immunohistochemical analysis of intracardiac …
TRANSCRIPT
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LITHUANIAN UNIVERSITY OF HEALTH SCIENSES
MEDICAL ACADEMY
FACULTY OF MEDICINE
INSTITUTE OF ANATOMY
ADINA HAIMOV
IMMUNOHISTOCHEMICAL ANALYSIS OF INTRACARDIAC GANGLIA IN RABBIT
ATRIA
Master thesis
Thesis supervisor:
Lekt. Hermanas Inokaitis
Kaunas, 2018
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TABLE OF CONTENT
1. SUMMARY .........................................................................................................................................2
2. ACKNOWLEDGEMENTS ................................................................................................................3
3. CONFLICT OF INTREST ................................................................................................................3
4. ETHICS COMMITTEE APPROVAL ..............................................................................................6
5. ABBREVIATIONS .............................................................................................................................7
6. INTRODUCTION ...............................................................................................................................8
7. AIM AND OBJECTIVES OF THE STUDY ....................................................................................9
8. LITERATURE REVIEW .................................................................................................................01
8.1 Neural regulation of the heart ................................................................................................................. 01
8.2 Extracardiac nervous system of the heart .............................................................................................. 00
8.3. Intracardiac nervous system of the heart ............................................................................................... 00
8.3.1 Ganglia .................................................................................................................................................... 01
8.4 Heart conduction system ........................................................................................................................... 02
8.5. The sinoatrial node innervation .............................................................................................................. 03
9. MATERIALS AND METHODS .....................................................................................................06
9.1 Material ...................................................................................................................................................... 06
9.2 Whole-mount atrial preparation .............................................................................................................. 06
9.3 Microscopic examination, measurements ............................................................................................... 06
9.4 Statistical analysis...................................................................................................................................... 07
10. RESULTS ........................................................................................................................................18
10.1. Distribution and architecture of intracardiac neurves in whole rabbit hearts ................................. 18
10.2. ChAT and nNOS-immunoractivity in neuron structure .................................................................... 18
10.3. ChAT and TH-immunoracactivity in neurons structure .................................................................... 19
10.4. Size of neuron in respect to chemical properties ................................................................................. 20
10.5. Shape of neuron in respect to chemical properties .............................................................................. 21
11. DISCUSSION ..................................................................................................................................22
11.1. Quantity Population, size, neural phenotype and appearance of neurons ........................................ 22
11.2 Population ................................................................................................................................................ 22
11.2. Size and appearance of neurons ............................................................................................................ 23
12. CONCLUSION ................................................................................................................................24
13. REFFERANCE ...............................................................................................................................25
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1. SUMMARY
Author: Adina Haimov
Title: Immunohistochemical analysis of intracardiac ganglia in rabbit atria
Scientific Supervisor: Hermanas Inokaitis, Institute of Anatomy, Medical Academy, Lithuanian
University of Health Sciences, Kaunas.
BACKROUND: The intrinsic neural plexuses and the immunohistochemical properties of neurons in
the rabbit heart have not been fully investigated, despite the extensively use of this model in
experimental cardiology therefore, the aim of the present study was to identify the neurochemical
properties of ganglionic cells from the rabbit atria.
AIM AND OBJECTIVE: The purpose of this study was to determine the structural organization of
the rabbit intracardiac ganglia, to identify the distribution of cholinergic, adrenergic and nitrergic
neural structures in the whole-mount rabbit’s atria heart preparations using double
immunohistochemical labeling and to determine the correlations between neurons and neurochemical
phenotype and somata size.
MATERIAL AND METHODS: 6 juvenile rabbit hearts were used in this study. Two combinations
of double labeling for choline acetyltransferase + nitric oxide synthase (ChAT + nNOS) and ChAT+
tyrosin hydroxylase (TH) were analyzed in order to identify: neuronal somata purely positive for
ChAT, TH, nNOS and biphenotypic simultaneously positive for ChAT+TH and ChAT+TH. The
quantity of neurons were calculated and then expressed in percent for each picture. Size and shape
were also examined.
RESULTS: Atrial ganglionic cell bodies were positive for all applied neuronal markers. Biphenotypic
neural cells positive simultaneously for ChAT+nNOS, and ChAT+TH were discovered as well.
Ganglionic cell bodies positive for TH or nNOS were in minority compared with other types, while
ChAT positive somata predominated absolutely in rabbit atrial ganglia. Biphenotipic cells found to be
the largest among other distinct phenotype, and expressed large long axis with small short axis
compered to ChAT, nNOS and TH positive neurons.
CONCLUSION: The rabbit atrial ganglia showed high heterogeneity of neural cells that can be
comparable with intracardiac ganglia of other mammalians including human, therefore rabbit heart is
suitable model for further cardiac experiments.
Key words: Immunohistochemical, ChAT, TH, biphenotipic neuron, ganglia
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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.
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3. CONFLICT OF INTREST
There is not conflict of interest.
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4. ETHICS COMMITTEE APPROVAL
Animal Research Center license number LT-61-19-004, certified by the State Service for
Food and Veterinary 2015.12.02.
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5. ABBREVIATIONS
AVN – atrioventricular node
ChAT – choline acetyltransferase
ECI – extrinsic cardiac innervation
GP – ganglionic plexus
HCN4 – hyperpolarization activated cyclic nucleotide-gated potassium channel 4
HH – hilum of the heart
ICNS – intrinsic cardiac nervous system
INP – intrinsic neural plexus
IR – immunoreactive
nNOS – nitric oxide synthase
PGP 9.5 – protein gene product 9.5
RA – right atria
RRVC – rabbit right cranial vein
SAN – sinoatrial node
TH – tyrosine hydroxylase
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6. INTRODUCTION
Neural regulation of the heart can be divided to extrinsic and intrinsic neural system.
Historically the autonomic effect was confined to centrally derive extrinsic inputs from sympathetic
excitatory and parasympathetic inhibitory, apparently neurocardiac control is more complex due to
intrinsic neurons constituting numerous plexuses and ganglia spread widely over the epicardial layer,
giving the idea of a 'little brain' influencing cardiac function [1].
Over the years from Scarpa in 1794 where mammalian intrinsic cardiac neurons first
identified till modern days [2] the location of ICNS and the mediastinal nerves remained poorly
understood despite the fact that the anatomy of this nervous system has been the subject of scrutiny for
over the century [3].
This system is heterogeneous population of neural elements, including preganglionic and
postganglionic nerve fibers, and cardiac ganglia containing parasympathetic, sympathetic, afferent and
local circuit neurons with a wide range of neurotransmitter phenotypes [4]. Anatomically the intrinsic
neural plexus (INP) can be ranged up to 7 subplexuses according to their location in the heart hilum,
and it is differ from species to species [5-9].
This has been developed from experimental studies on different mammalian hearts including
rabbit. The neuroanatomy of the rabbit heart is not well examined therefore study was aimed to
examine the topography, structural organization, immunohistochemical characteristic of INP of rabbit
heart [8-10].
Studies show neurochemical heterogeneity, with pour distinct subpopulation of intrinsic
neuron identified ChAT found abundantly but not the only [11]. While some nerves and neural bundle
are mixed most express either adrenergic or cholinergic markers. TH was another neurotransmitter to
be found responsible for the production of the sympathetic nerve neurotransmitter noradrenalin, when
ChAT immounoreactivity (IR) are more abounded (83%) then TH-IR (4%) [10]. nNOS, for both
sympathetic and parasympathetic activity found to be in ganglionic plexuses of rabbit mammalian
model [1].
The present study was undertaken to determine the structural organization of intracardial
atrial ganglia, size and the number of ganglionic cells and the neurochemical phenotype of the nerve
cells.
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7. AIM AND OBJECTIVES OF THE STUDY
Aim of the study: The aim of the present study was to determine the neurochemical and
morphological properties of neurons located in rabbit intracardial atrial ganglia in whole mount atria
preparations.
The objectives of the study:
1. To ascertain the structural organization of the rabbit intracardiac ganglia.
2. To identify the distribution of cholinergic, adrenergic and nitrergic neural structures in the whole-
mount rabbit’s atria heart preparations using double immunohistochemical labeling.
3. Determine the correlations between neurons neurochemical phenotype and somata size.
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8. LITERATURE REVIEW
8.1 Neural regulation of the heart
The cardiac neuronal hierarchy can be represented as a redundant control system made up of
spatially distributed cells stations comprising afferent, efferent, and interconnecting neurons. Its
peripheral and central neurons are in constant communication with one another such that, for the most
part, it behaves as a stochastic control system [12]. The milieu of diverse cardiac regions, the coronary
vasculature, as well as major intrathoracic and cervical vessels, is continuously transduced by
mechano- and/or chemosensing afferent neurons. This ‘little brain’ on the heart is comprised of
spatially distributed sensory (afferent), interconnecting (local circuit) and motor (adrenergic and
cholinergic efferent) neurons that communicate with others in intrathoracic extracardiac ganglia, all
under the tonic influence of central neuronal command and circulating catecholamines (Fig.1). In wide
speaking term the autonomic nervous system of the heart is comprised of the extrinsic and intrinsic
cardiac control [13].
Fig.1. Hypothetical model of the cardiac neuronal hierarchy, with emphasis being placed on its peripheral
neuronal components. Cardiac sensory information is transduced by afferent neuronal somata in intrathoracic
intrinsic and extrinsic cardiac ganglia via intrathoracic local circuit neurons to cardiac motor neurons. Cardiac
sensory information is also transduced centrally to generate longer-loop medullary and spinal cord reflexes.
Bottom right box indicates that circulating catecholamines exert direct effects on the intrinsic cardiac nervous
system to affect cardiac motor output to the heart. CNS, central nervous system. Taken from Armour JA,
2004[12].
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8.2 Extracardiac nervous system of the heart
The cardiac nerve system may be grossly divided into extrinsic and intrinsic parts [14-16].
The extrinsic cardiac innervation (ECI) collectively can be subdivided into sympathetic and
parasympathetic componenets. This system goes along the spinal cord and their axons (e.g. the
vasosympathetic trunk) en route to the heart, along arterial routes between the aorta and pulmonary
trunk directly onto the ventricular epicardium by the left and right coronary subplexuses [14,15].
Extrinsic nerves may also synapse with cell bodies of ICNS [1]. The intrinsic cardiac innervation
consists of four subplexuses that are epicardial- consists of the autonomic ganglia and axons located on
the heart itself or along the great vessels in the thorax, myocardial, endocardial, and coronary vessels
[5]. The sympathetic efferent preganglionic neurons largely originate in the cervical spinal cord
projecting axons to post ganglionic neurons in paravertebral ganglia [17], while parasympathetic fibers
arise from the vagus nerve originating in the dorsal motor nucleus of the medulla (Smith DC 1970,
Randall WC 1972, 12-13D, Mcallen RM, 1976) (16-18), with efferent regulation (efferent
preganglionic neuron in the medulla) reaches the heart by 3 branches of the vagus nerve (superior,
inferior and thoracic) to intrinsic cardiac parasympathetic efferent postganglionic neuron [21,12]. The
sympathetic input consists of pre- and postganglionic fibers, with the former originating in the spinal
cord and the latter in the stellate ganglion [18-19]. The nerves supplying the heart joining the cardiac
plexus, an accumulation of mixed neurons located cranial and dorsal to the heart. In the human heart,
left and right-sided cardiac plexuses surround the brachiocephalic trunk and the aortic arch,
respectively, and form part of a larger cardiac plexus that lies between the aorta and pulmonary trunk
[21,3,5].
8.3. Intracardiac nervous system of the heart
There is a rich intrinsic innervation of the heart that includes cardiac ganglia, collectively
termed ganglionic plexuses (GP) [22]. As was investigated by Yinglong at 2007 [15] the GP function
as the interacting centers that modulate the autonomic interaction between the intrinsic and extrinsic
cardiac autonomic nervous system (ANS) and has a potential to affect cardiac function independently
[1]. As was showed previously by Armour 1997 [3], extrinsic cardiac neurons access the heart through
the heart hilum, from the arterial part of the hilum to ventricle and from venous part of heart hilum
along the root of the right cranial vein to left atria and dorsal left ventricle [5,9], intrinsic nerves are
usually grouped into defined subplexuses routes projecting to different effector sites, with up to seven
subplexuses that are species dependent- human, dog, ovine hearts through seven subplexuses, two
arose from arterial part and five from venous part [5-7,9]. Although other study revealed 5 epicardial
routes for innervation in rabbit heart [9], two subplexuses in mouse heart [8], it was established that
topography of epicardial subplexuses are consistent from heart to heart [3,5]. ICNS is crucial for
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regulating the heart rate, contractility features, conduction and the coronary blood flow [4-5, 7-8]. In
experimental physiology of rabbit heart, nerves approaching the heart innervate the atria, interstitial
septum and ventricle by five nerve subplexuses are left and middle dorsal, dorsal right atrial, ventral
right and left atrial subplexuses [1,9]. In general, the human right atrium is innervated by two
subplexuses, the left atrium by three, the right ventricle by one, and the left ventricle by three
subplexuses. The highest density of epicardial ganglia was identified near the heart hilum (HH),
especially on the dorsal and dorsolateral surfaces of the left atrium. Nerves that passed onto the heart
through the venous part of the HH could be grouped into five pathways: by the (1) anterior interatrial
sulcus and (2) sulcus situated dorsally amid the roots of superior vena cava (SVC) and RSPV nerves
proceeded mainly to the right atrium; while by the (3) left atrial nerve fold to the lateral left atrial
surface; and by the (4) ventral, and (5) dorsal surfaces of the left atrium epicardiac nerves coursed to
the left atrium and dorsal wall of the left ventricle [5]. Cardiology review the atrial location of ganglia
on heart and it is include the: superior right atrium, superior left atrium, posterior right atrium,
posteromedial right atrium and the inferolateral aspect of the posteromedial left atrium, meanwhile, the
ventricular location of ganglionated plexuses appear to have a preference for the fat surrounding the:
1) aortic root 2) the origin of the left and right coronary arteries of the posterior descending artery 4)
the origin of the right acute marginal coronary artery [3,5,23] (fig2).
8.3.1 Ganglia
In epicardial tissue adjacent to the SAN node are situated several large ganglia and nerve
bundles, which in certain specimens (ex. dog, pig and human) may involve up to 1,500 ganglia of
various size [6, 24-25]. The largest number of epicardial ganglia (about 75%) was concentrated at the
dorsal heart region, while ventral ones accumulated only 25% of all ganglia [5]. The right sided
cluster of ganglia located within this plexus on the left atrium at the right pulmonary vein is the sole
source of epicardial nerves extending toward the sinoatrial node (SAN) region [26]. In epicardial tissue
adjacent to the node are situated several large ganglia and nerve bundles [25]. Large ganglia,
possessing numerous protein gene product 9.5-immunoreactive cell bodies (PGP), were situated in the
epicardial tissues and in the peripheral nodal regions, often in close proximity to prominent nerve
trunks [27]. Ganglionic neural somata of different chemical phenotype identified in the SAN region
were ChAT, nNOS and biphenotypic [28]. Based on previous study the SAN receives the AChE
positive tiny epicardial nerves from the ganglionated nerve plexus on the heart as the nerve plexus of
the heart hilum [8]. Epicardial ganglia were vary in their shape and size when most of the ganglia are
more or less oval [5], the size of the ganglia was from those that were observed with confocal
microscope to ganglia that were easily discernible with the naked eye, both distribution and size of
some mammalians were dependent on the age of the animal [6], the number of neurons in epicardiac
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ganglia ranged from few to more than 400 [5], the largest number of epicardial ganglia was
determained on the root of the SVC [6].
Fig. 2 Morphological pattern of distinct epicardial nerve subplexuses from rabbit hearts as seen from the ventral
(a) and dorsal (b) views of the pressure-distended heart stained histochemically for acetylcholinesterase (AChE).
The clusters of intrinsic cardiac neurons (ICNs) (drawn in red) were outlined from the whole-mount stained
histochemically for AChE. Dotted lines demarcate limits of the heart hilum. Black arched arrows indicate the
course of nerve subplexuses on the rabbit heart surface. Red polygonal triangled areas indicate the location of
neuronal clusters and epicardial ganglia. Ao, ascending aorta; CS, coronary sinus; CV, caudal vein; DRA, dorsal
right atrial subplexus; ICNs, intrinsic cardiac neurons; LAu, left auricle; LC, left coronary subplexus; LCV, left
cranial vein; LD, left dorsal subplexus; LNC, left neuronal cluster; LPV, left pulmonary vein; LV, left ventricle;
MD, middle dorsal subplexus; MPV, middle pulmonary vein; PT, pulmonary trunk; RAu, right auricle; RC,
right coronary subplexus; RCV, right cranial vein (superior caval vein); RNC, right neuronal cluster; RPV, right
pulmonary vein; RV, right ventricle; VLA, ventral left atrial subplexus; VRA, ventral right atrial subplexus.
Taken from Saburkina et al, 2014 [9].
8.4 Heart conduction system
When determining the organization and distribution of the ICN that supply the cardiac
conduction system (CCS) the result are four microscopic epicardial nerves orientated toward the SAN
region derive from both the dorsal right atrial and right ventral nerve subplexuses. The atrioventricular
region is typically supplied by a single intrinsic nerve derived from the left dorsal nerve plexuses at the
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posterior interatrial groove [26]. The relative density of innervation is greater in the central region of
the SAN than in the peripheral region. Nerve densities are also higher in the transitional region of the
AVN compared with its compact region [25]. Functionally the CCS initiates and coordinates the
electric signal that causes the rhythmical and synchronized contraction of the atria and ventricles. In
higher vertebrates, this system compromises the SAN and AVN and the "wiring" of the ventricles [29].
The SAN of healthy humans is the primary pacemaker of the heart, expresses a unique set of ion
channels necessary for the generation and propagation of the action potential. Over the years the
localization of SAN in the heart was defined as a "small condense area of tissue, just where the cava
sank into the auricle", in following studies it was defined as a wonderful structure in the right auricle
[30]. Investigating the development studies in molecular genetics showed the need of transcriptional
factors for the formation of SAN and AVN as Tbx5 and Nkx2-5 as well as Tbx3 expression delineates
the SAN region, which runs a gene expression program that is distinct from that of the bordering atrial
cells, data identify a Tbx3-dependent pathway for the specification and formation of the SAN, and
show that Tbx3 regulates the pacemaker gene expression program and phenotype [31-33]. SAN role is
the primary pacemaker of the heart and generates the initial electrical impulse that rapidly spreads
through the atria. The electrical impulse slows as it enters the AVN and then is propagated rapidly
through the bundle of His, the right and left bundle branches, and the peripheral ventricular conduction
system [34]. The distinct components of the CCS of the heart are essentially myocardial [35-36] that is
why the cardiomyocytes are the essential cells for the generation and propagation of the impulse [9].
The components of the adult mammalian heart conduction system are morphologically well defined,
although species differences exist. Some animals have well developed structures, other have poorly
developed ones, and some of them are somewhere in between [37].
8.5. The sinoatrial node innervation
When determine the relative distribution of autonomic and sensory nerves in the cardiac
conduction tissues of calves, IR to the general neuronal marker (PGP 9.5) demonstrated that all regions
of the conduction system possessed a higher relative density of total nerves when compared with the
surrounding myocardial tissues [27]. No significant differences were observed between the densities of
the total PGP 9.5-immunoreactive innervation throughout the component structures of the conduction
system, with the nodal tissues displaying similar percentage stained nerve areas to the ventricular
conduction tissues. No significant difference was found in the total percentage stained area of PGP
9.5-immunoreactive nerves between the central and peripheral nodal regions. AChE-positive nerve
trunks and fibers represented approximately 65% of the total PGP 9.5-immunoreactive innervation,
being by far the most dominant subpopulation within the node [38].
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Study on mouse shows adrenergic, cholinergic nerve fibers together with hyperpolarization
activated cyclic nucleotide-gated potassium channel 4-positive (HCN4) cardiomyocytes using primary
antibodies for TH, ChAT, and the HCN4 channel respectively. Additionally novel electron microscopy
data revealed that the mouse SAN contained exclusively unmyelinated nerve fibers, in which the
majority of axons possess varicosities with clear mediatory vesicles that can be classified as
cholinergic [39]. Similar study done on rabbit heart was found that a dense and complex ganglionated
neural network of both autonomic and sensory nerve fibers (NFs), closely related to SAN cells which
spread widely on the rabbit right cranial vein (RRCV), where the main mass of SAN cells are positive
for HCN4, extend as sleeves of these cells toward the walls of the rabbit right atrium. The dense
complex ganglionated neural network contained adrenergic (positive to TH), cholinergic (positive to
ChAT), nitrergic (positive to nNOS), and possibly sensory (positive to substance P) NFs [28]. The
distribution and density of nerves fibers in the SAN region was mainly in the epicardium, larger nerves
in adventitia of nodal artery, endocardium and near SAN cell in rabbits heart, on the medial anterior,
lateral and even posterior sides of the root of the right cranial (superior caval) vein in murine CCS,
density in human heart varied in different region being SAN >AVN> penetrating bundle and bundle
branch [25-26,28]. In other study the relative density of innervation was greater in the central region of
the SAN than in the peripheral region. Nerve densities were also higher in the transitional region of the
AVN compared with its compact region [25]. Both nodal and transitional cells are AChE positive and
are associated with a rich plexus of AChE- containing nerves [24-25]. The node possessed a threefold
higher density of PGP 9.5-immunoreactive nerve trunks and fibers than did the surrounding
musculature of the right atrium [27].
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9. MATERIALS AND METHODS
9.1 Material
6 rabbit’s hearts of either sex, 4-8 week of age were used. After thoracotomy the hearts were
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 atrial preparation
Following animal euthanasia, hearts were dissected from the chest, cleaned with 0.01 M
phosphate buffer saline (PBS) and the coronary vessels were retrogradely perfused with PBS.
Afterwards, the atria were dissected from the ventricles. Then, the atria were flattened and pinned in a
Petri dish with a silicone bottom. The flattened specimen was fixed for 40 min at 4◦C in 4%
paraformaldehyde solution in 0.01 M phosphate buffer (pH = 7.4). In order to decrease background
light for laser scanning microscopic examination, tissues were bleached using a dimethyl sulfoxide and
hydrogen peroxide solution, and dehydrated as reported previously [40]. Subsequently, whole-mount
preparations were rehydrated through a graded ethanol series (in each for 10 min), washed 3×10 min in
0.01 MPBS containing 0.5% Triton X-100 (Serva, Heidelberg, Germany). Non-specific binding was
blocked for 2 h in PBS containing 5% normal donkey serum (Jackson Immuno Research Laboratories,
West Grove, PA, USA). Afterwards, the specimens were incubated with an appropriate combination of
primary antisera for 48 – 52 hours 4◦C (Table 1). Afterwards, whole-mounts were washed 3 times for
10 min in 0.01 M PBS and incubated with an appropriate combination of secondary antisera for 4 h at
room temperature (Table 1). During the pilot studies neuronal somata simultaneously positive for TH
and nNOS were not observed. Only two combinations of double labeling for ChAT+nNOS and
ChAT+TH were used in this study in order to identify: neuronal somata purely positive for ChAT, TH,
nNOS and biphenotypic simultaneously positive for ChA+TH and CHAT+TH. 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), cover slipped and sealed with clear nail
polish.
9.3 Microscopic examination, measurements
Microphotographs in which immunohistochemical reactions showed the most well defined
contrast were used for quantitative analysis. Neural structures were examined and digital images were
acquired by using a microscope LSM 700 (Carl Zeiss, Jena, Germany). Randomly taken pictures of
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ganglia from six rabbit’s hearts were (three labeled for ChAT+TH and tree for – ChAT+nNOS).
Opened access feature FIJI used for calculating and measuring neural somata.
9.4 Statistical analysis
The quantity of neurons were counted and then expressed in percent for each picture. In
overall number of 393 neurons in the group of ChAT+nNOS were analyzed taking into account the
difficulty assessing number of neurons due to clarity of their border, and we analyzed 615 neurons
that were immunoreactive for ChAT+TH. The size of clearly visible neuronal somata was measured on
digital images and expressed as the mean of their long and short axes. Data in tables are presented as
percent of absolute numbers (n), means (m) and standard errors (SE). Statistical analysis performed
using the two-sample independent t-test with the aid of Ms Excell software (2010). Differences were
considered significant when p < 0.05.
Table.1 Primary and secondary antisera used in the study
Antigen Host Dilution Company Catalog
number
Primary
ChAT GOAT 1:100 Chemicona
AB144P
TH MOUSE 1:400 Chemicona
MAB318
nNos MOUSE 1:400 Sata Cruizb
biotenologies
SC-5302
Secondary
GoatCy3
Donkey 1:300 Chemicona
AP180C
MouseCy3
Donkey 1:300 Chemicona
AO192C
a Chemicon International, Temecula CA, USA
b Santa Cruz Biothechnology, Dallas, TX, USA
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10. RESULTS
We aimed to analyze the structural organization of the intracardiac ganglia in rabbit atria, size
and the chemical phenotype of the neurons.
10.1. Distribution and architecture of intrinsic cardiac neurons in whole rabbit hearts
Based on Saburkina et al 2014 and similar in this study, when analyzing the
immunohistochemical preparations, ICN were found either in single way or formed into cardiac
ganglia where there are a number of cell bodies collected together, or in neural clusters where there
were large area occupied by nerve cells. The nerve cells of the rabbit heart that were examined, found
within the venous part of the heart hilum on epicardial surface at the root of pulmonary trunk. Extrinsic
cardiac nerves access the rabbit heart through arterial and venous part of the HH selectively extends
towards the anterior surface of the left and right ventricles.
10.2. ChAT and nNOS - immunoreactive neural structures
3 heart rabbit were examined by immunohistochemistry for different neurotransmitter and
neuromodulators in intrinsic network. When comparing between ChAT positive (Fig.3) and nNOS
positive nerves, cholinergic cell bodies were predominate (52.8±5.2%) than nNOS positive neurons
(31.9±4.0%) and biphenotipic neurons, co-localization of ChAT+nNOS in rabbit atria were the least
(15.2±3.2%) but still present (Graph.4).
Fig.3. (A, B, C) several neural somata purely positive for ChAT (in red and marked with x), nNOS (in
green), and simultaneously for both neural markers (biphenotipic neural somata is marked with
asterisk) in the ganglia of rabbit atria. Arrowheads point to purely nNOS positive neurons. A: Long
and short axis of the three type of neurons.
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Fig.4 The mean population of 3 types of neurons, ChAT, nNOS and biphenotipic neurons that display
both type of chemical staining showing differences that *statistical significant compared with ChAT
positive cells, nNOS and biphenotipic neurons.
10.3. ChAT and TH-immunoreactive neural structures
In contrast when comparing between ChAT positive (Fig.6) and TH positive neurons,
cholinergic cell bodies were predominate (ChAT-36.4±4.4%) than TH positive neurons that relatively
rare (TH - 12.8±2.7%) but the biphenotipic cells (ChAT+TH) were present and predominant from both
ChAT and TH neurons (ChAT+TH - 48.2±4.6%) (Fig.5). Small intensity florescent cells (SIF) were
found.
Fig.5 The mean population of 3 types of neurons, ChAT, TH and biphenotipic neurons that display
both type of chemical staining; there are population differences that are *statistical significant
compared with ChAT positive neurons, TH positive neurons and biphenotipic cells. The largest
population is biphenotipic positive cells compered to TH neurons.
0
10
20
30
40
50
60
70
ChAT nNOS biphenotipic
*
*
*
%
0
10
20
30
40
50
60
ChAT TH Biphenotipic
%
*
*
*
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Fig.6 Whole-mount preparation of the rabbit atria showing ganglionic neurons positive to ChAT
(marked with x) and SIF cells (marked with arrows) positive to TH (A). Purely positive ChAT neurons
(B), SIF cells (C). biphenotipic positive both to ChAT+TH (marked by asterisk) (D), arrowheads
points to purely TH positive neurons (D,F).
10.4. Size of neuron in respect to chemical properties.
The size was measured by long and short axis of each neuron (Table.2); Size differences
between neural somata positive for distinct neural markers weren't statistically significant. Mean long
axis of ChAT positive neuron is 29.4±0.7µm and mean short axis is 18.5±0.5µm, nNos positive
neurons and biphenotipic neurons with similar values when the short axis of biphenotipic neurons is
larger (mean 20.7±0.6) (Fig.3). So the largest neuron is in average of 25.09±0.6 µm of the biphenotipic
neurons (ChAT+nNOS), no significant difference between average size of ChAT positive neurons and
nNOS positive neurons. Mean average of ChAT group is 23.8±0.4µm and mean average of nNOS
group is 23.5±0.5 µm, as we see no significant differences when comparing between these two groups
at p value of 0.05.
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We observed size differences in preparation group of ChAT+TH but they weren't statistically
significant. The group with the largest long axis size of 35.4±0.6µm was the biphenotipic positive
neurons and the smallest long axis size of neuron was of the TH positive cell bodies of 28.8±1.4µm
and also the smallest short axis of 17.4±0.7µm. ChAT size is similar to biphenotype neurons. Similar
to the other group of neurons the largest group of neurons examined is of the biphenotipic type
(ChAT+TH) of neuron with average size of 28.1±0.4µm and the smallest neuron is from TH group
cells with average size 23.1±1.01µm. ChAT neurons are intermediate in size 25.3±0.4µm (Table.2).
Compering average between the two groups the mean average of ChAT positive cells is 25.3±0.5 and
of the TH positive cells is 23.1±1.0 here again no significant statistical difference at p value of 0.05.
10.5. Shape of neuron in respect to chemical properties
The intrinsic cardiac neurons in the rabbit heart varied in shape and we can see that by the
long and short axis of neuron, long axis was larger than the short axis. Majority of ChAT and nNOS
group of cells expressed big long axis mean size 29.4±0.7µm when short axis 18.5±0.4µm. (Fig.3).
biphenotipic cells expressed the same shape to ChAT and nNOS cells group.
No significant differences between this group of neurons and ChAT+TH group of neurons.
Big long axis is predominant especially in biphenotipic group of neuron (ChAT+TH) with long axis of
35.4±0.6µm and short axis of 21.7±0.3µm. ChAT and TH positive cells expressed similar size of axis
but shorter than the biphenotipic cells (Fig.4).
Table.2 The percentage of neural somata and the average size of different chemical phenotype
identified in the rabbit atria
Average size
µm
SD SE Mean
%
Chemical
phenotype
32.8µm 3..2 2.3 52.8% ChAT
32.5µm 12.31 ..4 21.2% nNOS
32.09µm 12.. 2.3 12.3% ChAT+nNOS
32.2µm 3..1 ... 2...% ChAT
32.1µm 11.1 3.1 13..% TH
3..6µm 32.. ... ...32% ChAT+TH
22
11. DISCUSSION
Our study analyzed the neural chemical phenotype of ganglionic neurons in whole mount
preparations that were described by previous studies and analyzed the neuroantomy of the rabbit heart
intrinsic nerve plexus using standard histochemical method for AChE staining [9]. The main results
are summarized below. Our findings of extrinsic cardiac nerves accessing the rabbit heart through
arterial and venous part of the heart hilum that were identified between the ascending aorta and the
pulmonary trunk, selectively extend towards the anterior surface of the left and right ventricle are
similar in other studies that done on rabbit, human, dog, and ovine hearts [5-7, 9].
11.1. Quantity Population, size, neural phenotype and appearance of neurons
We evaluate approximately 1008 intrinsic cardiac neurons by immunohistochemical method,
from ganglia neurons reside within the rabbit heart examined by Saburkina et al, 2014 [9], based on
that there is impressive differences in number of neurons between different species dogs - 58,000,
human-28,000 [6] in contrast to Armour 1997 [3] 7,000 in dog and 14.000 in human, rat-4000 - 7000
when in tissue section and 1000-2000 in heart preparation [41-42], pig - found 3,848 ventricular
neuronal somata per heart [43], as we can see this discrepancies may be due to type of species, the
methodology used, and subplexuses involving intrinsic ganglia.
11.2 Population
Two groups of preparations from 6 rabbit hearts were analyzed for size, shape and phenotype,
3 heart were used for ChAT+nNOS and 3 heart used for ChAT+TH. In the first group preparation
both cholinergic, nitrergic and biphenotipic neurons were found but most somata were ChAT –
immunoreactive (IR), prior studies have shown similar findings in mouse [10], rat [41], and in pig the
absolute neuronal somata in ventricular ganglia were positive for ChAT [43] as previously described in
Kieran 2015 [1] the intrinsic neuron are mostly cholinergic therefore playing as inhibitory role in
cardiac regulation. Despite the fact that majority of neuron in our study are IR to ChAT, neurons were
also found to be positive to nNOS but less in number with the same findings as in Kieran 2015 [1]. In
rat two small population of neuron were identified 2 types of nNOS- IR that may serve different
physiological functions [41]. 34% percent of neuronal somata of rabbit atria were biphenotypic for
ChAT/nNOS, much larger percentage compared with rabbit ventricles [44]. In pig ventricle almost
40% of them were biphenotypic for either ChAT/nNOS [43], as in other study analyzed the neurons in
SAN, neuronal somata were positive solely for ChAT and nNOS cells or for both neural markers, but
23
neuronal somata positive for nNOS were more frequent than those positive for ChAT, suggesting a
preferential and dominating nitrergic innervation in this region [28].
We found that ChAT positive neurons were dominant together with biphenotipic cells
ChAT+TH (47%) more than the TH positive cells. It is widely accepted that the cholinergic phenotype
is prominent, but estimates of biphenotypic neurons from various authors are conflicting, 37% of
neurons were biphenotypic for ChAT/TH in pig ventricle [43], yet only 14% of neurons were
biphenotypic in the mouse heart [8], the percentage of ChAT/TH, biphenotypic neurons was 11% in
rabbit ventricle [44]. In rat no TH-IR neurons were found [41] but positive SIF cells were found. No
SIF cells in SAN [8] in contrast; SIF cells were distributed within or close to ganglia on the root of the
pulmonary trunk as showed in [44]. We found SIF cells in the second group of preparation positive to
TH as in mouse [10] and they smaller than TH-IR neurons. We can see that atria possess more
cholinergic neuron in contrast to ventricle that posse more adrenergic neurons [26, 44].
11.2. Size and appearance of neurons
When measuring the size of neuron, we found that there is differences between distinct
phenotypic neurons but no statistically significant. ChAT - IR cells were the largest from nNOS and
TH neurons but the biggest neurons were of the biphenotipic neurons of ChAT+TH,
ChAT+TH>ChAT+nNOS>ChAT>nNOS>TH, as we see the smallest neurons are TH positive. In
Inokaitis et al, 2016 [28] size of intrinsic neural somata were depended on their distribution and
chemical phenotype, in SAN region were smaller than in neurons in left atrium, and the solitary
biphenotipic neural somata were significant larger compared with positive for ChAT. Intrinsic
ventricular neuronal somata (ChAT+nNos and ChAT+TH) of pig were small [43], somata of intrinsic
nerve cells from the rabbit arterial cone and pulmonary trunk root were about 25 µm in diameter,
nNOS-positive neurons were significantly smaller than all other groups in the rabbit ventricular
ganglia only 22 µm in diameter [44]. Difference in size of neuronal somata could be attributed to the
age of newborn rabbit. We demonstrate that the shape of neurons is expressing large long axis and
small short axis. Long in shape is predominant especially in biphenotipic group of neuron (ChAT+TH)
similar in other studies [6, 8].
24
12. CONCLUSIONS
1) The structural organization of the rabbit intracardiac atrial ganglia is similar to other
mammalian species, including humans.
2) Cholinergic, nitrergic and adrenergic neurons together with biphenotipic cells were found in
the rabbit atria. Majority of neurons were positive to ChAT. Moreover ChAT+TH were
predominant than TH-IR and ChAT+nNOS neurons. Most of neurons expressed cholinergic
activity and play an inhibitory role in cardiac regulation.
3) We observed size difference between the distinct phenotypic neurons that wasn't statistically
significant. The largest neurons were the ChAT positive neurons and biphenotipic ChAT+TH
positive neurons.
25
13. REFFERANCE
1. Brack K. The heart's ‘little brain’ controlling cardiac function in the rabbit. Experimental
Physiology. 2014; 100(4):348-353.
2. Yuan B, Ardell J, Hopkins D, Losier A, Armour J. Gross and microscopic anatomy of the canine
intrinsic cardiac nervous system. The Anatomical Record. 1994; 239(1):75-87.
3. Armour J, Murphy D, Yuan B, MacDonald S, Hopkins D. Gross and microscopic anatomy of the
human intrinsic cardiac nervous system. The Anatomical Record. 1997; 247(2):289-298.
4. Hoover D, Shepherd A, Southerland E, Armour J, Ardell J. Neurochemical diversity of afferent
neurons that transduce sensory signals from dog ventricular myocardium. Autonomic
Neuroscience. 2008; 141(1-2):38-45.
5. Pauza D, Skripka V, Pauziene N, Stropus R. Morphology, distribution, and variability of the
epicardiac neural ganglionated subplexuses in the human heart. The Anatomical Record.
2000;259(4):353-382.
6. Pauza D, Skripka V, Pauziene N. Morphology of the Intrinsic Cardiac Nervous System in the Dog:
A Whole-Mount Study Employing Histochemical Staining with Acetylcholinesterase. Cells
Tissues Organs. 2002;172(4):297-320.
7. Saburkina I, Rysevaite K, Pauziene N, Mischke K, Schauerte P, Jalife J et al. Epicardial neural
ganglionated plexus of ovine heart: Anatomic basis for experimental cardiac electrophysiology
and nerve protective cardiac surgery. Heart Rhythm. 2010;7(7):942-950.
8. Rysevaite K, Saburkina I, Pauziene N, Noujaim S, Jalife J, Pauza D. Morphologic pattern of the
intrinsic ganglionated nerve plexus in mouse heart. Heart Rhythm. 2011;8(3):448-454.
9. Saburkina I, Gukauskiene L, Rysevaite K, Brack K, Pauza A, Pauziene N et al. Morphological
pattern of intrinsic nerve plexus distributed on the rabbit heart and interatrial septum. Journal of
Anatomy. 2014;224(5):583-593.
10. Rysevaite K, Saburkina I, Pauziene N, Vaitkevicius R, Noujaim S, Jalife J et al.
Immunohistochemical characterization of the intrinsic cardiac neural plexus in whole-mount
mouse heart preparations. Heart Rhythm. 2011;8(5):731-738.
26
11. Batulevicius D, Skripka V, Pauziene N, Pauza D. Topography of the porcine epicardiac nerve
plexus as revealed by histochemistry for acetylcholinesterase. Autonomic Neuroscience.
2008;138(1-2):64-75.
12. Armour J. Cardiac neuronal hierarchy in health and disease. American Journal of Physiology-
Regulatory, Integrative and Comparative Physiology. 2004;287(2):R262-R271.
13. Armour J. Potential clinical relevance of the ‘little brain’ on the mammalian heart. Experimental
Physiology. 2008;93(2):165-176.
14. Kapa S, Venkatachalam K, Asirvatham S. The Autonomic Nervous System in Cardiac
Electrophysiology. Cardiology in Review. 2010;18(6):275-284.
15. Hou Y, Scherlag B, Lin J, Zhang Y, Lu Z, Truong K et al. Ganglionated Plexi Modulate Extrinsic
Cardiac Autonomic Nerve Input. Journal of the American College of Cardiology. 2007;50(1):61-
68.
16. Kawashima T. Anatomy of the cardiac nervous system with clinical and comparative
morphological implications. Anatomical Science International. Anat Sci Int.2011 Mar;86(1):30-
49.doi:10.1007/s12565-010-0096-0. Epub 2010 Nov. Review. PubMed: 21116884.
17. Skok VI. Physiology of autonomic ganglia. Tokyo: Igaku Shoin, 1973.
18. Smith DC. Synaptic sites in sympathetic and vagal cardioaccelerator nerves of the
dog. Am J Physiol. 1970;218:1618 –1623
19. Randall WC, Armour JA, Geis WP, et al. Regional cardiac distribution of the
sympathetic nerves. Fed Proc. 1972;31:1199 –1208.
20. McAllen RM and Spyer KM. The location of cardiac vagal preganglionic
motorneurons in the medulla of the cat. J Physiol 258: 187–204,
1976.
21. Kawashima T (2005) Autonomic nervous system of the human heart with special
reference to the origin, course, and peripheral distribution of the nerve. Anat
Embryol 209:425–438
22. Emily Wake, Kiran Brack Dr, Department of Cardiovascular Sciences, Cardiology
Group, Glenfield Hospital, Groby Road, University of Leicester, LE3 9QP, United
Kingdom Leicester NIHR Biomedical Research Unit in Cardiovascular Disease,
27
Glenfield Hospital, Leicester LE3 9QP, United Kingdom, 2016
23. Singh S, Johnson PI, Lee RE, et al. Topography of cardiac ganglia in the adult
human heart. J Thorac Cardiovasc Surg. 1996;112:943–953
24. Anderson RH. The disposition, morphology and innervation of the cardiac
conduction specialized tissues in the guinea pig. J Anat. 1972;111:453-468.
25. Simon J. Crick, BSc; John Wharton, PhD; Mary N. Sheppard, MD, MRCPath;
Dervil Royston, MD, MRCPath; Magdi H. Yacoub, MD, FRCS; Robert H.
Anderson, MD, BSc, FRCPath; Julia M. Polak, DSc, MD, FRCPath, Innervation
of the Human Cardiac Conduction System, A Quantitative Immunohistochemical
and Histochemical Study, 2015
26. Pauza D, Saburkina I, Rysevaite K, Inokaitis H, Jokubauskas M, Jalife J et al. Neuroanatomy of the
murine cardiac conduction system. Autonomic Neuroscience. 2013;176(1-2):32-47.
27. Crick S, Sheppard M, Anderson R, Polak J, Wharton J. A quantitative assessment of innervation in
the conduction system of the calf heart. The Anatomical Record. 1996;245(4):685-698.
28. Inokaitis H, Pauziene N, Rysevaite-Kyguoliene K, Pauza D. Innervation of sinoatrial nodal cells in
the rabbit. Annals of Anatomy - Anatomischer Anzeiger. 2016;205:113-121.
29. Christoffels V, Moorman A. Development of the Cardiac Conduction System: Why Are Some
Regions of the Heart More Arrhythmogenic Than Others?. Circulation: Arrhythmia and
Electrophysiology. 2009;2(2):195-207.
30. Monferedi O, DOBRZYNSKI H, MONDAL T, BOYETT M, MORRIS G. The Anatomy and
Physiology of the Sinoatrial Node-A Contemporary Review. Pacing and Clinical
Electrophysiology. 2010;33(11):1392-1406.
31. Hoogaars W, Engel A, Brons J, Verkerk A, de Lange F, Wong L et al. Tbx3 controls the sinoatrial
node gene program and imposes pacemaker function on the atria. Genes & Development.
2007;21(9):1098-1112.
32. Moskowitz I, Kim J, Moore M, Wolf C, Peterson M, Shendure J et al. A Molecular Pathway
Including Id2, Tbx5, and Nkx2-5 Required for Cardiac Conduction System Development. Cell.
2007;129(7):1365-1376.
28
33. Bakker M, Boukens B, Mommersteeg M, Brons J, Wakker V, Moorman A et al. Transcription
Factor Tbx3 Is Required for the Specification of the Atrioventricular Conduction System.
Circulation Research. 2008;102(11):1340-1349.
34. Mikawa T, Hurtado R. Development of the cardiac conduction system. Seminars in Cell &
Developmental Biology. 2007;18(1):90-100.
35. Eichna L, DEHAAN R. Differentiation of the Atrioventricular Conducting System of the Heart.
Circulation. 1961;24(2):458-470.
36. Virágh S, Challice C. The development of the conduction system in the mouse embryo heart.
Developmental Biology. 1977;56(2):382-396.
37. Moorman A, CHRISTOFFELS V. Cardiac Chamber Formation: Development, Genes, and
Evolution. Physiological Reviews. 2003;83(4):1223-1267.
38. Crick S, SHEPPARD M, HO S, ANDERSON R. Localisation and quantitation of autonomic
innervation in the porcine heart I: conduction system. Journal of Anatomy. 1999;195(3):341-357.
39. Pauza D, Rysevaite K, Inokaitis H, Jokubauskas M, Pauza A, Brack K et al. Innervation of
sinoatrial nodal cardiomyocytes in mouse. A combined approach using immunofluorescent and
electron microscopy. Journal of Molecular and Cellular Cardiology. 2014;75:188-197.
40. Dickie R, Bachoo R, Rupnick M, Dallabrida S, DeLoid G, Lai J et al. Three-dimensional
visualization of microvessel architecture of whole-mount tissue by confocal microscopy.
Microvascular Research. 2006;72(1-2):20-26.
41. Richardson R, Grkovic I, Anderson C. Immunohistochemical analysis of intracardiac ganglia of
the rat heart. Cell and Tissue Research. 2003;314(3):337-350.
42. Batulevicius D, Pauziene N, Pauza D. Topographic morphology and age-related analysis of the
neuronal number of the rat intracardiac nerve plexus. Annals of Anatomy - Anatomischer
Anzeiger. 2003;185(5):449-459.
43. Pauziene N, Rysevaite-Kyguoliene K, Alaburda P, Pauza A, Skukauskaite M, Masaityte A et al.
Neuroanatomy of the Pig Cardiac Ventricles. A Stereomicroscopic, Confocal and Electron
Microscope Study. The Anatomical Record. 2017;300(10):1756-1780.
44. Pauziene N, Alaburda P, Rysevaite-Kyguoliene K, Pauza A, Inokaitis H, Masaityte A et al.
Innervation of the rabbit cardiac ventricles. Journal of Anatomy. 2015;228(1):26-46.