MORPHOLOGY AND INNERVATION OF SINOARTRIAL NODE IN SHEEP

MORPHOLOGY AND INNERVATION OF SINOARTRIAL NODE IN SHEEP

Sabahat Hussain Afzal Chaudhary BSc(Hons)

Institute of Anatomy

Medicine (MF VI)

Hermanas Inokaitis, PhD

Lithuanian University of Health Sciences

2017

CONTENTS

1. SUMMARY...3

2. FOREWORD...4

3. CONFLICT OF INTEREST...5

4. ETHICS COMMITTEE APPROVAL ...6

5. ABBREVIATIONS ...7

6. AIM AND OBJECTIVES OF THE STUDY...9

7. LITERATURE REVIEW ...10

7.1 ANATOMY/HISTOLOGY ...10

7.2 PHYSIOLOGY...11

7.3 HISTOCHEMISTRY...12

7.4 PACEMAKER CELL STRUCTURE ...13

7.5 INNERVATION OF HEART ...13

8. MATERIALS AND METHODS ...16

8.1 WHOLE MOUNT PREPARATIONS...16

8.2 QUANTITATIVE AND STATISTICAL ANALYSIS...17

9. RESULTS...18

9.1 THE DISTRIBUTION OF HCN4 POSITIVE MYOCYTES ...18

9.2 GANGLIA WITHIN SAN...18

9.3 SIZE OF NEURONS WITHIN SAN GANGLIA ...20

10. DISCUSSION ...21

11. CONCLUSIONS...23

12. BIBLIOGRAPHY...24

3

1. SUMMARY

The sheep heart has of yet not been thoroughly investigated in terms of the cardiac

conductive system (CCS) and specifically the sinoatrial node (SAN) of the sheep heart has not

yet been investigated. To that end, we, through this investigation hope to glean a more in-depth

understanding of the sinoatrial node using Potassium/sodium hyperpolarization-activated

cyclic nucleotide-gated channel 4 (HCN4) immunohistochemical labelling. The sinoatrial node

was measured to be 70.8±5.4mm

. The sheep (ovine) heart was shown to have 422±18

ganglia, each ganglia had an average of 21.0±2 neurons. The majority of the ganglia

investigated contained between 2 and 20 neurons. Overall average size of neurons had a short

axis size of 15.74±0.11µm and the long axis 24.11±0.15µm giving overall average size of

19.33±0.1µm.

2. FOREWORD

I want to thank everyone who I had the honor to work with in the Institute of Anatomy

and the whole of the medical faculty within Lithuanian University of Health Sciences. I would

also like to thank my family and friends for all their help and support during this time.

5

3. CONFLICT OF INTEREST

The author hereby certifies that he has no affiliations with entity of any financial interest

or non-financial interest in the subject matter or materials discussed in this thesis.

4. ETHICS COMMITTEE APPROVAL

All the procedures were perfomed in aproval with local and state guidelines for the care

and use of laboratory animals (Permission Nr. LT-61-19-004).

7

5. ABBREVIATIONS

AChE – Acetylcholine Esterase AV – Atrioventricular

AVN – Atrioventricular Node

CCS – Cardiac Conductive System ChAT – Choline acetyltransferase SAN – Sinoatrial Node

TH – Tyrosine Hydroxylase VF – Ventricular Fibrillation

HCN2 – Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 2 HCN4 – Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4 I

– Funny channel

PBS – Phosphate Buffer Saline

PGP9.5 – Protein Gene Product 9.5

FIJI – Image J2, image analyser

6. INTRODUCTION

The sinoatrial node holds a very important function in terms of regulating the cardiac impulses. From our already wide understanding, the sinoatrial node is known as the pacemaker of the heart [1]. It is located on the upper aspect of the atrium at junction of crista terminalis [2- 5] and regulates the initial impulse activity of the heart [6, 7]. By doing so, its role both for out wellbeing and our livelihood is crucial [8]. For this reason, our study was aimed at trying to understand the sinoatrial node in more depth.

A lot has already been done to determine the function of the sinoatrial node. Many mammals have been investigated in order to further our understanding of the heart, however, the mammals that were studied during these experiments e.g. rabbit, cats share less common traits with larger mammals [2-5]. Furthermore, larger mammalian animals are used for clinical trials and so, where results of this investigation would be applied to clinical trials in the future.

For these reasons, we chose to study the sheep heart to further our understanding of sinoatrial node in relation to the size of the mammal.

The conduction system is made up of differing levels of control over the cardiac cycle

and it all begins with the sinoatrial node (SAN), which itself is under the influence of the

excitatory sympathetic and inhibitory parasympathetic divisions of the autonomic nervous

system [9]. This is the first study specifically investigating sinoatrial node of the sheep through

the use of whole mount in situ preparations.

9

6. AIM AND OBJECTIVES OF THE STUDY

Overall aim of the study which is to determine the distribution of pacemaker cells within the SAN and their innervation in a sheep heart.

Objectives:

1. To find out the average size of the sinoatrial node in the sheep.

2. To find the average number of ganglia within the sinoatrial node.

3. To find the average number of neurons within the ganglia.

4. To determine the dominant ganglia group.

5. To determine neurons size within sheep sinoatrial node.

7. LITERATURE REVIEW

7.1 ANATOMY/HISTOLOGY

One of the main figures involved in identifying structure on a histological level with regards to the human heart was Sir. Arthur Keith. The research showed the sinoatrial node (SAN), a small pale region on the superior part of the right atria. Further, these small poorly demarcated structures were in fact myocytes with poorly developed sarcomere and sarcoplasmic reticulum [2,10-12]

Later on, during the century, Crick et al. examined human hearts with a keen interest in the SAN [5]. One of the first things that was noticed in all preparations was that a nodal artery was located inside the SAN structure [13], the sinoatrial nodal artery [14]. Secondly, under microscopic examination, the cells of SAN in comparison to the atrial myocytes show strict demarcation between the SAN and other myocytes. [13].

In most human hearts, the sinus node is a ‘oval shaped’ structure located in the

“subepicardial” [15] intercaval region of the heart at the junction of the superior caval vein and the right atrium, with a tail extending along the terminal crest [16-18].

Furthermore, Fedorov et al., 2009 [19], showed that the SAN exit pathways are superior and inferior in direction and that only handful in number, action potentials that originally travel in the transverse direction spread a lot more slowly than those that are traveling in the superior and inferior direction, eventually disappearing without ever leaving the node [19]. This organization of fibers shows good electrical insulation from the surrounding hyperpolarizing influences of the atrial muscle and that any damage to the exit pathways can easily lead to SAN exit block, bradycardia, syncope and sudden cardiac death.

In recent years Chandler et al., 2009 demarcated a new region surrounding the SAN with specialized cells which are characteristically different than cells within the SAN and atrial muscle, this area is known as the paranodal area. An example of these structural characteristics is their location. Atrial and sinoatrial (SA) myocytes are usually located both in the atria and connective tissue respectively [20]. In contrast myocytes of the paranodal area are found in adipose tissue surrounding muscle structures.

The paranodal area is described as being a large area surrounding the SAN myocytes

which run to parallel to the SAN along the terminal crest [20].

11 7.2 PHYSIOLOGY

Functionally these myocytes identify with different properties. Within the SAN, maximum diastolic potential becomes more negative, action potentials become shorter, action potential upstroke becomes faster and intrinsic pacemaker also becomes faster [21].

This further elaborates the theory that specialized and necessary myocytes other than the SAN are required to create a physiological response within the atrium [22].

One can speculate that through human-computer modeling, where high conclusive evidence suggests propagation of action potentials from the SAN through the paranodal area was occurring [23]. A further illustration of this theory is suggested by [24]; where action potentials in a rabbit model atrial muscle was ~70 cm/s and in SAN was much faster. This theory is thought to occur due to the fact that central nodal myocytes are isolated and protected from hyperpolarization from atrial muscles showing it’s pacemaker activity [25]. These all would indicate the importance of the paranodal area and its importance in research. Kalman et al.1998 [26] postulated that a “ cristal tachycardias”, it could be that these two areas could be one and the same, in that these specialized myocytes produce abnormal rhythms.

In another experiment, there was an assumption made that the paranodal area is made of a 50:50 ratio of nodal cells to atrial myocytes [27]. Then three-dimensional computer modeling was used to calculate the initiation as well as the conduction action potentials within the SAN, giving a theoretical ‘heart rate’ of 67 beats/minute [27]. It’s interesting to note as the paranodal area runs in parallel and its close proximity to the SAN yet at the moment no direct connection has been found between two [27].

Two models regarding organization of the SAN have been proposed [28-31]. The

gradient model proposes that only nodal cells are within the SAN, however there is a transition

in cell size and electrical properties from the periphery to the center. On the other hand, the

mosaic model suggests that 2 cell types within the SAN, both atrial and nodal, both have

uniform properties, i.e. cell size and electrical properties. Looking into the latter model there is

some variation in the percentage of atrial cells from the periphery to the center (63-41%)

[32,33], but the cells themselves are not uniform. On the basis of experimental data, it could

well be the case that a hybrid of the two theories actually exists. There is a slight problem with

the experiment noted by the author, and that is that the “ functional properties of the cells

correlate with the expression of the marker proteins used” [27].

7.3 HISTOCHEMISTRY

In the early experimental stages of SAN and cardiac conductive system (CCS) investigation, methylene blue impregnation of silver was used. However there was a basic problem, they not only stained nerves but also the fibrous tissues [34, 35]. Therefore AChE staining techniques were introduced which is used to visualize the innervation, this showed that there were nerves within all parts of the CCS demonstrating a rich distribution of cholinesterase activity within the sinus node, as well as the rest of CCS [34, 35]. Within the heart SAN there are a few fundamental types of cells, elongated spindle, spindle, and spider cells [36]. In the critsa terminalis, the majority of cells are the elongated spindle cells. No cell type has exclusivity of any one area of the SAN [16]

In Crick et al., 1994 used a different approach of visualization making use of neural marker enzyme PGP 9.5, again showing that this is a better method to distinguish CCS from the myocardium[13].

During the investigation, there was an increased amount of nerve markers within the central portion of the SAN, leading to their hypothesis that this was the " hotspot" similar to what was previously found in rabbits [6]. One of the problems with the above was that specimens had to be fresh, therefore it immunohistochemistry staining specifically to cardiac innervation was then carried out through studying the patterns of PGP9.5, Dopamine β Hydroxylase (DBH), TH (Tyrosine Hydroxylase) along with other peptides on specimens. This meant specimens were not required to be fresh, this was tested and shown to work successfully in specimens that had been left for 2-6 days post mortem [37].

HCN4, an ion gated channel found primarily within the conductive system of the heart (SAN, AVN, Purkinje fibers) [38-40]. When HCN2 is knocked out it leads to sinus dysrhythmia and importantly a decline in number of I

channels, at very least in the mouse models. Further labeling was along the outer cell membrane in all SAN cells [41], much more specific to cells that have nodal activity, instead of labeling proteins that may or may not be present at the time.

From an immunohistochemical point, choline acetyltransferase (ChAT) has been approved as the most reliable cholinergic marker for nerve fibers [42]. Because acetylcholine esterase (AChE) is abundant and available in and around cardiac fibers, it is possible to use ChAT as a marker since it is a required enzyme for the production of acetylcholine within cholinergic fibers [42].

There are a few reseasons to to choose the use of immunohistochemistry, one of the

13 investigated would be highlighted. Immunohistochemical techniques have been advanced to such a degree that they are able to establish distinction between myocytes CCS, to the point that advancements have led to findings of additional areas with the characteristics of conduction tissues [44].

7.4 PACEMAKER CELL STRUCTURE

When dealing with the SAN, it is important to understand its ultrastructure. As an action potential from the sinoatrial node, nodal myocytes are seen to potentiate electrical impulses carried through the atria. These cells have a lower amount of mitochondria in comparison to other myocytes, as well as “ poorly organized myofilaments” [45]. From examination, cells within the SAN and surrounding region show similar tendencies with other myocytes in and around the atrium. This is depicted best in Boyett [45] when it was shown that there were structural differences between peripheral nodal myocytes and central ones, showing more abundant and better myofilament organization in peripheral nodal myocytes which lead to the suggestion that a highly intricate and intrinsic system is working at hand [45]. This interconnectivity is further illustrated in Sanchez-Quintana et al., 2005 [46] where they showed clear interdigitations between the SAN and atrial myocytes. There are indications that these specific structures connecting the muscle fibers plays and integral role in correlating a potential electrical impulses from the SAN to the surrounding myocytes [47].

7.5 INNERVATION OF HEART

The extrinsic regulation of the heart is usually divided into two categories sympathetic and parasympathetic control. Both sympathetic and parasympathetic nerve fibers are found to be closely related to one another. They have shown to have opposite physiological responses on the heart [48]. Both efferent and afferent neurons work in close relations with other interneurons in the cardiac ganglion, which have been evident including dog [49], guinea pig [50] and rabbit [51].

Ganglia around the heart were found in separate areas of the atria in many test models,

surrounding the SAN, around the roots of the vena cava and pulmonary veins, interatrial

septum, and in the proximity of the AV node [52].

Intrinsically the cardiac control is attained by a complex mesh-like structure which is highly intercalated and found in all areas of the dorsal atrium and superior regions of the ventricle. In 1997, Armour et al. described atrial ganglia and their locations on the human heart.

He found that there were five distinct locations where ganglia for innervation were found: Atrium 1) the superior surface of the right atrium, 2) the superior surface of the left atrium, 3) the posterior surface of the right atrium, 4) the posterior medial surface of the left atrium (the latter two fuse medially where they extend anteriorly into the interatrial septum), and 5) the inferior and lateral aspect of the posterior left atrium.

In contrast to the ganglionic pattern within the atria, the ventricular ganglions were found in the surrounding adipose tissue. According to Armour et al., these ventricular plexuses were found 1) surrounding the aortic root, 2) at the origins of the right and left coronary arteries (the latter extending to the origins of the left anterior descending and circumflex coronary arteries), 3) at the origin of the posterior descending coronary artery, 4) adjacent to the origin of the right acute marginal coronary artery, and 5) at the origin of the left obtuse marginal coronary artery.

A further study by Pauza et. al 2000 [53], found that the human heart was innervated by seven subplexuses: two subplexuses on the right atria, three on the left atrium, one on the right ventricle and finally three subplexuses on the left ventricle.

On examination of a canine cardiac nervous system, the intrinsic control was found to be initiated by four atrial and three ventricular areas [54] The ones located in the atrium were found in different locations i.e. 1) central to dorsal surfaces of the right atrium 2) fat on the left atrial ventral surface 3) the mid-dorsal surface of the two atria, 4) inferior vena cava.

With regards to the ventricle, three ganglion plexus were located on the cranial surface of the ventricles. A ganglionated plexus that surrounded the aortic root was identified. This plexus extended to the right and left main coronary arteries and origins of the ventral descending and circumflex coronary arteries. Two other ventricular ganglionated plexuses were identified adjacent to the origins of the right and left marginal coronary arteries [54].

As with most of the models seen, there is great variance between the numbers of cardiac ganglia, 19 in the mouse [55], humans have 700 scattered across the heart [53].

There are commissural nerves that connect the left and right neuronal clusters. Further to this there are also connections between the left and right coronary subplexuses in the ventricle [56], giving rise to what could be an intrinsic local neural network of the heart.

An interesting phenomenon to note is when one side of the ANS increases it’s output

the other side is decreased, i.e. increase in sympathetic stimulation caused a decrease in

15 system was confirmed by use of hexamethonium which ended the vagal protection and caused VF.

While the majority of the ganglia are indeed on the epicardial surface, there are some

on the endocardial surface, the septal ganglia. Those located within the right atrium are most

concerned with modulation of the sinoatrial as well as the atrioventricular node conduction. It

should however be noted that these also have variation across the species [58,59].

8. MATERIALS AND METHODS

8.1 WHOLE MOUNT PREPARATIONS

Three Scottish Blackface newborn sheep (dead at 1

– 3

day) of either gender were

used in this study. Hearts were dissected from the chest from these dead sheep 1 – 2 hours

after their death by thoractomy. The following standard protocol was followed. Disected hearts

were washed and thoroughly cleaned with 0.01 M phosphate buffer saline (PBS), by inserting

syringe needle directly into the ascending aorta, and compressing it’s upper part to direct PBS

directly to the coronary vessels. The coronary vessels were also perfused with PBS. Following

sectioning of the atria from the rest of the heart, it was then flattened and pinned in a Petri dish

which had a silicone base. The sample was fixed for 40 min at 4

C in 4% paraformaldehyde

solution in 0.01 M phosphate buffer (pH = 7.4). Next, the sample was bleached with a solution

of dimethyl sulfoxide and hydrogen peroxide, and then dehydrated. Rehydration was carried

out by graded ethanol series (10 minutes each). After they were washed three times for 10 min

in 0.01 M PBS containing 0.5% Triton X-100 (Serva, Heidelberg, Germany). To stop any non-

specific binding, sample was then soaked in PBS containing 5% normal donkey serum

(Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 2 hours. The sample was

then incubated with antiserum A (Table 1) for a period of 48–72 h at 4

C. Later, the sample

was again washed three times for 10 minutes in 0.01 M PBS and then incubated with an

antiserum B (Table 1) for a further 4 h at room temperature. PGP9,5 and HCN4 were used in

this study. During the last stage, specimens were washed 3 times for 10 min in 0.01 M PBS,

mounted with Vectashield Mounting Medium (Vector Laboratories, Inc., Burlingame, CA, USA),

coverslipped and sealed with clear nail polish.

17 Table 1 – Primary and Secondary serum used

Antigens Host Dilution Company Catalogue

number Primary

PGP9.5 Mouse 1:200 AbD Serotec

7863-1004

HCN4 rabbit 1:100 Chemicon

AB5808

Secondary

Mouse

Donkey 1:300 Chemicon

AP192C

Rabbit

Donkey 1:100 Chemicon

AP182F

a

Chemicon International, Temecula, CA, USA.

c

AbD Serotec, Kidlington, UK.

8.2 QUANTITATIVE AND STATISTICAL ANALYSIS

Next the whole-mount which had been immunohistochemical stained through the

above described protocol, were then examined and digital images were acquired using

LSM700 microscope (Carl Zeiss, Jena, Germany). Firstly, each specimen SAN was measured

using FIJI to measure the extreme on all sides where ganglia were seen. Secondly the number

of ganglia within the demarcated area of SAN were then counted. Finally, all the neurons were

then measured, using the simple method of confirming the nucleus was visible within each

neuron, in this investigation 2636 neuron somata were measured. Then measuring the long

axis and short axis and making sure each both of the lines of measurement passed through

the nucleus, this ensured that the entire neuron was seen and measured as accurately as

possible, this can be seen in figures 1-3. Overall 2636 neuron somata were measured in this

investigation. Both axis measurement data points were then processed, separately, through

Microsoft Excel with data statistical analysis which gave summary statistics as shown in table

2. Finally, all data was processed to give an overall average size for the neuron somata. T test

was performed to see if there had been any statistically significant differences between the

means.

9. RESULTS

9.1 THE DISTRIBUTION OF HCN4 POSITIVE MYOCYTES

The mean area using a circular cursor was found to be 70.8±5.4mm

, the overall size of the atria was 2.38±0.36cm

, therefore the sinoatrial node occupies and overall area equating to 30% of the atria. Both minimum and maximum areas were also measured from 53.8mm

to 96.2mm

as demonstrated in the table 2 below.

Table 2 – Tabulated results SAN Size

(mm

)

Number of Ganglia in SAN

Neurons within ganglia

Neuron Size (Long Axis, µm)

Neuron Size (Short Axis, µm)

Mean 70.8 421 21 24.11 15.744

Standard Error

5.4 18 2 0.15 0.11

Standard Deviation

16.3 31 20 7.50 5.47

Minimum 53.8 398 1 5.89 3.43

Maximum 96.2 457 100 69.05 42.14

9.2 GANGLIA WITHIN SAN

Of the three sheep hearts that were investigated, the mean number of the ganglia

within SAN is 422±18. The first preparation had 398 individual ganglia within the SAN. The

second preparation was slightly higher in comparison with 410 ganglia. Finally, the last

preparation contained 405 ganglia within the SAN. Overall there is a high confidence in these

results with a standard deviation of 31.18. This is further illustrated in the graph 1 below.

19 Graph 1 – Distribution in number of ganglia in specimens

Overall in the sheep heart, 60.9% of neurons were grouped in small ganglia which consisted of between 2-20 neurons. The overall number of ganglia that were measured was

0 50 100 150 200 250 300 350 400 450 500

Sheep 1 Sheep 2 Sheep 3

Nu m be r o f G an glia

Distribution in number of ganglia in specimens

Figure 1 - Composite image of PCP9.5 and HCN4 of SAN, outline of the SAN in white

Figure 1 - Ganglia (PGP9.5)

Figure 3 – Long axis measurement (1), Short axis measurement (2)

127. Then these ganglia sizes were grouped according to number of neurons per ganglia, graph 2.

Graph 2 – Distribution of ganglia size in the sinoatrial node

* Statistically different from ganglia that consisted of 2-20 neurons in sheep SAN (p<0.05)

▼ Statistically different from ganglia that consisted of 21-40 neurons in sheep SAN (p<0.05)

■ Statistically different from ganglia that consisted of 61-80 neurons in sheep SAN (p<0.05)

To determine what the dominant group was in order to understand the nature of the SAN better. As seen from the graph 2 the most dominant group of ganglia were of a smaller ganglion, i.e. ganglia with 2-20 neurons within.

9.3 SIZE OF NEURONS WITHIN SAN GANGLIA

A number of 2636 neurons were analyzed in total. To be able to understand the size and relative relation to location, measurements of both long and short axis of each individual neuron. The measurements were taken and an estimated mean length of neurons for the short axis was 15.74±0.11µm and the long axis 24.11±0.15µm. Standard deviation was 5.47 for the short axis and 7.50 for the long axis. By measuring both short and long axis we are able to calculate the overall size of the neurons. Overall the average size of the neuron somata was measured to be 19.33±0.1µm with a standard deviation of 7.78.

0 10 20 30 40 50 60 70

1 2-20 21-40 41-60 61-80 81-100

Nu m be r o f ga nglia (% )

Neurons per ganglia

Distribution of ganglia size in sinoatrial node

*

*

*

*

*

▼

21

10. DISCUSSION

Overall we notice a few important factors regarding the sheep sinoatrial node. Firstly, this study showed that HCN4 cells occupied a large area that almost equated to one third of the entire area of the whole right atria. Previously there had been similar findings was observed in investigations into the mouse, rabbit and pig [60].

During this investigation three individual hearts were prepared and analyzed. The part that stood out was that there was little variation in number of ganglia within the sinoatrial node.

As seen in the results, there is not much variation between the numbers of ganglia present in each member of the same species indicating that each individual mammal species has different variations of a similar structure. Prior worked has showed that higher vertebrates further down the evolutionary timeline have additional neural regulation components within sinoatrial node.

This is exemplified in the work of Inokaitis et al. 2014, which show that in mice there were no ganglia within the sinoatrial node, the were a few within the rabbit and many more within the pig [60]. In the present study average number of ganglia was 422 and the area of SAN was 70.8mm

. Rabbits and pig sinoatrial nodes are 18mm

and 52mm

[61], thereby showing that the sinoatrial node is much larger in the sheep heart by 75% and 25% respectively.

Furthermore, this shows that 1 mm

of SAN area is effected by 5.9 ganglia versus 1.8 ganglia in pig SAN [61]. From the above works cited, it may seem as though the larger the mammal, the larger the amount of ganglia would be present. However, when comparing the pig and sheep hearts, the pig’s is larger than sheep. Yet there are more ganglia within the sinoatrial node of the sheep and therefore does not support this hypothesis. This therefore means that there is another reason behind why there are more ganglia. It is not within the purview of this investigation to understand the reasoning behind this, it should be therefore left to those who will come after to understand as to why this is.

The next step was to group the ganglia based on the number of neurons located within each of them. We found that the group with the highest abundance was the ganglia with 2-20 neurons inside with roughly 75 out of 127 ganglia, over half of all calculated ganglia. Again this is significant as previous studies have shown from Inokaitis [61] that the smaller sized ganglia dominate typically within the sinoatrial node of mammals such as the rabbit and pig.

Moreover, both long and short axis of each individual neuron were measured. We

found that the mean short axis 15.74±0.11µm and the long axis 24.11±0.15µm. After

calculating all axis sizes we notice that the short axis is almost 66% of the long axis across the

board, which indicates a 2:1 ratio of long axis versus short axis (can be seen more graphically

in graph 3). Again the neuron somata were larger when compared to that of the rabbit (16.5±0.2µm) and those of the pig (19.2±0.13µm) [61]. This roughly equates to +33% and +11% size difference. Again the sheep heart shows larger size of neurons when compared to that of the pig, even though the overall size of the heart is smaller. Both would be considered to be on par with each other on the evolutionary timeline, yet they differ in terms of neurocardiology of the sinoatrial node.

Finally, with both long and short axis lengths available, one is able to calculate the

overall area of each individual neuron within the ganglia. The average area size of neurons

within the sinoatrial node was 405.92±4.76 µm

with a minimum size of 34.8µm

and a

maximum size 2113.1µm

. Assuming the average size of the neuron is 405.92±4.76 µm

, this

would mean that the area occupied by pacemaker cells is only 1.07mm

, showing that only

1.5% of the sinoatrial node is made of up pacemaker cells.

23

11. CONCLUSIONS

1) The right atria in the sheep heart measured at 2.38±0.36cm

. The sinoatrial node was found to measure at 70.8±5.4mm

occupying 30% of the area in the right atrium.

2) The average sinoatrial node area contained 422±18 ganglia.

3) The average ganglia were composed of 21.0±2 neurons.

4) The most abundant group of ganglia within the sinoatrial node consisted of between 2-20 neurons.

5) The mean size of neuron was 19.93±0.1µm.

a) Neuron short axis measured at 15.74±0.11µm

b) Neuron long axis measured at 24.11±0.15 µm.

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