD subplexus, 2544.048 µm. The number of nerves was also slightly greater, 21 nerves, originating from the RV subplexus in comparison to that originating from the LD subplexus 19 (table. 1).
This slight difference in the morphology of the neurons derived from the RV and LD subplexuses, has not shown to be of any statistical significance (P-value>0.05).
a
3.3. The morphological differences between ventral and dorsal surfaces of the heart
It was previously shown that there was no significant findings when comparing the average width of nerves supplying the ventricles of the heart. However, comparing the sum of the width of nerves and the average number of nerves between the ventral surface and that of the dorsal surface of the heart, we find that there was a statistically significant difference between them (P-value<0.05).
The average sum of the width of nerves innervating the ventral surface of the heart is 1717.610645.9630µm, which was significantly greater than that of the neurons innervating the dorsal surface, 1051.649545.7985µm (P-value<0.05). Additionally, when comparing the average number of neurons we find that it is higher in the ventral surface 12.54.2, than in the dorsal surface, 8.23.7 (P-value<0.05)(table. 1).
Therefore, the ventral surface of the heart exhibits a significantly higher innervation from the epicardial plexus than the dorsal surface (fig. 7).
3.4 The morphological differences between the surfaces of the right and left ventricles
The thickness of the nerves on the surface of the left ventricle is 1807.3043, which shows to be higher than that of the right ventricle, which is 961.9551µm. That great difference is of statistical significance (P-value<0.05)(table. 1).
The results also show a significant difference between the neuron average count on the surfaces of the left and the right ventricle. The average count on the surface of the left ventricle is 13 nerves higher than the count on the surface of the right ventricle 7.62.9. there is a substantial difference between the number of nerves distributed on the surfaces of the left and right ventricles (P-value<0.05)(table.1).
Figure. 7. Showing the number of nerves entering ventricular epicardium comparing the right ventricle with the left ventricle, the ventral surface with th dorsal surface.
Surfaces of
ventricles/subplexuses
Average width of nerves (µm)
Average sum of width of nerves (µm) per
heart
Average number of neurons (n) per
heart
Dorsal left ventricle 136.110.5 1415.4698520.7505 10.44.0
Dorsal right ventricle 114.68.9 687.8286261.0964 6.01.9
Ventral left ventricle 139.26.5 2199.1388472.2410 15.82.2
Ventral right ventricle 134.49.0 1236.0816369.0086 9.22.9
Dorsal surface 128.27.5 *1051.649545.7985 *8.23.7 Ventral surface 137.45.3 *1717.610645.9630 *12.54.2 Left ventricle 135.37.0 #1807.3043624.6878 #13.14.1 Right ventricle 132.45.4 #961.9551417.5069 #7.62.9 RV subplexus 138.95.9 2944.422500.7569 21.23.1 LD subplexus 128.56.5 2544.048464.5983 19.83.9 Whole surface 133.84.5 11026.991811.875 40.43.2
* - statistical significance comparing the dorsal and ventral sufaces (P-value<0.05). # - statistical significane comparing the left and right surfaces (P-value<0.05).
0 2 4 6 8 10 12 14
Right ventricle Left ventricle ventral surface dorsal surface
Number of nerves
3.5. Topography and morphology of the intrinsic cardiac ganglions in porcine ventricles
The number of the ventricular ganglia varied from 6-72 ganglia per heart. The ganglionic count the average and the average sum of the ganglionic area in was calculated. Some of the heart preparations tested negative for AChE and did not reveal ganglionic disposition. However, the ganglia were regularly found on 3 different regions of the ventral surface of the ventricles, the ganglia appeared regularly on 3 different regions, the ventral left ventricle (VLV), the ventral left coronary sulcus (VLCS) and the region laying next to the conus arteriosus (PreCa), no appearance of intracardiac ganglia on the dorsal surface of the ventricles.
The average ganglionic number was the highest on VLV 56 ganglions; slightly lower on VLCS 50 ganglions and the lowest on Pre Ca 34. Considering the number of ganglions a comparison between the average ganglionic area among all 3 regions, shows that there is a statistical significance, where VLV contains the smallest average ganglionic area 0.02273929 mm2, VLCS contains the largest average of ganglionic area 0.041542 mm2, while the average ganglionic area in Pre Ca stands between the two previous values at 0.03895882 mm2, the difference between the average ganglionic area on the different regions was of statistical significance (P-value<0.05)(table. 2).
In respect to the average sum of the ganglionic areas per heart was the highest on the Pre Ca region 0.6623 mm2. On the other hand, it was the lowest in the VLV region 0.25468mm2.
We can conclude that among all the ventricular regions, VLV contains the highed number of ganglions however; the size of the ganglions is considerably smaller than the ganglions on VLCS and Pre Ca.
Table 2. The number, area and sum of areas of the ganglia revealed histochemically for AChE and distributed onto the surface of the porcine ventricles.
PreCa VLCS VLV Average ganglionic number 341.2 503.7 564.1 Average ganglionic area (mm2) ^0.03895882 0.06968635 ^0.04154200 0.03732940 ^0.02273929 0.02055350 Average sum of ganglionic areas (mm2) per heart 0.3461833 0.3360055 0.6623000 0.7553315 0.2546800 0.2447560
4. Discussion
4.1 The distribution of ganglia and nerves on the ventricles of the porcine heart
The results of previous studies done on the porcine epicardial plexus suggested that the left ventricles is innervate by three subplexuses the RV, LD and MD, while the right ventricle innervation was thought to be done by only two subplexuses, the RV and MD [17]. The results of this study concur, and we add that the ventral and lateral surfaces of the porcine heart are innervated by three subplexuses (RV, MD and LD), while the dorsal surface is innervated by only two subplexuses (LD and MD).
The cardiac nerves did not access the ventricles of the heart between the ascending aorta and the pulmonary trunk [17], those results were consistent with the results of our study. Unlike the porcine nerves, in human, dog and rabbit hearts the cardiac nerves gained access to the ventricles passing between the ascending aorta and the pulmonary trunk [14,15,64].
It was stated that the porcine right ventricle is more densely innervated than the left one [12,69], however other studies revealed that the left ventricle was more densely innervated compared to the right one [12]. This study complements the finding published [12], we found that the left ventricle was significantly more innervated than the right ventricle.
Studies noted that the largest ganglionic clusters of the porcine ventricular ganglia are distributed on the basis of the pulmonary trunk, along the origin of the right and left coronary arteries [13,17], they estimated number of intrinsic cardiac ganglia between 200-700, the majority of the ganglia were found to be in the atria of the porcine heart [13,17]. This study focusing more on the ventricles we find that the ganglionic number range was 6-72, confirming that the minority of the ganglia is found in the ventricles. Based on the results of this study a hypothesis can be made, the ganglia adjacent to the atrium are thicker than those disposed further in the ventricles.
considerably heavy distribution of LD subplexal nerve on the dorsal surfaces of the left and right ventricles where the epicardial nerves spread abundantly.
The results of our study reveal that the highest and thickest distribution of epicardial nerves in a porcine heart were found on the left ventricle rather than the right. That is consistent with the results found in a rabbits heart [64].
4. Conclusions
1. The RV and LD subplexuses penetrated the heart through the venous part of the heart hilum, the RV subplexal entered at the root of the cranial caval vein, the LD subplexal nerves entered at the root of the left azygos vein.
2. The RV subplexal nerves spread on the ventral and lateral wall of the left and right ventricle, while the LD subplexal nerves spread on the dorsal surfaces of the left and right, and spread to innervate a small region on the ventral and lateral sides of the left ventricle.
3. The intracardiac ganglia are distributed on the ventral surface of the ventricles, intracardiac ganglia are the larger on PreCa and VLCS, but more numerous on the VLV.
4. The porcine intracardiac nerves have the heaviest distribution on the ventral surface of the ventricles, the left ventricles contains thicker and more numerous nerves than the right ventricle.
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