LITHUANIAN UNIVERSITY OF HEALTH SCIENSES
MEDICAL ACADEMYFACULTY OF MEDICINE INSTITUTE OF ANATOMY
RAMI AOUF
INNERVATION OF CARDIAC VENTRICLES IN SHEEP
Master thesis
Thesis supervisor Dr. Kristina Rysevaite-Kyguoliene
TABLE OF CONTENTS
1. SUMMARY ... 3
2. ACKNOWLEDGEMENTS ... 5
3. CONFLICT OF INTEREST ... 6
4. ETHICS COMMITTEE APPROVAL ... 7
5. ABBREVIATIONS ... 8
6. INTRODUCTION ... 9
7. AIM AND OBJECTIVES OF THE STUDY ... 10
8. LITERATURE REVIEW ... 11
8.1 Extrinsic innervation of the heart and intracardiac accesses of the mediastinal nerves . 11 8.2 Innervation of cardiac ventricles ... 12
8.4 Distribution and morphology of cardiac ganglia ... 13
8.5 Immunohistochemistry of nerves and cardiac neurons ... 14
9. MATERIALS AND METHODS ... 16
9.1 Materials ... 16
9.2 Whole-mount ventricular preparations ... 16
9.3 Fluorescent immunohistochemistry on ventricular preparations ... 16
9.4 Microscopic examinations, measurements and statistical analysis ... 17
10. RESULTS ... 19 10.1 Epicardial nerves ... 19 10.2 Intrinsic ganglia ... 22 11. DISCUSSION ... 25 11.1 Nerves ... 25 11.2 Ganglia ... 26 12. CONCLUSION ... 28 13. BIBLIOGRAPHY ... 29
1. SUMMARY
Master in AnatomyInnervation of cardiac ventricles in sheep Rami Aouf
Supervisor Kristina Rysevaite-Kyguoliene
Lithuanian University of Health Sciences, Medical Academy, Faculty of Medicine, Institute of Anatomy. Kaunas.
Aim of the study: The aim of our present study is to determine the innervation of cardiac ventricles in the ovine heart, using immunohistochemistry of nerve fibres and neurons located within intrinsic neural plexus.
Objectives: To identify the size of nerves within the whole mount preparations obtained. To assess the ganglion size and neuron groups. To analyse neuron size within ganglia of the ovine ventricles.
Method: 6 black-faced new-born lambs (3±0.5 kg) of either sex, were used in accordance with local and state guidelines for the care and use of experimental animals (permission no. 0206) and the EU directive. After thoracotomy the hearts where removed from the chest and perfused with 0.01M phosphate-buffered saline (PBS). Following perfusion, fixation with 4% paraformaldehyde (PFA) solution in PBS was proceeded. Tissue samples were placed with the epicardium face up, then stretched and pinned on a silicone elastomer (sylgard) pad. Afterwards, immunohistochemical reactions were performed using general, adrenergic and cholinergic markers. Preparations were analysed and images were acquired using a fluorescence microscope AxioImager Z1 with a digital camera AxioCamMRm and an ApoTome (both from Carl Zeiss, Gottingen, Germany).
Results: Findings show that the mean overall size of nerves within the epicardial layer of the ovine heart was 65.3 µm. We notice that the size of the nerves examined in the slides decreased as they descended the heart e.g. the base nerves were measured to have an average width of 108.8 µm in comparison to the apex which had 29.9µm. The largest measurement of a ganglion was taken on the anterior surface of the heart around the left epicardium with a measurement of 52.8µm. In comparison the smallest measurement was found on the posterior surface of the heart at the area of bottom apex at 1.4µm. We notice that the most abundant group of neurones found in the slices were numbers between 1-10
neurons per ganglia. As the group size increased the number of ganglia decreases and continued to decrease.
Conclusion: We notice with regards to nerve size, the largest diameters as shown in the results where on both aspects of the heart, anterior and posterior 189.5µm and 236.6µm respectively. The smallest results in comparison, were all shown to be on the posterior heart with a measurement of 2.17µm. The total overall difference between the largest and the smallest 234.43µm, showing that the diversity within the heart for the nerve fibres shows a high profile. The average size of a ganglion was 231.42± 2.69 µm2 .It was established that the largest mean ganglia size was 281.6µm2 and was located at the apex, base, middle slice. The lowest mean area of a ganglia was in the left epicardium with a measurement of 145.1µm2. We found 13 ganglia with 1-10 neurones, 8 ganglia with 11-20 neurones, 1 ganglion with 21-30 and 1 with 31-40 neurones respectively. The largest measurement of neuron length within the ganglia was taken at the anterior surface of the heart left epicardium with a measurement of 52.8µm. In comparison the smallest measurement was found on the posterior surface of the heart at the area of bottom apex measuring at 1.4µm. The overall mean length of neuron within a ganglia was 15.2±0.1 µm.
2. ACKNOWLEDGEMENTS
I would like to thank the Anatomy institute and LSMU for your continued pursuit of excellence. I would also like to thank my family for their support during this period.
3. CONFLICT OF INTEREST
There are no conflicts of interest.4. ETHICS COMMITTEE APPROVAL
Animal Research Center licence number LT-61-19-004, certified by the State Service for Food and Veterinary 2015.12.02.
5.
ABBREVIATIONS
ACh – acetylcholineAChE – acetylcholinesterase
ChAT – choline acetyltransferase
HCN4 – hyperpolarization activated cyclic nucleotide-gated potassium channel 4
NNOS – Neuronal nitric oxide synthase
PGP 9.5 – protein gene product 9.5
SAN – sinoatrial node
SIF – small intensively fluorescent cell
SP – substance P
TH – tyrosine hydroxylase
6. INTRODUCTION
Understanding the autonomic nervous system has a played a crucial and integral role in the progression of science. Clinically, when discussing arrhythmias, more and more therapy relies on our understanding of autonomic regulation of the heart. Other aspects of clinical research also apply specifically when discussing the effects of drugs on cardiac function. With more and more necessity for information regarding the cardiac innervation, we endeavoured to review this further.
Due to the nature of the cardiac nervous stimulation, we found that the majority of research publications focused on atrial innervation more than ventricular. This could be because both the sinoatrial node (SAN) and the atrioventricular node (AVN) are found in these locations. It could also be because we know that the nervous impulses are then passed down through the interventricular septum and eventually spreads across the ventricles. This study focused on ventricular conduction. Information available regarding this topic is scarce and unverified [1] and it is crucial from a clinical perspective that the information provided for the care of patients is adequate. A few studies have used immunohistochemistry in aiding their understand of the cardiac neuroanatomy in other mammalian hearts [2, 3] with great success. Applying a similar methodology on a different species of mammal will allow us to confirm any constants found in our results with previous studies and add to the data collection of the cardiac innervation.
Functionally, the ventricles are tasked with the most strenuous activity. This anatomical study conducted on ovine ventricular innervation, will validate any previous study with similar outcomes and aid in practical use of this knowledge. Also, having the knowledge of location of nerves and ganglia can allow us to understand how the neuroanatomy is related to function, finally this allows us to compare validity with other studies and recognise trends or faults in them.
7. AIM AND OBJECTIVES OF THE STUDY
The aim of our present study is to determine the innervation of cardiac ventricles in the ovine heart, using immunohistochemistry of nerve fibres and neurons located within intrinsic neural plexus.
Our objectives are:
1) To identify the size of nerves within the whole mount preparations obtained. 2) To assess the ganglion size and neuron groups.
8. LITERATURE REVIEW
8.1 Extrinsic innervation of the heart and intracardiac accesses of the mediastinal nerves
To thoroughly understand the extrinsic innervation of the heart, one must first understand how the central nervous system is responsible for regulating our cardiac cycle. In a wide speaking term, the autonomic nervous system of the heart is comprised of intrinsic and extrinsic cardiac control. This is best described in the picture below (figure 1).
Figure 1 – Model for neuronal hierarchy of the heart (taken from Armour JA, 2004 [37]).
When discussing the hearts innervation, it is important to mention that extrinsically, chemicals and hormones found in the surrounding tissues effect muscle behavior in the heart. Having the heart encased in fat, it is important to understand how cardiac output is matched to changing body blood flow demands in different physiological states [4]. This is known as the reciprocal thesis of cardiac control. It was first described by Kollai & Koizumi in
cholinergic efferent preganglionic neuron activity. Increased cardiac sympathetic efferent neuronal tone increases cardiac chronotropism, dromotropism and inotropism [7]. Parasympathetic efferent preganglionic neurons have the opposite effect [6, 8]. Intrinsic cardiac nerves extend from the epicardium to ganglion plexus on the heart, innervating the atria, interatrial septum and the ventricles [9]. The left coronary and right coronary subplexus extend from the arterial region of the hilum between the pulmonary trunk and the aorta to the respective left and right ventricles sites [10].
8.2 Innervation of cardiac ventricles
In recent years, mammalian ventricles have been studied in more depth. Examples of these are humans [9], rabbit [10,11,12], dog [13], cat [14], sheep [15,16] and guinea pig [17]. Historically, mammalian ventricles were believed to be exempt of ganglia and no innervation from the intracardiac innervation through the nervous system. This was later refuted by Gagliardi et al. (1988) who described ganglia present on human ventricular myocardium at locations anterior to the coronary groove and also around the region of the conus arteriosus[18].
What is shown from the studies is that in larger mammals that where tested, ventricular ganglia are clearly evident [9,13]. In contrast, smaller mammals appear to have a reduced number of ganglia in the ventricular region [14]. Innervation of the ventricles begins by ganglia found in the region opposite the aortic root as well as the pulmonary trunk root. Smaller regions of innervation are found around the area of the inter-ventricular sulcus and also other ganglia similarly, but located around the left atrioventricular groove [9,19]. The pattern of innervation in mammalian rabbit experiment suggests that it originates as described previously through two pathways. One is accessing mediastinal nerves entering arterially around the aorta and pulmonary trunk [10, 12]. The second is venous and it travels around the roots of the pulmonary vein on the heart hilum. The numbers of ganglia present around the region of the coronary artery alters exponentially between different mammalian heart specimen usually with numbers from 11 to 220 [12].
Recent studies done on mammals such as mice [2] and rats [3] have shown a different picture completely. Nervous ventricular supply originates almost completely from the atria with the ventral surface of the ventricles being principally supplied by the sub-plexus route located at the right ventral atrial area. This in comparison with larger mammals such as dogs and sheep [20] shows a great difference where the left and right coronary sub-plexuses
for almost all ventricular innervation [3,20]. The fact that sub plexus neuronal projections branch onto the ventricles, emphasizes the role of the intra-cardiac nervous system on ventricular control and research data suggests evidence from a number of different species ganglia that there is a physiological role in ventricular control [21]. This further illustrates the neuronal complexity of the heart with alternative mammals.
8.4 Distribution and morphology of cardiac ganglia
The internal workings of the cardiac nervous system comprise of collections of neuronal somata, with connecting nerve fibres known as ganglionic plexuses. These somata are in size 15 and 20–45 μm [2]. They are found as isolated scatters, gathered into ganglia containing anywhere between two to one thousand five hundred neurons. They are also found in clusters with a number of smaller groups of cell bodies [22]. The majority of somata reside on supra-ventricular region lying directly on the surface of the epicardium but also founds within surrounding adipose tissue in the area of the heart hilum [2,22]. Ganglia are primarily found on the dorsal atrial surface around the base of pulmonary artery or aorta ventral to the pulmonary veins and on the anterior ventricular surface. [2,22]
From all anatomical studies analysed, one thing was common in all mammal specimens. All ganglia are closely connected by minute commissural nerves [3,9,23,24]. Having these nerves here can suggest and further elaborate the theory that ganglia and even individual neurons within ganglia have the ability to communicating with one another. This can show evidence that the heart in providing autonomic feedback to the brain. It may also suggest that this can be a way of making sure that the heart is beating accurately and in a healthy manner allowing for normal physiological activity.
In the past, to get a true histological understanding of the specimens was complicated to say the least. Methylene blue was the choice of staining and was used to identify neurons from varying parts of the body. However, with the dye and chemical fixation, the specimens were less visible and often unstable [25]. For this reason, it would not have been wise to use the same method with relation to intrinsic cardiac nervous system. During the mid-sixties, a new, more reliable and most importantly cost effective methodology was implemented. It was used to identify nerves and their cell bodies using histochemical stain using acetylcholinesterase (AChE) [36].
8.5 Immunohistochemistry of nerves and cardiac neurons
Originally, our understanding of the intrinsic ganglia was that it worked as a communication between parasympathetic inputs from the nervous system. This would mean, by hypothesis, that the majority or even all markers would show the presence of independent cholinergic markers such as choline acetyltransferase (ChAT) which is responsible for the synthesis of acetylcholine (ACh). Having said this, new discoveries in recent years have indicated another theory. Using immunohistochemical staining, a variety of neurochemicals where found to be visible. Both neuro-modulators and transmitters where shown to be highly reactive to the staining. This opened up the doors for more detailed analysis and the following where found to be present. ChAT was found abundantly however was not the only one [26]. Tyrosine hydroxylase (TH) was another neurotransmitter found. Its purpose is the production of noradrenalin in sympathetic system [23] neuronal nitric oxide synthase (NNOS) deals with the production of nitrous oxide for both sympathetic and parasympathic activity with branching to non-adrenergic and non-cholinergic nerves [3]. Vasoactive intestinal peptide (VIP) which is synthesized and released simultaneously with ACh and Substance P (SP) was also present [26]. These finding show us one very important fact, that the hearts ganglia are not only receiving information from the central nervous system through the Vagus nerve through a cluster of greatly specialized and intricate structures but suggests that with the aid of the above neurotransmitters and neuromodulators as well as their location next to sensory nerves, the heart is both regulated extrinsically, but also deeply on an intrinsic aspect.
Studies from Horackova et al., 2000, Hoard et al., 2007, Rysevaite et al., 2011 and Richardson et al., 2003 [2,3,27,28] have shown that almost all of cardiac ganglia are ChAT immunoreactive. This is widely accepted and re identified in research projects. However, a main discussion point in terms of neuromodulator is TH responsible for the production of the sympathetic nerve neurotransmitter noradrenaline. The research tried to assess the total TH immunoreactivity in experimental models. Moreover, a more in-depth analysis of cells to determine TH presence was also performed. Results were at both ranges of the spectrum from many to a few [2, 3, 27, 29, 30] showed ganglionic neurons of diameters from 20-40μm and others like Richardson et al., 2003 which described no evidence of such cells. However, the relationship between ChAT and TH cells in terms of immunoreactivity demonstrated that around 20% of all neurons have both ChAT and TH representations [2, 31].
Another substance is identified as a neurotransmitter and has a very vital role in the intrinsic nervous circulation of the heart. That substance is NNOS. This deals with the production of NO for both sympathetic and parasympathetic activity as well as branching to non-adrenergic and non-cholinergic nerves. This was found to be in ganglionic plexuses of model mammals including rabbit [32], mice [33] and guinea [34]. Richardson et al., 2003 [3], Hoover et al., 2009 [29] and Herring et al., 2002 [35] characterized that neuronal nitric oxide synthase are usually being in co existence with ChAT. These studies suggest that the neuronal oxidase synthase has a functional role as a co-transmitter. This allows for the vagus nerve to control the hearts ventricals. Maifrino et al., 2006 [34] described neuronal oxidase synthase to be from around 7 up to 67% imunno-reactive. More currently, Pauziene et al., 2016 [12], further illustrated the co-existance of the neuronal oxidase synthase with ChAT in ventricles of rabbit models.
9. MATERIALS AND METHODS
9.1 Materials
6 black-faced newborn lambs (3±0.5 kg) of either sex, were used in accordance with local and state guidelines for the care and use of experimental animals (permission no. 0206) and the EU directive. After thoracotomy the hearts removed from the chest and perfused via both coronary arteries by a syringe with room temperature 0.01M phosphate-buffered saline (PBS) until the tissues turned pale. The composition of the PBS was (in mM): NaCl, 137; KCl, 2.7; Na2HPO4, 10; KH2PO4, 2.
9.2 Whole-mount ventricular preparations
Following perfusion with PBS, fixation with 4% paraformaldehyde (PFA) solution in PBS was proceeded. After prefixation, hearts again were washed with PBS, then the pericardium and atria was removed and the walls of cardiac ventricles were carefully dissected from the interventricular septum. Ventricular walls were pinned on a special dissection dish with a silicone pad, and the epicardium were carefully separated from the largest mass of myocardium using Sprinbow dissecting microscissors. Only thin layer of myocardium was left. Tissue samples were placed with the epicardium face up, then stretched and pinned on a silicone elastomer (Sylgard) pad. Afterwards, immunohistochemical reactions were performed.
9.3 Fluorescent immunohistochemistry on ventricular preparations
To decrease tissue autofluorescence, the flattened tissues were dehydrated through a graded ethanol series as reported by Dickie et al. [27], permeable using ethanol with 20% dimethylsulfoxide (DMSO), bleached with hydrogen peroxide in ethanol (6%, H2O2) and DMSO during the night. Next day whole-mount preparations were rehydrated with 10-minute successive washes through a descending alcohol series, washed with PBS, and permeabilized with 0.5% Triton X-100 (Carl Roth, Karlsruhe, Germany) in PBS containing. Nonspecific binding was blocked for 2 hours in PBS containing 5% normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Preparations were subsequently washed (3 × 10 min) in PBS and incubated in a mixture of two primary
the whole-mount preparations during the night at 4oC. After whole mounts were washed 3 times in PBS, and incubated in an appropriate combination of secondary antibodies for 4 hours in a dark humid chamber on a shaker stage at room temperature (Table 1). All antibodies were diluted in 0.01M PBS. On the last stage, the whole-mount tissue preparations were carefully stretched on a microscope slide, cover- slipped using a mounting medium (Vectashield, Vector Laboratories, USA) and sealed with clear nail polish.
Table 1 – Primary and Secondary Antibodies
Antigens Host Dilution Company Catalog number
Primary
CHAT Goat 1:100 Chemicona AB144P
TH Mouse 1:400 Chemicona MAB318
PGP9.5 Rabbit 1:500 AbD Serotecb 7869-0504
Secondary
GoatCy3 Donkey 1: 00 Chemicona AP180C
MouseFITC Donkey 1:300 Chemicona AP192F
MouseCy3 Donkey 1:300 Chemicona AP192C
RabbitFITC Donkey 1:300 Chemicona AP182F a Chemicon International, Temecula, CA, USA.
b AbD Serotec, Kidlington, UK.
9.4 Microscopic examinations, measurements and statistical analysis
Preparations were analysed and images were acquired at 20x and 40x magnification employing either a fluorescence microscope AxioImager Z1 with a digital camera AxioCamMRm and an ApoTome 2 supplement (both from Carl Zeiss, Gottingen, Germany), or a confocal laser-scanning microscope LSM 700 with ZEN 2010 software (Carl Zeiss, Jena, Germany). The location of any neuronal structure were analysed and photographed with AxioVision software Rel. 4.8.2 (Carl Zeiss, Gottingen, Germany). Using FIJI, we are able to asses the length of each structure using curser and measurement tools on the software. Taking a long and short axis can also enable us to measure the total area of a structure. The results where then compiled on excel and formatted for statistical evaluation. The mean,
Then, the next step was analysing the nerves within the whole mounts. Over two thousand nerves were analysed throughout this experiment and the findings were divided in terms of its location with its relation to the heart. Sections were made and their representations are as follows: Anterior aspect of the heart; 1) Left epicardium, 2) Middle epicardium, 3) Right epicardium, 4) Anterior epicardium, 5) Posterior epicardium. The Posterior aspect of the heart had: 6) Base and Middle epicardium top half, 7) Apex base bottom slice, 8) Apex base middle different location.
When analyzing the nerves, it was important to calculate the nerve diameter. We did this by first determining where the largest diameter and shortest diameter were located in order to further interpret results. To do this, the curser measurement tool on the FIJI application was used and axon diameter was taken at several points. These where then calculated and put on a table in Microsoft Excel (2013) for further statistical analysis.
10. RESULTS
10.1 Epicardial nerves
Findings show that the mean overall size of nerves within the epicardial layer of the ovine heart was 65.3 µm. We notice that the size of the nerves examined in the slides decreased in size as they descended the heart e.g. the base nerves were measured to have an average width of 108.8 µm in comparison to the apex which had 29.9µm.
The results also show that the size of the nerve decreased based on anterior posterior locations. Using the reference from chapter 9.4, the largest nerves that were found were located on both the anterior and posterior surfaces of the heart. The highest recorded figure was 236.6µm which was located on the posterior aspect at the base and middle, top slice (6). The second highest figure 189.5µm located on anterior left surface of epicardium (1). Finally, the third largest figure was also on the anterior surface of the heart, the middle section i.e. the middle epicardium (2) at a size of 173.4µm.
Figure 2 –Average nerve sizes in different ventricular locations
Above figure 2, the average size of nerves from each section were as follows: 1)108.8µm, 2)100.9µm, 3)47.3µm, 4)72.4µm, 5)74.9µm, 6)90.8µm, 7)71.7µm, 8)29.9µm, 22.0µm, 33.9µm.
In comparison the three smallest results were all found on the posterior aspect of the heart. The apex of the heart showed to have the smallest size nerve of 2.17µm with the base nerve measuring at 2.19µm. The third smallest was found in the middle posterior aspect of
When comparing nerve sizes with relation to anterior or posterior location, we find that the average nerve size located on the anterior surface was 82.4µm in comparison to 53.9µm size nerve found on the posterior surface. This is further illustrated in figure 3.
Figure 3 – Average nerve size base on orientation
When comparing nerves sizes with relations to its location i.e. the anterior or posterior surface of the heart, we notice that the smallest average size of a nerve on the anterior aspect was approximately two folds that of the smallest average size of a nerve on the posterior surface, i.e. 47.3µm right epicardium (3), 22.0µm base epicardium (8).
Table 2 – Nerve analysis
Left Middle Right Anterior Posterior Base middle top
Back base
Apex Base Middle Mean 108.79 100.92 47.27 72.43 74.86 90.84 71.68 29.88 22.01 33.86 Standard Error 16.60 9.06 6.59 6.081 4.55 4.46 7.98 1.35 0.65 0.86 Standard Deviation 52.50 41.53 30.21 28.52 26.55 38.67 29.87 22.91 18.04 27.01 Minimum 30.62 34.11 15.99 29.35 26.56 18.42 0.43 2.17 2.18 2.91 Maximum 189.49 173.41 104.54 133.92 135.54 236.63 131.99 125.98 108.36 171.23
Figure 4 – Image of nerve fibre under microscopy in
Middle size. Showing high diversity index with regards to nerve size and connectivity.
Figure 5 - Image of nerve fibre under microscopy in
Base, where the largest length nerve size was located measuring at 236.6µm.
Figure 6 - Image of nerve fibre under microscopy
Apex. The size of the nerves examined in the slides decreased in size as they descended the heart e.g. the base nerves were measured to have an average width of 108.8 µm in comparison to the apex which had 29.9µm.
10.2 Intrinsic ganglia
Throughout all the cross sections that were analysed, a total of 75 ganglia were noted. Measurements were taken on FIJI of both the long and short axis of each neurone and were accumulated and analysed on Microsoft Excel (2013). Shown below in figure 7 are the average lengths of the neurons within the ganglia.
Figure 7 – Neuron axis length in different ventricular sections
After analysing the average lengths of neuron axis, both the standard error and standard deviation were calculated as shown in table 3. The largest measurement was taken on the anterior surface of the heart left epicardium (1) with a measurement of 52.8µm. In comparison, the smallest measurement was found on the posterior surface of the heart at the area of bottom apex (7) measuring at 1.4µm. From these results, the mean measurements were taken across the samples and the mean long axis measurement was 21.8µm and was found on the posterior aspect of the heart base and middle top slice (6). In contrast, the mean short axis measurement was found also on the posterior surface of the heart at the apex, base and middle level (8) with a length of 12.9µm. The overall mean length of neuron within a ganglia was 15.2±0.1 µm.
Then the mean number of ganglia that were measured where statistically analysed to give an overall average measurement of 75. These were then grouped by size in their respective locations based on the number of neurons located within.
Table 3 – Neuron axis length in different ventricular sections Left Epicardium Middle Epicardium Base Middle, Top slice Base Epicardium, Bottom slice Apex, Base, Middle Long 11.49 17.719 21.81 19.08 21.03 Long (SE) 0.63 0.36 0.16 0.27 0.18 Long (SD) 12.56 5.87 6.11 5.54 5.87 Short 6.17 10.37 10.76 9.17 12.88 Short (SE) 0.33 0.23 0.14 0.23 0.12 Short (SD) 6.72 3.77 5.57 4.71 4.01
From the results we notice that the most abundant group found in the slices were numbers between 1-10 neurons per ganglia. As the group size increased, the number of ganglia decreased and continued to decrease. This is further illustrated when quantified as shown below table 3.
Figure 8 – Ganglia grouped by neuron number
Table 4 – Groups of ganglia by neuron number
Left Epicardium Middle Epicardium Base Middle, Top slice Base Epicardium, Bottom slice Apex, Base, Middle 0 -10 65 13 51 29 65 11-20 20 8 41 14 20 21-30 9 1 13 2 9
Having done these reading, we can then multiply the long and short axis, allowing us to calculate the overall area of each ganglia. This is illustrated in the graph below, figure 9. The highest mean area of ganglia was found at 281.6µm2 and was located at the apex, base, middle slice (8). The second highest figure was located in the base middle, top slice (6) with an area of 239.2µm2. In comparison, the lowest mean area of a ganglia found was in the left epicardium (1) with a measurement of 145.1µm2. The total difference between the highest located value 281.6 µm2 at the apex, base, middle slice (8) and the lowest measurement of 145.1µm2, located at the left epicardium (1) was 136.5µm2 in sum. This was almost two folds the size of the smallest ganglia which gives us further indication as per function. The mean area of ganglia was found to be 231.42± 2.69 µm2
Figure 9 – Area of Ganglia in different ventricular location. ∗Statistically different from ganglia of Left Epicardium, (p<0.05)
⊗ Statistically different from ganglia of Base Middle, Top Slice (p<0.05)
◊ Statistically different from ganglia of Apex, Base, Middle (p<0.05)
Figure 10 – Ganglia (PGP9.5), arrow showing single neuron somata. Measurements were taken from a long and short axis through the nucleus and
⊗ ◊
*⊗ ◊
* ◊
*⊗ ◊
11. DISCUSSION
11.1 Nerves
In this study, our aim was to find the best possible understanding of the ovine ventricular system particularly the intrinsic neuronal pathways. We did this using immunohistochemical whole-mount heart preparations. Then these images were analysed under FIJI. These where then analysed based on structure i.e. nerve or ganglia and the results that were found are as follows.
When analysing the nerves, it was important from a statistical point of view to calculate the nerve diameter. The first important finding was to determine where the largest diameter and shortest diameter located in order to further interpret results.
The largest diameters of nerve as shown in the results where on both aspects of the heart (anterior and posterior). The largest reading being on the posterior aspect at the base and middle, top slice (6) with 236.6µm with the second largest being 189.5µm on the left surface of epicardium and (1). This indicated that the difference between the largest diameter and the second largest was 47.1µm. The smallest results, in comparison were all shown to be on the posterior heart. The smallest axon size in diameter was 2.17µm (8). The second smallest was 2.19µm±0.02µm between them. When comparing them to other results such as that of a rabbit e.g. Basal width: 36.4µm±1.6µm, middle: 36.2µm±2.7µm, apical 44.0µm±4.9µm [13]. The total overall difference between the largest and the smallest 234.43µm, showing that the diversity within the heart for the nerve fibres share a high profile. This shows the direct correlation between the different areas of the heart that are supplied by different size nerves i.e. areas of high excitability and areas of low excitability. This is further reiterated when we calculated the average size of the nerves from each section.
We found that the area with the highest average of nerve size was the left ventricle on the anterior surface (1), which correlates with the left ventricle’s high excitability in terms of cardiac physiology. Another example of this was the second highest nerve size figure which was located middle epicardium anterior surface (2) at a size of 100.9µm, which has a functional relation to ventricular excitability. In comparison the smallest average sizes of nerves were all located on the posterior surface around the apex, base and middle (8) with readings of 29.9µm, 22.0µm, 33.9µm. Another important analysis that was made was that, when we calculated the nerve sizes with relation to their specific locations either on the anterior surface of the heart or the posterior surface of the heart. We found that the smallest
right epicardium (3) in comparison to 22.0µm, base epicardium (8). This was almost 2.2 times the average size difference between the nerves anterior and posterior aspects of the heart’s epicardium. This with mapped images of the heart can give us an indication as to why the nerves are slightly smaller [36].
11.2 Ganglia
After analysing the nerves and axons it was important to quantify the size and intrinsic structure of ganglia in the epicardial slides to determine functional importance. Similar to the results found in the nerves the largest ganglia length was at the level of the left epicardium (1) measuring at 52.8µm and the smallest was 1.4µm at the area of bottom apex (7). This supports the theory that the left epicardium requires a higher electrical stimulation for increased action potential to contract the left ventricle as it’s function its crucial physiologically, with comparison to the bottom apex which hold a relatively low physiological significance apart from the separation of nerve fibres that circumvent into the ventricular walls [38].
The next step that was taken was grouping the ganglia based on individual number of neurons within them. Overall 75 ganglia were measured and interpreted. We then grouped the ganglia based on the number of neurons within them e.g. group 1 was 1-10 neurons per ganglia, group 2 11-20 neurons per ganglia etc. as shown in graph 3. From our understanding of the intrinsic cardiac nervous system, most ganglion plexuses originate with close relation to major blood vessels. Having said this, our results showed that the two largest ganglia groups (based on their number of neurons per ganglia) were located at the left epicardium (1) and the base middle top slice posterior (6), which correlates with the major ganglionated plexuses associated with this region with respect to the major blood supply. However, the most numerous group of ganglia were the smallest i.e. 1-10 neurons per ganglia, with major influence in the left epicardium (1) 65 ganglia and apex, base and middle (8) 65 ganglia. This shows physiologically the high interconnectivity between the major ganglionic plexuses and the smaller ganglia surrounding the cardiac tissue. As the ganglia interconnect with other groups, they create a more intricate and complex set of nervous pathways that allow for full correct physiological functioning of the heart.
The overall lowest amount of ganglia with regards to location was found in the middle epicardium (2). 13 ganglia with 2-10 neurons, 8 ganglia with 11-20 neurones, 1 ganglia with 21-30 and 31-40 neurones respectively. This supports the theory that the interventricular
septum is comprised mainly of collagen fibres and connective tissue rather than nerve fibres [39].
Finally, based on the average sizes of neurons, we calculated the overall mean area of ganglion and displayed visually in graph 1.4. We can see that the largest ganglia were found in the posterior heart apex, base and middle (8) with a size of 281.6µm2. When comparing this to average areas of other mammals, e.g. guinea pig 585±22µm2, dog 578±29µm2, human 627±20µm2, we notice a large interval between sizes [9]. In comparison the smallest average area of ganglia was located in the left epicardium (1) at a size 145.1µm2.
Overall, throughout the entire investigation, it was key to understand our hearts intrinsic nervous system function with respects to its structure. With that being said, the majority of our results support the evidence already out there with relation to ventricular innervation. There are however some variations which are less documented and therefore gives us the understanding that every mammal has a unique structure for ventricular activity. Based on our findings, it is clear to say that the most contractile area of the ovine ventricle with relation to nerve size and ganglia amount was the left surface epicardium. This is further supported by our understanding of the left side of the heart and its increased contractile requirement. Also, but not new, we found that in general, larger axons and groups of ganglia where found in places which required increased activity of the heart. All in all, I think that the results to this experiment correlate well with others from similar investigations.
12. CONCLUSION
1) The nerve sizes in the sheep ventricles varied with respects to locations. The total overall difference between the largest and the smallest nerves where 234.43µm. 2) It was found that the ganglion size on average was 231.42± 2.69 µm2. The most
abundant ganglion where those that contained 1-20 neurones.
3) There was a big difference between the largest length of neuron within the ganglia and the smallest, showing great diversity in size. The overall mean length of neuron within a ganglia was 15.2±0.1 µm.
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