ELECTRON MICROSCOPIC STUDY OF RABBIT’S CARDIAC GANGLION GANGLION Prabhu Muthu

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ELECTRON MICROSCOPIC STUDY

OF RABBIT’S CARDIAC GANGLION

GANGLION

Prabhu Muthu

Institute of Anatomy

Medicine

(MF VI)

Supervisor: Prof. Neringa Paužienė

Lithuanian University of Health Sciences

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1. SUMMARY

Through this ultrastructural study of the rabbit cardiac ganglion, we have described the ultrastructure and compared it with other mammalian species. Three types of ganglia cluster organizations were found in the atria. The cluster of closely packed ganglia with a well-defined perineurium, blood vessels and collagen fibers was described as first type. Second type was compact, big ganglion with a well-defined perineurium. Third type was small ganglion with well-defined perineurium and numerous nerve fibers in neurophil. Two types of satellite cells have been found in the same neuron. Similar satellite cells have been reported in porcine cardiac ganglia. First type was with a thin, dark, dense cytoplasm and second type with thick less dense cytoplasm. Three types of axon terminals have been identified. First type was exclusively made of small, spherical clear core vesicles. Second type had the same small, spherical, clear core vesicles in addition they had few large dense cored vesicles. Third type of axon terminals were exclusively made of large dense cored vesicles. Axodentritic synapses have dominated compared to axosomatic, which is in contrast to the previous rabbit ultrastructural studies. The mean ganglia and neuron area are 13993.14±3475.09 21µm² and 781.845±39.21µm² respectively. The mean satellite cell thickness and perineurium thickness are 0.76±0.09 µm and 0.32±0.26 µm. It is recommended to visualise the ganglia location by some technique before sample collection. This would have provided accurate and good

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4 quality samples. Immunohistochemical staining of the thin sections is recommended. This would help in identifying the functional type of the neurons.

1. SANTRAUKA

Šio tyrimo tikslas buvo ultrastruktūriškai ištirti triušio širdies mazgus. Buvo nustatyti trys skirtingi mazgų išsidėstymo tipai. Pirmuoju atveju stambūs susigrupavę mazgai buvo susitelkę į „klasterį“, kuriame mazgai buvo atskirti perineuriumo sluoksniu, o tarp jų įsiterpusios kraujagyslės ir kolageno skaidulos. Antrasis tipas - tai kompaktiški, dideli mazgai, apsupti gerai išreikšto perineuriumo. Trečiajam tipui buvo priskirti maži mazgai, apdengti gerai matomu perineuriumo sluoksniu ir neuropiliu tankiai užpildytu gausiomis nervinėmis skaidulomis. Satelitinės ląstelės dengiančios atskirus neuronus buvo dviejų tipų. Pirmosios buvo tamsios ir plonos, antrosios – šviesios ir storos. Buvo nustatytos trys aksonų terminalių tipai. Pirmuoju atveju terminalės buvo užpildytos mažomis skaidriomis sinapsinėmis pūslelėmis. Antruoju – be tokių pačių mažų skaidrių sinapsinių pūslelių papildomai buvo keletas didelių tamsia šerdimi pūslelių. Trečiojo tipo aksonų terminalės turėjo tik dideles, tamsia šerdimi sinapsines pūsleles. Aksodendritinės sinapsės buvo dominuojančios lyginant su aksosomatinėmis sinapsėmis ir šis rezultatas yra priešingas anksčiau atliktiems triušio širdies mazgų ultrastruktūriniams tyrimams. Vidutinis mazgo plotas buvo 13993,14 + 3475,09 μm². Vidutinis neurono plotas buvo 781,845 + 39,21 μm². Vidutinis satelitinės ląstelės storis buvo 0.76±0.09 µm. Vidutinis perineuriumo ląstelės storis buvo 0.32±0.26 µm.

Intrakardinių nervinių mazgų pavyzdžių ultrastruktūriniams tyrimams paėmimas būtų efektyvesnis, jei pradžioje būtų atliekamas mazgų vizualizavimas visoje širdyje.

Tolimesniam, neurofunkciniam triušio intrakardinių mazgų tyrimui būtų tikslinga atlikti imunohistocheminį tyrimą ultrastruktūriniame lygmenyje.

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2. ACKNOWLEDGEMENT

I would like to thank Prof. Neringa Paužienė for her guidance throughout the research. Ms.Aistė Masaitytė and Mrs.Jurgita Šventoraitienė for their technical assistance and everyone from the Institute of anatomy whom I had the privilege to work with.

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3. CONFLICT OF INTEREST

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4. ETHICS COMMITTEE APPROVAL

Approved by: State Food and Veterinary Service Kaunas Food and Veterinary Service Chief – State Veterinary Inspector

Permission issued for: LSMU VA Animal Research Center

Permision number: LT-61-19-004 given 2015-12-02, by the order No. 33ĮV-638 Full document attached in annexes.

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5. ABBREVIATIONS

AV – Atrioventricular

AVCA- Atrioventricular conduction axis DRA – Dorsal Right Atrial

GF’s – Ganglia Fields GN’s – Ganglionic Nerves

LSPV- Left Superior Pulmonary Vein LNC – Left Neuronal Cluster

LC – Left Coronary subplexus LD – Left Dorsal subplexus

LDCV – Large Dense Cored Vesicles MD – Middle Dorsal subplexus PBS – Phosphate- Buffered Saline PB – Phosphate-Buffer

RSPV – Right Superior Pulmonary Vein RCV – Right Cranial Vein

RNC – Right Neuronal Cluster RC – Right Coronary Subplexus RER – Rough Endoplasmic Reticulum SVC – Superior Vena Cava

SC – Satellite Cell

VRA – Ventral Right Atrial subplexus VLA – Ventral Left Atrial subplexus

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6. INTRODUCTION

Rabbits are often used as animal model in various cardiac disease studies. They are convenient because of their size, cost, maintenance and their genetic makeup. Also they are the biggest animals in small animal model group [1]. Both the intrinsic and extrinsic cardiac nervous system of the mammals plays a major role in the development of arrhythmias [2]. In addition, it was found that, these systems have the ability to cause arrhythmias independently [3,4] . Thus understanding of the normal ultrastructure of rabbit’s cardiac ganglia would provide data to compare in pathologic rabbit models. R.E. Papka [5] in the 70s studied the ultrastructure of rabbits in pre and post natal rabbits. Since then, many animals ultrastructure have been studied. This study was conducted to study the rabbit ganglia ultrastructure in adult rabbits and compare that with other mammals. Many similarities with other mammals and contrasting findings with previous studies on rabbit are presented.

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6. AIM AND OBJECTIVES

The aim of the study is to describe the ultrastructure of the rabbit’s intrinsic cardiac ganglia.

OBJECTIVES

1. To describe histological structure of rabbit ganglia by light microscopy. 2. To describe the ultrastructure of the rabbit cardiac ganglia:

a) To define ultrastructural features of neurons; b) To describe the types of intraganglionic synapses; c) To determine ultrastructural character of neuroglial cell;

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7. LITERATURE REVIEW

According to D.H. Pauza, the part of heart hilum around aorta and pulmonary trunk is arterial hilum, and the part of hilum around pulmonary veins and vena cava is venous hilum. This is crucial for understanding the accessing location of extrinsic cardiac nerves and topography of the intrinsic cardiac nervous system [6].

7.1 SOURCES OF INNERVATION

The parasympathetic innervation of the heart is achieved by left and right vagal trunk whose axons or preganglionic fibers are from nucleus ambigus and dorsal motor nucleus of the medulla oblongata [7, 8]. These parasympathetic preganglionic projections innervate multiple intrinsic cardiac ganglionic plexuses located within atrial and ventricular tissues [9].

The sympathetic preganglionic neurons originate in the intermediolateral column of the spinal cord. According to P. Hanna et al. [8] and J.L. Ardell [9], they project out through cervical 7 to thoracic 6 spinal cord segments. But J.H. Coote et al. and T. Kawashima described projections are confined to thoracic 1 to thoracic 6 spinal cord segment [10, 11]. These nerves synapse with the postganglionic sympathetic neurons located in the superior and middle cervical, cardiothoracic, mediastinal, and intrinsic cardiac ganglia [3,4]. The postganglionic nerve fibers from postganglionic neurons project through superior, middle, inferior and thoracic cardiac nerves. The names of the nerves correspond to their origin [11]. The sensory information of the heart is relied to upper centre by the vagal nerve. It has been found that 80% of vagal nerve fibers are afferent, carrying sensory information. The cardiac related vagal afferent neurons have cell bodies located in the nodose ganglia that project sensory information onto secondary afferent neurons in the nucleus solitarii located in the medulla. Sympathetic afferents arising from dorsal root ganglion, also input to the nucleus tractus solitarius through spinoreticular projections [9].

The extrinsic cardiac nerves access the heart hilum through both the arterial and venous hilum. In humans, extrinsic cardiac nerves enter the arterial hilum between and around the aorta and pulmonary trunk [12]. In the venous hilum, the access is at the left atrial nerve fold, between right superior pulmonary vein (RSPV) and superior vena cava (SVC), between the RSPV and LSPV (left superior pulmonary vein) and in between the pulmonary veins [11, 12]. In dogs, the access of extrinsic cardiac nerves is similar to that of humans, but extrinsic cardiac nerves access at the interatrial sulcus in addition [13]. In rabbit, the extrinsic cardiac nerves enter the venous hilum at the root of right cranial vein and caudal vein. In the arterial

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12 hilum, the entry is at the fat pad located at the bifurcation of pulmonary trunk [14]. In pig and mouse the access was at the right and left cranial veins [15, 16].

7.2. TOPOGRAPHY OF THE GANGLIA FIELDS

The nerves entered did not directly penetrate the myocardium in spite entered the epicardium and coursed towards the ganglia fields (GFs). The nerves coursing to the GFs are thin. They are termed as preganglionic nerves and those that proceed after the GFs are termed as postganglionic nerves [12]. The location of the GFs in most of the mammals (human, rabbit, dog, mouse, pig, rat, sheep) were confined to the venous hilum. In all the above mentioned mammals, quantitative analysis showed a high number of ganglia in the venous hilum compared to the para aortic and ventricular region. Some animals had different pattern of GFs distribution. In frog, majority of ganglia are in the interatrial septum [17]. In bat, the hilum was devoid of any ganglia. [18].

In rabbit, mouse and rat the GFs around the right cranial vein (RCV) or cluster of neurons on the right side of venous hilum were termed right neuronal cluster (RNC) and those around the left cranial vein (LCV) or clustered around the left of venous hilum and ventral to the pulmonary vein were named as left neuronal cluster (LNC). In these animals extrinsic nerves accessing the hilum through the right and left cranial vein entered directly into right and left neuronal clusters respectively [13, 14, 19].

On rabbit’s ventricular studies [20], 94% of ventricular ganglia are distributed near the root of the pulmonary trunk or slightly above the semilunar valve. The remaining 6% of the neurons were singularly disseminated on the conus arteriosus. In the pig cardiac ventricles [21], the distribution was totally different. Most of the ganglia were distributed in the anterior and posterior interventricular grooves. In pigs cardiac ventricles, ganglia scattered along the epicardial nerves were found to be oblongated. Those ganglia’s found farther away from the large epicardial nerves were more spherical.

Sometimes near the coronary sulcus and in the ventricles, postganglionic nerves entered subsequent cluster of epicardiac ganglia. The density of these GFs was less compared to that of the ones in venous hilum [12]. The GFs are interconnected by thin commissural fibers. In animals with RNC and LNC type of GFs, right and left NC were interconnected by these thin commissural fibers [13, 14, 19].

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7.3. PLEXUS OF THE HEART

The postganglionic fibers leaving the GFs were gathered into seven subplexuses as described in previous studies [12, 13, 14, 16, 22] namely right coronary (RC) and left coronary (LC) subplexus, ventral right atrial (VRA), ventral left atrial (VLA), left dorsal (LD),middle dorsal (MD), dorsal right atrial (DRA). Out of the seven subplexus, RC and LC originate from the arterial hilum and the remaining five originate from the venous hilum. Here the subplexal structure of the human, dog and rabbit will be described as it summarizes the basic subplexal structure.

The right and left coronary subplexuses has its ganglion in arterial hilum. The RC subplexus extend along the right coronary sulcus, innervating the ventral, lateral surface of right ventricle and right half of conus arteriosus. The LC subplexus course towards the apex giving branches to the ventral, lateral and dorsal ventricle, left side of conus arteriosus, inferior part of left auricle. On the dorsolateral surface of the left ventricle, LC and LD subplexus overlap.

VRA subplexus has its GFs in the root of RCV. The subplexus extended to the right auricle, ventral right atrium as well as to the left atria. VLA subplexus extended to the left ventroatrial surface and to the left atrial appendages. DRA subplexus extended into the dorsal and lateral surface of the right atrium. At the inferior margin of the right ventricle the DRA, VRA and RC converge to form a fine ganglionated meshwork. Innervation of sino-atrial node is achieved by DRA and VRA subplexus in addition to the right auricle [12, 14, 17, 22, 23].

The preganglionated nerves of LD subplexus entered the heart at left atrial nerve fold or at the LNC. Giving off some branches to the left auricle, other nerves entered the GFs around the coronary sinus, crossing the coronary groove onto the left dorsal ventricular surface. Some postGNs extended obliquely into the middle dorsal subplexus. MD subplexus had its ganglions in LNC located above MPV or between the pulmonary veins. The course was along the coronary sulcus to the crux cordis and then to the caudal surface of right atrium, another part along dorsal surface of the right ventricle, and the last penetrated into the dorsal aspect of the interatrial groove. Also the MD subplexus spreads dorso-ventrally as endocardial nerve along the right side of interatrial septum becoming thinner and ramified at the AV node [14].

In dogs, it has been documented that VRA, DRA, LD, MD, LC extend in the direction of the canine AV node within the interatrial septum [13]. The atrioventricular conduction axis (AVCA) was supplied by nerves and neural bundles that extended from the atrioventricular nodal region along the His bundle. Many thin nerve fibers also coursed towards the AVCA

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14 from the dorsal and ventral side of the interventricular septum in the endocardium. Another observation in the ventricle was that the epicardial nerves size and width decreased gradually from the base to the apex [24].

7.4. ULTRASTRUCTURE OF GANGLIA

The cardiac ganglia population is presumably made of parasympathetic postganglionic neurons that receive input from both the vagal nerves. In addition there may be at least three more types of neurons sensory neurons, interneurons and sympathetic neurons [7, 25]. There are studies reporting the ultrastructure changes in the ganglia structures following pathology. [26] Reported an increase in size of sympathetic and parasympathetic neuron after atrial fibrillation. Morphological changes have been seen post myocardial infarction. Rajendran PS et al. showed hypertrophied neurons with edematous pale neuropil in humans and canine with heart failure and hypertensive rat models [27]. Therefore it is of importance to study the basic normal structure of the neurons, neuropil of the ganglia in experimental animals.

7.4.1. GANGLIA STRUCTURE

The intracardiac ganglia appear as solitary or cluster of nerve cell bodies surrounded by nerve cell process. The ganglia content is made up of neuronal and non-neuronal type cells. The non-neuronal cells are fibroblasts, macrophages, collagen and blood vessels. The neuronal content of the ganglia are cell body, axons, dendrites, axon terminals and nerve bundles [25, 29].

7.4.2. NEURON BODY

The neuron cytoplasm is made of numerous organelles and euchromatic nuclei. The nuclear envelope is smooth with regular outline with indentation sometimes. The neuroplasmic organelles include long cylindrical mitochondria, Golgi complex, condensation of ribosomal rosettes and rough endoplasmic reticulum (RER) embedded in neurofibrills and microtubules. Multivesicular bodies, pinocytic, coated dense core vesicles are seen sometimes. In humans lipofusin granules are aggregated to one side [25, 29]. In mouse, the plasma membrane showed indents [29].

7.4.3. SATELLITE CELL

The neuronal cell body is covered by sheath of satellite cells. In humans, ultrastructure study by N.Pauziene, showed neuronal somata with single and multilayered sheath of satellite cells [25, 29]. Places of neuronal plasma membrane devoid of the satellite cell sheath were also seen. The satellite cells nuclei is oval heterochromatic with cytoplasm made of

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15 mitochondria, RER, Golgi apparatus and gliofilaments. [25, 29]. Ellison and Hibbs in mammals described that neuron and its process are covered by sheath of satellite cells [30]. In humans the dendrite as a whole was covered by satellite cell, part of the dendrite was not covered and had direct contact with the basal lamina [25, 30]. In human cardiac ganglia, primary cilium was found on the surface of the satellite cells. Also, presence of primary cilium was found in the enteric neurons [31].

7.4.4. DENDRITE

The dendrite cytoplasm is similar to that of the neuronal cell body as it is the direct extension. The dendrite is attached to the cell body by a narrow stack. The proximal part is made up of microtubules, neurofilaments with ribosomes in between them. The distal part of the dendrite has ribosomal particles, mitochondria and cistern of smooth ER. Multivesicular bodies, lipofuscin granules, pinocytotic vesicles [25].

7.4.5. AXON

The other processes of the neuronal somata are the axons. The unmyelinated and myelinated axons are in the ganglion neuropil, of which the unmyelinated axons dominate in number. Both types of axons are embedded in Schwann cell cytoplasm or its process. The axoplasmic content is made of evenly distributed microtubule and microfilaments with some mitochondria and cisterns of agranular endoplasmic reticulum [25, 32].

Unmyelinated axons exhibited some varicosities which are rich in neurofilaments, microtubules, mitochondria, vesicles and glycogen particles. Unmyelinated axons axoplasmic content is rich compared to its counterpart. Axon terminals are located in the neurophil as well. The content of which are small mitochondria with lamellated dense bodies and vesicles in their cytoplasm [25, 30].

7.4.6. SYNAPTIC PROFILES

The type of synaptic connection varies among experimental animals and humans. Human cardiac ganglia are mostly made of axosomatic and axodendritic synapses [25]. Whereas in rabbit it was found to have more axosomatic synapses. The synaptic terminals are grouped based on the type of vesicles found in the terminals. R.E. Papka [5] described two types of synaptic terminals, first type with small, clear vesicles and considered cholinergic. The second type is made of small, dense large cored vesicles and considered as adrenergic. N.Pauziene has grouped the terminals into three in humans. The first ones are the most common with abundant small agranular, scarce, large dense cored vesicles. They are

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16 considered cholinergic. The second type had all the features of the first with glycogen like particles. The third type had large pleomorphic clear with or without dense cored vesicles, small clear vesicles, mitochondria, dense axoplasm and multivesicular bodies [25]. Studies done by Ellison and Hibbs [30] on rats, guinea pig, kittens and monkey, also reported to have similar presynaptic profile as in humans.

Other type of cell found in rabbit ganglia [2] are the small granule containing cells. They are also called as small immunofluorescent cells because of their characteristic appearance in immunohistochemistry. These cells are considered to be interneuron’s due to the fact that they received preganglionic afferent synaptic terminal and give efferent synaptic contacts to the postganglionic neurons.

8. MATERIALS AND METHODS

8.1. MATERIALS

Two New Zealand female rabbits of age 2 and 3 months were used in this study. Both the animals were euthanized. Old resin blocks from 10 adult rabbits, which were previously studied were also used.

8.2. FIXATION

Thoracotomy was performed in the euthanized animals to reveal the heart. The blood was washed out from the organ with phosphate-buffered saline (PBS) of 4ºC and ph-4. The heart was placed in 2.5% glutaraldehyde for 1.5 hours.

Tissue blocks of 1mm thickness were taken using dissection microscope, fine scissors and tweezers. The location from which the specimen’s were collected is illustrated in Fig. 1.Primary fixation of the collected tissue samples was done in 2.5% glutaraldehyde at 4ºC overnight for 15 hours. The specimen was washed in PB, 2 times for 10 minutes. Post fixation of the specimen was done with 1% osmium tetroxide in 100mM phosphate buffer for 1.5 hours and then washed in PB for 10 minutes.

8.3. EMBEDDING

Then the specimens were dehydrated through a series of different concentration of ethanol in 30%, 50%, 70%, 95%, ethanol for 10 minutes each. Followed by, 100% ethanol 2 times for 10 minutes. Finally with 100% acetone 2 changes each, each of 10 minutes. The specimens were oriented for transverse sectioning in flat embedding moulds under a stereoscopic microscope Stemi 2000CS (Zeiss, Gottingen, Germany). The specimens were

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17 infiltrated with resin using the following mix 2:1 mix of acetone: resin for 17 h, then in 1:2 mix of acetone: resin for 29 h. And at last, in 100% resin for 24 h. The tissue specimens were embedded in moulds with fresh resin and labelled using the number coded when taking the samples. The resins were left to polymerise at 60ºC overnight.

Fig. 1. Illustration of rabbit heart with tissue sampling location. (a). Ventral view of rabbit heart. Rectangles representing the places of tissue sampling. (b). Dorsal view of rabbit heart. Squares representing the places of tissue sampling. Image adapted from histochemistry study by I. Saburkina [14].

8.4. STAINING FOR LIGHT MICROSCOPE

In total, 44 resin blocks were made and 103 resin blocks were used from the old studies. The newly made resin blocks and old resin blocks from previous studies were made into pyramids in a trapezoid shape with a stereomicroscope. Semithin sections of 1000nm were cut using Leica EM UC7 ultra‐microtome (Leica Mikrosystem Handelsges.m.b.H., Viena, Austria) with a glass knife. Cut semithin sections were placed in labelled glass slides. The staining was done with methylene blue. The semithin sections were assessed for the presence of ganglia using a light microscope. The respective pyramids of the semithin sections in which the ganglia were found were taken out. The semithin section in which ganglia were not found was cut deeper and stained. By repeated semithin sectioning and staining, we were able to find 14 resin blocks which had ganglia. The perimeter of the ganglia containing pyramid’s trapezoid part was downsized in such a way the ganglia are left out for thin sections. The ones which were found to have ganglia were prepared for thin sections.

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8.5. STAINING FOR ELECTRON MICROSCOPE

Thin sections of thickness 70nm were cut with diamond knife using Leica EM UC7 ultra‐microtome (Leica Microsystems Handelsges.m.b.H., Vienna, Austria). The thin sections were placed on a grid. Staining of thin sections was done with uranyl acetate and lead acetate for 10 minutes using lab protocol. All the ganglia studied in detail are atrial as the ganglia from arterial hilum i.e. ventricular ganglia were lost during thin sectioning.

8.6. IMAGE AND STATISTICAL ANALYSIS

Images of semithin section containing ganglia were taken using Axio camera in different magnifications. The thin sections were analysed using Tecnai BioTwin Spirit G2 transmission electron microscope (FEI, Eindhoven, Netherlands). Electron micrographs were photographed at grid bar intersections using various magnifications from low to high, and analyzed with AxioVision Rel. 4.8.2 (Carl Zeiss, Jena, Germany).

The ganglia and neuron area in semithin sections were measured using “Image J” software’s free hand tool. Only the neurons with visible nucleolus were measured. Also the satellite cell thickness and perineurium thickness was measured using “Image J” straight line measurement tool. Data was recorded in “Microsoft excel 2010” for statistical analysis. The results of which are presented in Table. 1.

9. RESULTS

9.1. SEMITHIN SECTION RESULTS

The ganglions are located in the subepicardial layer with adipocytes often present in the vicinity of the ganglion. The ganglia appeared as a cluster and in some thin section as a compact single structure. So, we have grouped the organization of ganglia cluster into three groups. First group was made of two to five individual ganglia, clustered together with nerves and collagen in between them (Fig. 2A). Second group had a big, compact ganglia with a well-defined perineurium(Fig. 2B). Third group had small ganglia with numerous nerves in the neuropil (Fig. 2C). Each ganglion had a thin rim of perineurium surrounding it, which was confirmed by electron microscopy. The shape of the neurons varied from circular, oval and triangular. The neurons had centrally or eccentrically located round nucleus with prominent nucleolus. The ganglion and neuron areas were measured. The results are tabulated in Table .1.

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Table. 1.

Tabulated results of ganglion area, neuron area, satellite cell thickness and perineurium thickness.

Light microscope Electron microscope Ganglia area (μm²) Neuron area (μm²) Satellite cell thickness (μm) Perineurium thickness (μm) n 19 44 25 30 mean 13993.14 781.845 0.76 0.32 Standard error 3475.09 39.21 0.09 0.26 Standard deviation 1514.59 260.136 0.47 0.14 Minimum 1681.36 356.55 0.15 0.11 Maximum 57272.76 1559.159 1.52 0.64

9.2. THIN SECTION RESULTS

9.2.1. THE GANGLION

In thin sections, the ganglia appeared as solitary or cluster of neurons. Each neuron was covered by a thin sheath of satellite cell. In between the satellite cells, neuron processes like axon, dendrite, axon terminals and Schwann cell nucleus were seen. The ganglion was covered by a single to multilayered perineurium. The space between the perineurium and neuron often had collagen fibers and other non-neuronal cells in between them. Each component of the ganglia was analyzed in detail and the results are presented.

9.2.2. THE NEURON

The ganglia examined had either grouped or solitary neurons. Majority of the neuron’s nuclear membrane were smooth. While neurons with irregularly shaped nuclear membrane were also observed. The nucleus was prominent and euchromatic. The cytoplasmic organelles were typical to that of autonomic neurons. The constituents observed were normal and enlarged mitochondria, Golgi apparatus, endoplasmic reticulum, pinocytotic vesicles (Fig. 3A). Lipofuscin granules, multivesicular bodies and lamellated bodies were seldom found (Fig. 3B, 7A). Membrane specialisations were observed between the neuronal plasma membrane

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Fig. 2. Semithin section of cardiac ganglia clusters showing 3 types of organizations. A: First group with many individual ganglia clustered together with nerves and collagen in between them. Ganglia (g) with neurons (stars) in the periphery of the ganglia, covered by perineurium (black arrows). Myelinated axon fibers (black arrowheads) with dark myelin are located away from the neuron and unmyelinated axon fibers (white arrowheads) located close to the neuron body. Abundant adipocytes (ad) and blood vessels (b) are noted in the ganglia vicinity. Ganglia made of single neurons (s) with nerve fibers in the neurophil. B: Second group with single compact ganglia (g) with neurons (stars) located peripherally. C: Third type of ganglia (g) with individual solitary neuron and numerous nerves in the neutrophil. Scale bar: 50 µm in A, B and C.

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21 and satellite cells (Fig. 5A). Small granule containing cells, similar in structure to a neuron was also found (Fig. 4A). They were differentiated from the neuron by the presence of numerous large dense cored vesicles and poor cytoplasmic organelles. The round vesicles had a dark core and clear ring of plasma between the vesicular membrane and dark core. The plasma membrane of the small granule containing cells was smooth (Fig. 4A).

9.2.3. SATELLITE CELL

All the neurons examined were covered by thin sheath of satellite cells. The neuronal satellite sheath was single layered all over the neuron, except in places with neuronal process and satellite cell nucleus. SC around the neuronal process was lamellated i.e. multilayered and engulfing.SC cytoplasm had mitochondria, rough endoplasmic reticulum (RER), microfilaments. The cytoplasmic constituents were loosely packed and placed parallel to the neuronal plasma membrane (fig.5A). Some neurons had SC with different cytoplasm i.e. half of the SC sheath in cross sections were dark and thin. Whereas the other half had lighter cytoplasm and thicker (fig.5B).The thickness of the satellite cell cytoplasm was measured and the results are tabulated in Table. 1.

9.2.4. DENDRITE

The observed dendrites were present in the vicinity of the soma and covered by SC sheath. The examined dendrites had long cylindrical mitochondria, microtubules, ribosomes and clear white large vesicles. Large dense cored vesicles (LDCV) and pinocytotic vesicles were seldom present. The dendritic contents were somewhat similar to that of the cell body, as they are the direct extension of the cell bodies (Fig. 6A).

9.2.5. AXON

The neuropil had both unmyelinated and and myelinated axons. Those unmyelinated axons, that are present near the soma were solitary and were ensheathed by SC. Unmyelinated axons present away from the soma were grouped in bundles. Both unmyelinated and myelinated axons were embedded in Schwann cell process. Both type of axons had collagen surrounding them. The axoplasmic content includes microtubules, neurofilaments, mitochondria, glycogen. Axon terminals with small clear vesicles and large dense cored vesicles were also found (Fig. 6A). Large axon terminals with closely packed mitochondria, microtubules, multivesicular bodies, clear and dense cored large vesicles were noticed.

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Fig. 3. Electron micrograph showcasing the neuronal cell body contents. A: Low magnification electron micrograph of a typical cardiac neuron. Nucleus (n) of the neuron less electron dense compared to the cytoplasm with uneven nuclear membrane. Golgi apparatus (g), lipofuscin granules (marked by black arrows), mitochondria (m) can be noted as well. B: Electron micrograph of different neuronal cytoplasm with satellite cell (s) on the right top corner, enlarged mitochondria (m), rough endoplasmic reticulum (r), large dense corvesicles (arrowheads), multivesicular body (arrow). Notice the absence of lipofuscin granules in B. Scale bar: 2µm and 1µm respectively.

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Fig. 4. Electron micrograph showing small granule containing cells with large dense cored vesicles and an axon terminal with large dense cored vesicles. A: Small granule containing cell (sg) with numerous dark dense cored vesicles (arrows), nucleus (n) and satellite cell (s) around it. B: Axon terminal filled (Ax) with large dense cored vesicles (black arrows). Scale bar: 1µm in A and B.

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Fig. 5. Electron micrographs showing satellite cell content. A: Neuron cell body (n) covered

by satellite cell sheath ( course of satellite cell is represented by stars). Between the satellite cell andneuron body membrane specializations (white arrow heads) are shown. Satellite cell contents mitochondria (m), endoplasmic reticulum (r), nucleus (n) and gliofilaments in-between them are seen. Axons (ax), dendrite(d) making synaptic contact.Notice the lamellations (arrows) formed by satellite cells around the axon and dendrite. B: Shows a triangle shaped neuron (n) surrounded by satellite cell sheath. Satellite cell sheath which is thin and have dark, dense cytoplasm ( arrows). satellite cell sheath which has thick and less dense cytoplasm (arrowheads). Neuron (n). Scale bar: 1µm and 5µm respectively.

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9.2.6. SYNAPTIC TYPE AND VESICLES

Axodendritic synapse dominated in all the ganglia. Three types of axon terminals have been reported in this study. First type was exclusively made of small, spherical clear core vesicles. Second type had the same small, spherical, clear core vesicles in addition they had few large dense cored vesicles (Fig. 6A). Third type of axon terminals were exclusively made of large dense cored vesicles (Fig. 3B).Of all the ganglia examined only one axosomatic synapse was found (Fig. 6B).

9.2.7. PERINEURIUM

Ganglia with clustered neurons and solitary neurons were covered by a sheath of perineurium. The perineurial contents observed were mitochondria, ribosomes, endoplasmic reticulum. The gap between the neuron and perineurium was often filled with collagen. The inner side of the perineural membrane had numerous caveolea (Fig. 7A). Fibroblasts were observed above and below the perineurium (Fig. 7B). The number of perineural layers varied from 1 to 5 (Fig. 7C). The mean thickness of the perineural membrane was measured and the results are presented in table. 1.

9.2.8. NON NEURONAL CELLS

Nonneuronal cells of the ganglion neurophil included, fibroblasts, blood vessels and collagen fibers (Fig. 7A, 7B).

Fig. 6. Electron microscope showing different synaptic contacts. A: Axodendritic synapse. Axon(ax), dendrite(d) making an axodendritic synapse (black arrowhead) are shown. In the axoplasm,

m

r

ax

d

A

B

s

p

n

c

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mitochondria (m), large dense cored vesicle (black arrow) and small clear vesicles (white arrows) are clearly seen. In the dendrite (d), mitochondria (m), large dense cored vesicles (black arrows) and ribosomes (r) are shown. B: Axosomatic synapse. Shows neuron (n) and an axon (ax) making axosomatic synaptic contact (black arrow). Large dense cored vesicle (white arrow) is seen inside the axon. Secretory vesicles on the neurons membrane (white arrowhead). Satellite cell (sc) ensheathing the axon is seen. Collagen (c) outside the satellite cell and perineurium are shown. Scale bar: 1µm, 500nm respectively.

Fig. 7. Electron micrograph showcasing perineurium and some neuron content. A: Perineurium (P) which is single layered having mitochondria (black arrowheads), caveolae (black arrows), ribosomes (white arrow). Beneath the perineurium, large amount of collagen (c) is seen. Neuron body (n) with lamellated bodies (white arrowheads) are seen in the neuron cytoplasm. Thin satellite cell sheath (s) around the neuron. B: Fibroblasts (f) are seen above the perineurium (P). Notice the large indentation (black arrows) in the neuron body. C: Shows multiple perineurium layers (P1, P2, P3), collagen (c). Scale bar: 500nm in A and 1 µm in B and C.

A

B

C

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c

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f

P1 P2 P3

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10. DISCUSSION

Light microscope and electron microscope observations showed features, which have been established in ultrastructure studies and confirms the data from histochemistry. The ganglia observed in semithin sections were clustered in groups. Solitary neurons with perineurium covering and nerve fibers inside were also noted. These observations are in accord with the data we have on histochemistry of rabbit’s atrium. I. Saburkina [14] in the histochemistry study, stated that the neurons were congregated in groups and solitary neurons were found in the dorsal part of the venous hilum.

Each ganglion had a thin rim of perineurium, which was confirmed by electron microscopy. The neuron bodies inside these ganglia are located in the periphery, with nerve fibers in the center. Similar distribution of neurons has been described in canine and porcine cardiac ganglia [33, 34]. In the cardiac ganglia ultrastructure studies done on mouse and human [15, 25], the neurons were evenly distributed inside the ganglia. This shows the uniqueness in the ganglia architecture despite the size of the animal.

We have grouped the organization of ganglia cluster into three groups. First group was made of individual ganglia clustered together with nerves and collagen in between them. Second group had single compact, big ganglia with well-defined perineurium. Third group showed individual solitary neuron with numerous nerves in the neuropil. According to the data from histochemistry, ganglia are located in the epicardium. Majority of the ganglia were located in epicardium except few from the first ganglia cluster group, situated in the superficial myocardium [14].

The ganglia and neuron structure were in accord with the previous studies done in rabbit and other mammals namely pig, human, mouse, dog [15, 25, 33, 34]. To summarize, the neuron had rich cytoplasm made of mitochondria, Golgi apparatus, endoplasmic reticulum, pinocytotic vesicles, lipofuscin granules, multivesicular bodies and lamellated bodies with a euchromatic nucleus. Variation and similarities between mammals ganglia ultrastructure have been observed. We have observed two types of satellite cells in the same neuron. First type was with a thin, dark, dense cytoplasm and the second type with thick less dense cytoplasm. These variations in satellite cells have been previously described by R.C. Arora [34] in porcine cardiac ganglia. He has also stated that the first type of satellite cells were present at the origin of neuronal dendrites. The majority of the synapses observed in this study were axodendritic and only one axosomatic synapse was seen. This finding is similar to human, dog and mouse’s cardiac ganglia which had axodendritic synapses as majority. But R.E. Papka [5] in his study on rabbit mentioned the dominance of axosomatic synapses which is in

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28 direct contrast with our findings. This could be because animals used in this study were adults. Whereas R.E.Papka [5] used rabbits of age, ranging from 18 days of gestation to day 35 postpartum. In this study we have also discussed about the perineurium structure in detail. The perineurium was multilayered with a minimum of 1 layer and maximum of 5 with collagen in between these layers. This structural similarity of perineurium has only been described in the mouse cardiac ganglia.

We were able to find the small granule containing cells in the thin sections examined. They were structurally similar to a neuron body with a euchromatic nucleus and a satellite cell sheath but with poor cytoplasmic organelles. These cells have been reported in previous rabbit study [35] and in cardiac ganglia of rat, guinea pig [30]. Rabbit ventricle studies have also reported these cells at the root of pulmonary trunk [20]. Studies on human cardiac ganglia haven’t reported these cells [25].

Three types of axon terminals have been reported in this study. First type was exclusively made of small, spherical clear core vesicles. Second type had the same small, spherical, clear core vesicles in addition they had few large dense cored vesicles. Third type of axon terminals were exclusively made of large dense cored vesicles. This implies that cholinergic, adrenergic and biphenotypic nerve fibers can be expected in immunohistochemical studies.

In thin sections, with large dense cored axon terminals i.e. type 3 terminals and small granule containing cells, capillaries were consistent in the vicinity. R.E. Papka [5] also noted such associations in the rabbit and proposed that, small granule containing cells might be serving two functions. First is that they are secretory and can have influence in the cardiac muscles through blood circulation. Second function is that of chemoreception as they are present in structures resembling glomus like bodies.

RECOMMENDATIONS

1, It is recommended to visualise the ganglia location by some technique before sample collection. This would have provided accurate and good quality samples.

2, Immunohistochemical staining of the thin sections is recommended. This would help in identifying the functional type of the neurons.

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11. CONCLUSSIONS

1. Light microscopic findings show:

a. Three types of ganglia – (1) clusters of ganglia, (2) compact, big ganglia with a well-defined perineurium (3) small ganglia - were found in the rabbit heart;

b. Peripheral position of neurons and centrally located nerve fibers inside the ganglia.

2. Ultrastructure of intracardiac rabbit ganglia is characterized by the:

a. Rabbit intracardiac neurons displayed general characteristic for autonomic ganglion;

b. Three types of axon terminals have been identified. First type with small clear core vesicles, second type with small clear core vesicles and large dense core vesicles and third type with purely large dense core vesicles;

c. Axodentritic synapses were dominating compared to axosomatic, which is in contrast to the previous rabbit ultrastructural studies;

d. Two types of satellite cells with a thin, dark cytoplasm and thick light cytoplasm were found;

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ELECTRON MICROSCOPIC STUDY OF RABBIT’S CARDIAC GANGLION GANGLION Prabhu Muthu