University of Otago

RESEARCH REPORT

Assessing the translational potential of oxytocin receptor blockade as a novel therapy for cardiac sympathetic activation following acute myocardial infarction.

Glenys Loke

A thesis submitted in partial fulfilment of the Degree of Bachelor of Biomedical Science with Honours. University of Otago, Dunedin, New Zealand

October 2020 ABSTRACT

Abnormal elevation of cardiac sympathetic nerve activity (cSNA) is a major contributor to the high mortality rate associated with acute myocardial infarction (MI). Increase in cSNA results in the propagation of ventricular arrhythmias, worsening the severity of damage to the heart and increasing the risk of sudden death. It was recently discovered that immediate administration of oxytocin receptor blockers (OTB) after a MI prevented increases in cSNA and improved survival outcomes. However, it is unknown whether the efficacy of OTBs would be maintained if administered at a later stage when cSNA would already be elevated. Thus, the purpose of this study was to investigate whether delayed administration of OTBs would allow for the reduction of elevated cSNA back to normal pre-infarct levels.

CSNA was recorded pre- and post-infarct for four hours using surgical and in vivo electrophysiological techniques on 22 urethane-anesthetised rats. In vivo surgical procedures were conducted in order to obtain haemodynamic and electrophysiological data. A MI was induced via physical ligation of the left anterior descending coronary artery. One hour post- infarct, a proportion of MI rats were treated with Atosiban (ATB), an OTB, to determine if there were any alterations to cSNA levels when compared to an untreated MI. CSNA was also recorded in sham rats without inducing a MI.

All rats treated with ATB (n = 6) survived throughout the four hour recording, compared to the

50% survival rate of untreated MI rats (n = 10). As expected, MI rats exhibited increases in cSNA, with a significant difference from the sham group occurring from 120 minutes (34 ±

11%, P = 0.0417) onwards. However, the cSNA levels of ATB treated rats did not present any significant decreases and continued to increase over time, following a similar trend to the untreated MI group.

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These findings suggest that delayed administration of ATB may not significantly reduce elevated cSNA levels, but could possibly lessen the overall increase in cSNA over time. Further investigation is needed to determine whether or not OTBs can be used as a therapeutic strategy in treating acute MI.

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ACKNOWLEDGEMENTS

Despite the interesting and peculiar events experienced during this academic year, the completion of this research project has been a largely rewarding and enlightening experience due to the help and support of many people, to whom I am very grateful for.

First of all, I would like to express my deep appreciation to my supervisor, Dr. Daryl Schwenke for accepting me as his Honours student and providing expert advice, guidance and humour throughout the year. His encouragement and patience have been indispensable in influencing the development of my research and surgical skills as well as furthering my interest in the health science field. I would also like to thank my advisor, Dr. Sheila Skeaff, for providing support and checking in on me during the lockdown.

A special thanks to Dr. Zoe Ashley for providing useful tips and information on surgical troubleshooting as well as sharing interesting lab stories. It was a pleasure working alongside the members of the Lamberts lab, especially Valeria and Yann, for the humorous and engaging discussions we would have during the times we were in the lab which definitely helped the long recording times pass by quickly. To Bernard, who helped me look after my rat when I went to get a tetanus shot after getting bitten, and to which we developed a friendly rivalry as we competed to see who Daryl’s favourite student was, Thank you for the much needed entertainment and witty conversation throughout the year.

To the Biomedical Sciences Administration and the Department of Physiology, thank you for taking care of all of us honours students throughout the year and providing assistance and resources to allow me to complete my research.

Finally, I will be forever thankful to my parents for their constant support, encouragement and love. Thank you for motivating me, reminding me of my goals and believing in me. Thank you for the opportunities you have given me which has shaped me to be who I am today.

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TABLE OF CONTENTS

ABSTRACT ...... i ACKNOWLEDGEMENTS ...... iii TABLE OF CONTENTS ...... iv LIST OF FIGURES ...... vi LIST OF TABLES ...... vii ABBREVIATIONS ...... viii 1. INTRODUCTION ...... 1 1.1. Regulation of the cardiovascular system ...... 1 1.2. Sympathetic cardiac regulation ...... 2 1.3. Acute myocardial infarction ...... 5 1.4. Current therapeutic strategies: ...... 7 1.5. Oxytocin and autonomic regulation ...... 8 1.6. The oxytocin receptor ...... 9 1.7. Oxytocinergic cardiac sympathetic innervation: ...... 9 1.8. Oxytocin’s involvement in acute myocardial infarction: ...... 10 1.9. Oxytocin receptor blockade as a therapeutic strategy for acute MI ...... 10 1.10. Aims and hypothesis ...... 11 2. MATERIALS & METHODS ...... 12 2.1. Animals ...... 12 2.2. Non-recovery surgical procedures: ...... 12 2.3. Experimental Protocol ...... 25 2.4. Data collection and statistical analysis ...... 26 2.5. Infarct size analysis ...... 27 3. RESULTS ...... 29 3.1. Baseline data ...... 29 3.2. Mortality ...... 30 3.3. Arrhythmic episodes ...... 31 3.4. Infarct size analysis ...... 34 3.5. CSNA response to acute myocardial infarction and atosiban ...... 35 3.6. Haemodynamic responses to acute MI and atosiban ...... 37 4. DISCUSSION ...... 39

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4.1. Mortality ...... 39 4.2. Sympathetic responses to MI ...... 40 4.3. The effect of Atosiban on elevated cSNA ...... 41 4.4. Study limitations ...... 42 4.5. Future directions ...... 46 4.6. Conclusions ...... 47 REFERENCES ...... 48

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LIST OF FIGURES

Figure 1.1. Schematic diagram of the cardiac sympathetic pathway ...... 3 Figure 1.2. Schematic diagram of cardiac parasympathetic innervation ...... 4 Figure 1.3. Diagram of an infarcted heart and thrombotic occlusion of the left anterior descending coronary artery ...... 5 Figure 1.4. Flowchart of the oxytocinergic pathway from the PVN ...... 8 Figure 2.1. Outline of ICV surgical procedure ...... 13 Figure 2.2. Evans blue stain on brain slice ...... 14 Figure 2.3. Schematic diagram of surgical set up ...... 15 Figure 2.4. Outline of tracheostomy procedure ...... 17 Figure 2.5. Outline of femoral artery catheterisation procedure ...... 19 Figure 2.6. Left thoracotomy procedure ...... 21 Figure 2.7. Successful ligation of the left anterior descending (LAD) coronary artery ...... 22 Figure 2.8. Image of the left cardiac sympathetic nerve branching from the stellate ganglion...... 23 Figure 2.9. Experimental protocol timeline ...... 25 Figure 2.10. Staining of heart with 2,3,5-triphenyl tetrazolium chloride (TTC) ...... 28 Figure 3.1. Kaplan-meier survival curves depicting survival rates ...... 30 Figure 3.2. Typical Labchart trace depicting arrhythmic episodes between periods of normal cardiac beats following acute myocardial infarction ...... 32 Figure 3.3. Arrhythmic episodes in hourly intervals ...... 33 Figure 3.4. Infarct sizes of MI and MI + ATB groups ...... 34 Figure 3.5. Percentage change in cardiac sympathetic activity over time following acute MI or sham ...... 36 Figure 3.6. Changes in haemodynamic parameters following acute myocardial infarction or sham ...... 38

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LIST OF TABLES

Table 3.1. Baseline electrophysiological and haemodynamic parameters ...... 29

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ABBREVIATIONS

ABP Arterial blood pressure ANS Autonomic nervous system ATB Atosiban β-AR Beta - adrenergic receptors BBB Blood brain barrier BP Blood pressure BPM Beats per minute Ca2+ Calcium CAD Coronary artery disease CSN Cardiac sympathetic nerve CSNA Cardiac sympathetic nerve activity DVN Dorsal vagal nucleus GPCR G protein coupled receptor HR Heart rate ICV Intracerebroventricular IML Intermediolateral cell column MABP Mean arterial blood pressure MI Myocardial infarction NTS Nucleus tractus solitarius OT Oxytocin OTB Oxytocin blockers OTR Oxytocin receptors PVN Paraventricular nucleus RLCV Right lateral cerebral ventricle ROS Reactive oxygen species RVLM Rostral ventrolateral medulla SNA Sympathetic nerve activity SNS Sympathetic nervous system SON Supraoptic nucleus SPN Sympathetic preganglionic neurons TTC 2,3,5-triphenyltetrazolium VP Vasopressin VPR Vasopressin receptors

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1. INTRODUCTION

As the most common cause of death globally, the high mortality rate of an acute myocardial infarction (MI) can be attributed to the disproportionate increase of sympathetic activity following shortly after an infarct (1). Overactivation of cardiac sympathetic nerve activity

(cSNA) results in propagation of ventricular arrhythmias associated with increased mortality, exacerbating cardiac damage and severity of myocardial injury (2-4). The causation for this sympathetic increase is still not fully understood, although, current investigations show indications of an emerging correlation between oxytocin (OT) release and post-infarct stimulation of cSNA (5-8). An initial study has determined that the immediate administration of oxytocin receptor blockers (OTB) in response to acute MI have identified signs of improving prognosis, exhibiting promising results (7). However, further insight into clinical translatability remains unelucidated as the optimal window of time for OTB administration remains unknown.

Thus, this study aims to explore the applicability of delayed OT inhibition using atosiban (ATB) to investigate whether an OTR blockade one hour post-infarct exhibits potential as a therapeutic strategy to effectively reduce elevated cSNA back to pre-infarct levels.

1.1. Regulation of the cardiovascular system

Regulation of cardiac function is controlled by intrinsic and extrinsic mechanisms. Intrinsic regulation arises in the heart from the sinoatrial node which transmits spontaneous bursts of excitation potentials through the electro-cardiac pathways to stimulate contraction and relaxation of the heart (9). Extrinsic cardiac regulation on the other hand, is largely influenced by mechanisms involving endocrine and autonomic pathways (10). Endocrine regulation of the cardiovascular system is generally interlinked with the primary extrinsic regulator, the autonomic nervous system (ANS), in eliciting changes in contractility and blood pressure (11).

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1.2. Sympathetic cardiac regulation

As a sub-branch of the peripheral nervous system, the ANS itself has two distinct pathways which regulate excitatory and inhibitory signals from the central nervous system to the heart, known as the sympathetic and parasympathetic nervous systems, respectively (12).

The Sympathetic nervous system (SNS) is a fundamental homeostatic regulator of normal cardiovascular function. associated with the fight-or-flight response, the SNS responds to physical changes in the body, the environment, and stress, by influencing increases in heart rate and blood pressure in order to maintain cardiac output (13, 14).

Sympathetic cardiac innervation

CSNA relies on afferent input from baroreceptors and chemoreceptors (15, 16). These afferent fibres synapse in the nucleus tractus solitarius (NTS), relaying a signal to the paraventricular nucleus (PVN) (17-19). Located within the hypothalamus, the PVN is comprised of magnocellular and parvocellular neurons which project to various neural regions involved with endocrine and sympathetic regulation (20). It is a distinct subpopulation of parvocellular neurons in the PVN that activates cSNA by projecting to sites which regulate efferent sympathetic signalling, either directly via the intermediolateral cell column of the spinal cord

(IML), or indirectly from the rostral ventrolateral medulla (RVLM) which also projects to the

IML (17, 21). Sympathetic preganglionic neurons from the IML in the superior thoracic region

(T1-T4) synapse at the stellate ganglia to the post-ganglionic fibres which sympathetically innervate the heart (22) (Figure 1.1). The release of noradrenaline from the post-ganglionic nerve terminals allow for a sympathetic response by activating β-adrenergic receptors (β-AR) which generally promotes positive chronotropic and inotropic responses via their associated

G-protein coupled receptor (GPCR) mediated pathways (23, 24). β-ARs have 3 distinct 2 subtypes known as β1, β2 and β3, with β1-ARs being expressed more prominently in cardiac cells than β2 in normal conditions (25). β3-AR expression, however, is associated with negative inotropic effects and is upregulated in pathological states associated with myocardial dysfunction (25).

Figure 1.1. Schematic diagram of the cardiac sympathetic pathway. Afferent fibres project from the heart to the nucleus tractus solitarius (NTS), which is relayed to the paraventricular nucleus (PVN). The PVN can directly stimulate the intermediolateral cell column of the spinal cord (IML) or indirectly via the rostral ventrolateral medulla (RVLM). The IML projects sympathetic preganglionic fibres into the stellate ganglion (SG), synapsing with the postganglionic fibres in the cardiac sympathetic nerve to stimulate cardiac sympathetic nerve activation. Loke 2020, original work, created on Biorender.com.

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Parasympathetic cardiac innervation

The parasympathetic nervous system mediates negative chronotropic, dromotropic and inotropic effects on cardiac function (26). Activation of this pathway is initiated by the dorsal vagal nucleus (DVN) and nucleus ambiguus which both project down the vagus nerve (27, 28).

These parasympathetic preganglionic fibres synapse at the cardiac ganglia where postganglionic fibres emerge, stimulating a parasympathetic response (27) (Figure 1.2).

Figure 1.2. Schematic diagram of cardiac parasympathetic innervation. Diagram depicts the dorsal vagal nucleus (DVN), also referred to as the dorsal motor nucleus, and the nucleus ambiguus projecting down the vagus nerve/ganglion, synapsing at the cardiac ganglia where the postganglionic fibres emerge to innervate the heart. Image adapted from Rajendran et al., 2017 (28).

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1.3. Acute myocardial infarction

Figure 1.3. Diagram of an infarcted heart and thrombotic occlusion of the left anterior descending coronary artery. Diagram illustrates how accumulation of plaque around the arterial lumen obstructs the passage of red blood cells to the heart. Loke 2020, original work.

The cardiovascular system regulates essential delivery of oxygen and nutrients to the various organs and tissues of the body, including the heart itself (29). Coronary artery disease (CAD) exhibits the highest prevalence in mortality rates world-wide, attributing to approximately 85% of all cardiovascular related deaths (30). CAD is typically caused by accumulation of atherosclerotic plaque in the walls of the coronary arteries which narrows the vessel, resulting in decreased blood circulation (31, 32). Over time, plaque instability elicits erosion or rupture of the fibrous cap, exposing the plaque to the lumen which instigates platelet aggregation and clotting resulting in development of a thrombus and consequential obstruction of essential circulation to cardiac tissue (31, 33) (Figure 1.3). As the heart requires a constant supply of blood in order to allow for normal cardiac function, the inability to receive oxygen to supply cardiac tissue creates a hypoxic environment where the heart can no longer maintain cardiomyocyte viability (34). As a consequence, an acute MI is initiated, invoking pathological myocardial modifications such as: 5

• Elevation of reactive oxygen species (ROS) levels and oxidative stress which adversely

affects mitochondrial function and energy synthesis (35).

• Autophagy, apoptosis and recruitment of inflammatory cytokines in response to

increased ROS and damaged cardiomyocytes (36).

• Increased cellular Ca2+ influx and increased acidity levels from lactate production due

to the conversion to anaerobic glycolysis, a non-oxygen energy synthesis pathway to

compensate for reduced energy production (37).

The effects of acute myocardial infarction

In response to the pathological changes occurring due to a MI, the heart sends afferent distress signals to the CNS which elicits an increase in cSNA (38). The stimulation of cSNA is comparatively faster and larger compared to the activation of other sympathetic sites in the body when responding to an MI (2), increasing within an hour of the infarct and reaching maximal levels at the second and third hour, resulting in sustained elevation over a duration of days to months (39, 40). The elevation in cSNA is caused by stimulation of superior thoracic

SPNs in response to the myocardial modifications associated with acute MI, leading to a considerable increase in cardiac noradrenaline (NA) levels and therefore increased sympathetic tone (38, 41).

The increase in cSNA results in arrhythmogenesis which occurs in the first hour after the onset of an MI, increasing the risk of tachy-arrhythmic incidences and therefore sudden cardiac death as arrhythmias weaken ventricular ejection due to an irregular increased heart rate caused by cardio-electrical misfiring, thus resulting in impaired cardiovascular circulation (4, 42).

At the site of the infarct, affected cardiomyocytes begin to necrotise, accompanied by acute inflammation (43). The intrusion of macrophages in response to necrosis enhances the progression of intracellular oedema, resulting in swelling of cardiac tissue (43).

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Cardiac remodelling manifests throughout the first few weeks after the infarct, expressed by the thinning of ventricular walls due to cell loss and rearrangement of subendocardial muscle fibres, distorting the morphology of the heart (44). Afferent cardiac fibres are also affected, leading to impaired signal transduction and desensitised responses to neural input depending on the region of cardiac tissue affected (45).

1.4. Current therapeutic strategies:

There are a range of therapeutic strategies involving medications to treat the effects associated with acute MI and surgical procedures that aim to restore blood flow and remove coronary obstructions, in which both have greatly improved patient prognosis. Currently, the gold- standards in treatment of acute MI are β-blockers and reperfusion therapy. β-blockers act non- specifically on the β-ARs by improving ventricular function through negative inotropic and chronotropic effects (46). Despite the benefits of β-blockers, The eligibility criteria for access to this treatment strategy is suggested to be relatively high due to contraindications which prevents subpopulations like the elderly from receiving appropriate treatment (47).

Reperfusion therapy has also showed promising results in reducing occlusion of the affected artery, preventing further cardiac deterioration by reducing infarct size and sustaining ventricular function (48). Yet, achieving a better prognosis is highly time dependent, with the rate of treatment effectiveness decreasing rapidly within the first 30 minutes to 4 hours after the onset of an infarct (49).

As of now, effective and viable therapeutic strategies targeting the initial adverse increase of cSNA following an MI have not yet been identified and remains an area of active research.

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1.5. Oxytocin and autonomic regulation

Oxytocin is widely established for its role as a hormone involved with parturition and milk ejection, yet what is lesser known is OT’s association with the ANS, specifically, the cardiovascular system (50-52). OT release originates in the PVN as well as the supraoptic nucleus (SON) (52). Processes involving the posterior pituitary gland, and therefore release of

OT into the vascular system to stimulate parturition and milk ejection are regulated by magnocellular OT neurons found in the PVN and the SON (50, 52). Although the parvocellular division of the PVN is already strongly associated with autonomic and neuroendocrine control of cardiovascular function (53, 54), a distinct subset of OT-expressing parvocellular neurons located in the PVN was also found to project to the RVLM and its associated sympathetic pathways (55, 56) (Figure 1.4).

Figure 1.4. Flowchart of the oxytocinergic pathway from the PVN. Diagram illustrates the pathways of the magnocellular and parvocellular OT neurons from the paraventricular nucleus (PVN) and supraoptic nucleus (SON). (RVLM; rostral ventrolateral medulla). Loke 2020, original work.

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1.6. The oxytocin receptor

Oxytocin receptors (OTR) are recognised as class-A GPCRS, belonging to the same family as

β-ARs (23, 57). In typical cases of OT stimulation, OT binds to the Gq subtype of the GPCR family in order to induce contraction while stimulating the release of Ca2+ from intracellular stores (58). In association with cardiac regulation, OTRs have also been located in the cardiac regions, such as the aorta, atrioventricular sites and vena cavae (59).

Oxytocin receptor blockers OTBs such as atosiban (ATB) have been found to eradicate the adverse increase in cSNA following a MI (7). ATB is a competitive, reversible OTR antagonist with promising safety profiles (60). Currently in phase III clinical trials, ATB has been treated on pregnant women as a pre-term labour preventative strategy, showing effective results in reducing uterine contractions as well as reducing oxidative stress (61). Furthermore, ATB has been found to have no negative effects on cardiovascular function (62).

1.7. Oxytocinergic cardiac sympathetic innervation:

OT innervation in response to ‘distress’ signals such as physical pain or injury has been shown to trigger cSNA, provoking increases in heart rate (6, 63, 64). Additionally, from a neurochemical standpoint, in response to these ‘distress’ signals, increase in cSNA was identified alongside a seven-fold increase in OT from basal levels in the PVN (63).

In regards to the cSNA pathway, it was found that 79% of neurons projecting from the parvocellular PVN neurons to the RVLM were oxytocinergic as well as 38% of neurons from the PVN that projected directly to the IML (21). OT-expressing neuronal fibres at the IML have also exhibited the ability to directly innervate large numbers of superior thoracic SPNs to elicit a sympathetic response (56, 65). 9

1.8. Oxytocin’s involvement in acute myocardial infarction:

OT activation is a relatively unexplored aspect of the mechanisms associated with acute MI.

New research has found that in response to acute MI, parvocellular OT neurons from the PVN projecting to the RVLM are activated (7). Increased levels of OT and upregulation of OTRs have also been detected in the sympathetic cervical ganglia and heart alongside a post-infarct increase in cSNA (8, 59), while an immediate blockade of OTRs after an acute MI entirely prevented the pathological MI-associated increase in cSNA (7).

Furthermore, it is suggested that the pathological overexpression of cardiac OTRs may be involved with the associated adverse cardiac modifications exhibited in an acute MI such as increased intracellular Ca2+ levels, detrimental cardio-hypertrophic effects as well as apoptosis of cardiomyocytes through various GqPCR-associated signalling pathways (5, 66, 67).

1.9. Oxytocin receptor blockade as a therapeutic strategy for acute MI

To date, there is only one study which explores the use of OTBs as a therapeutic strategy for acute MI, to which they discovered that immediate administration of OTBs such as retosiban and ATB after an MI was able to lead to the abolishment of the adverse increase in cSNA (7), thus being the first study to discover the potential of OTBs as a therapeutic strategy for acute

MI. However, although it is known that immediate OTB treatment is effective in preventing cSNA increases, the impossibility of immediate treatment limits the clinical translatability of

OTBs. Therefore, it is imperative to investigate the therapeutic window of opportunity associated with OTB efficacy. As such, this study may be the first to examine the possibility of the use of OTBs in the reduction of elevated cSNA.

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1.10. Aims and hypothesis

This study intends to further explore the relationship between OT and the MI-associated cSNA increase as well as the clinical applicability of oxytocin receptor blockers. This will be conducted through investigation of whether a delayed 1 hour administration of ATB post-MI can effectively reduce elevated cSNA back to pre-infarct levels.

A 1 hour delay before administration allows for a conceptual test of viability for real-world application as a potential primary response strategy in reducing mortality risk. Thus, as OTBs have indicated therapeutic potential in the regulation of MI-associated cSNA, I hypothesised that it was possible to reduce the post-infarct associated increase in cSNA and expected to see a decrease in cSNA levels upon administration of OTB.

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2. MATERIALS & METHODS

2.1. Animals

All experiments were approved and conducted in accordance with the guidelines supplied by the University of Otago Animal Ethics Committee, New Zealand. Experiments were conducted on 6 – 8 week old male Sprague Dawley rats (350 – 500g). 22 rats were allocated into 3 groups;

Sham (n = 6), MI (n = 10), or MI + ATB (n = 6) and were housed in a temperature regulated environment (25˚C ± 1˚C) on a 12 hour light/dark cycle with ad libitum access to food (standard laboratory chow) and water.

2.2. Non-recovery surgical procedures:

Surgical anaesthesia Rats were deeply anaesthetized via intraperitoneal injection of urethane (1.5g/kg; Sigma-

Aldrich®, St Louis, MO, USA). Full anaesthesia was confirmed by complete loss of limb withdrawal reflex in response to a firm toe pinch. The neck, left axilla and chest, left groin and head were shaved once under full anaesthesia.

Intracerebroventricular (ICV) surgery A stereotaxic ear bar was secured on both sides of the skull at the ear cavity before the rat was fitted on a stereotaxic frame. Once secure, an incision was made at the midline of the scalp, starting at the base of the ears towards the eyes (Figure 2.1). The subcutaneous connective tissue adhering to the skull was removed to reveal the bregma, the site of intersection between the sagittal and coronal sutures. Once the bregma was identified, a 22 gauge guide cannula

(C313G-SPC, Bio-Scientific Pty. Ltd) was positioned and adjusted to locate the right lateral cerebral ventricle (RLCV) using coordinates from Paxinos & Watson (0.8mm posterior to

12 bregma, 1.5mm lateral to midline) (68). Using a drill (LEC-800, Nihon Seimitsu Co., Ltd), a

2mm diameter hole was made at the guide point. An additional 1mm hole was made to install a micro screw (specifications: M1.0 x 3mm) to provide scaffolding support as the cannula was inserted into position. The surgical site was then covered with dental cement to prevent displacement of the cannula. Once the dental cement had solidified, the animal was removed from the stereotaxic frame and placed onto a surgical board while a needle tipped catheter filled with either 10µL of atosiban (10µg/10µL, Sigma CAS Number 90779-69-4) or saline was inserted into the cannula. Confirmation of drug delivery was visualised at the end of each experiment via injection of 5µL of Evans blue dye through the cannula (Figure 2.2).

Figure 2.1. Outline of ICV surgical procedure. (A) A midline incision on the scalp, spanning from the ears towards the eyes. (B) Separation of connective tissue adhering to the skull via scalpel. (C) Haemostats used to retract scalp. (D) identification the bregma. (E) Identified location of RLCV, marked with pencil. (F) Drilled hole at RLCV on the right, with an additional hole drilled on the left for insertion of a screw. (G) Installation of cannula and screw. (H) Surgical site covered with dental cement.

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Figure 2.2. Evans blue stain on brain slice. Image depicts successful placement of cannula as seen by the infiltration of Evans blue in the ventricles of the brain (areas of blue accumulation in the middle of the brain).

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Placement of animal for surgery Upon completion of ICV surgery, the rat was transferred and placed in supine position on a heating pad (Figure 2.3). Body temperature was maintained at 37˚C using a rectal thermistor connected to a thermostatically controlled heating pad (BWT-100, Bio Research Centre Co.,

Ltd).

Figure 2.3. Schematic diagram of surgical set up. Image shows rat secured on the surgical board with rectal thermistor and heating pad. Shaded regions are used to visualise locations shaved for surgery. Original work, Loke 2020.

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Tracheostomy: A 15mm midline incision was made at the base of the jaw towards the sternum (Figure 2.4).

Visceral fat was excised to reveal the sternohyoid muscle which was blunt-dissected to access and isolate the trachea. Ligatures were tied loosely around the caudal and rostral regions of the trachea and a small incision was made anteriorly, allowing for insertion of a polyethylene tracheal cannula (1.7mm ID, 2.7mm OD). The cannula was positioned caudally to ~10mm and was held in place by tightening the caudal and rostral ligatures. The cannula was then attached to a rodent ventilator (M-7025, UGO Basile, Italy) to maintain normocapnic arterial PCO2.

Ventilator settings were adjusted according to the body weight of the rat (respiration rate: ~80 breaths/min, tidal volume: ~1mL/100g) and ~50% O2 enriched air (from compressed O2 tank).

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Figure 2.4. Outline of tracheostomy procedure. (A) Midline incision exposing subcutaneous fat. Caudal and rostral labels indicate positioning of rat. (B) Visualisation of sternohyoid muscles (outlined in blue), light blue line indicates site of blunt dissection to expose trachea. (C) Isolation of trachea. (D) Positioning of ligatures. (E) Radial incision of trachea (blue arrow). (F) Insertion of tracheal cannula. (G) Advancement of cannula. (H) Both ligatures and cannula secured.

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Catheterization of the femoral artery and vein: A skin incision originating 3cm above the medial malleolus of the left hindlimb was made. Via blunt dissection, skin was separated from the subcutaneous fat pad and connective tissue before the initial incision was extended towards the midline (Figure 2.5). The subcutaneous fat was electro-cauterized (Geiger Medical Technologies-150A, USA) to reveal the femoral artery and vein underneath. Using a stereo microscope (Leica M80, Germany), the femoral artery and vein were isolated from the surrounding connective tissues using blunt-tip tweezers and curved forceps.

Once the vessels were isolated, two pieces of surgical suture (4/0 silk USP, Resorba, Germany) were threaded underneath the femoral artery and positioned proximally and distally. The suture positioned at the distal region was firmly ligated. A microvascular clamp was placed above the proximal thread to prevent blood flow and the proximal suture was loosely tied. Using iris scissors, a small radial incision was made between the proximal and distal sutures, allowing for the insertion of an arterial catheter into the lumen of the artery which was advanced beyond the vascular clamp and inguinal ligament. The arterial catheter was connected to a deltran pressure transducer (Utah Medical Products Inc., Utah, USA) in order to monitor arterial blood pressure (ABP), and was filled prior to catheterisation with heparinised saline (40 I.U./mL, of

0.9% NaCl) to prevent coagulation of blood within the catheter.

The same process was repeated for the catheterization of the femoral vein, with the insertion of a saline-filled (0.9% NaCl) catheter connected to an electronic perfusion pump (PhD UltraTM,

Harvard Apparatus, Holliston, MA) to maintain hydration levels in the animal via constant infusion of saline (3ml/hr, 20mL Terumo syringe) throughout the duration of the experiment.

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Figure 2.5. Outline of femoral artery catheterisation procedure. (A) incision on left hindlimb exposing subcutaneous fat pad. (B) Visualisation of femoral vessels below fat pad. (C) Retractors positioned to provide further exposure of femoral vessels. (D) Isolated femoral vein and artery. Inguinal ligament (IL) is clearly visualised. (E) Distal ligature knotted. (F) microvascular clamp and loosely knotted proximal ligature in position. Incision made between ligatures, closest to distal ligature as seen by white arrow. (G) insertion of arterial catheter. (H) Proximal ligature tightened to secure catheter. (I) Catheter advanced up artery and proximal ligature firmly knotted.

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Left thoracotomy: A skin incision was made down the midline above the sternum and toward the xiphoid process

(Figure 2.6). To further expose the thoracic musculature, an incision was made perpendicular to the midline, from the xiphoid process to the left mid-axillary line. The skin flap was laterally reflected prior to removal of muscles found at the surgical site (pectoralis major and minor, rectus abdominis, and external oblique), via blunt dissection and electrocautery to prevent haemorrhage (Geiger Medical Technologies-150A, USA).

Two small incisions were made near the sternal border, one at the 2nd intercostal space and the other at the 3rd intercostal space on both sides of the 3rd rib. A ligature was pulled through these incisions using blunt-tip curved forceps. Further incisions were made towards the left mid- axillary line of the 3rd rib where the 2nd ligature was positioned. Prior to excision of ribs using a rib-cutter, the ligatures were both tightened and double-knotted to prevent blood loss from intercostal vessels, This procedure was repeated on the 2nd rib and 4th rib. All ligatures were pulled away from the body and taped to the surgical board so as to further open up the exposed area for access to the heart and cardiac sympathetic nerve.

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Figure 2.6. Left thoracotomy procedure. (A) midline incision exposing thoracic region. (B) Lateral reflection of skin flap. (C) Removal of Pectoralis major (PM) (dotted blue line indicates location of muscle), pectoralis minor is not shown in image, but was also removed. (D) removal of rectus abdominis (RA) from thoracic region. (E) Removal of external oblique (EO). (F) Insertion of curve forceps in area of incision for ligation. (G) Ligatures threaded through incisions. Numbers indicate rib arrangement of rat to be removed. (H) Ligatures in position and secured. Rib isolated and ready for removal. (I) Removed 3rd rib. (J) Removed 4th and 2nd rib. Ligatures positioned to expose thoracic cavity.

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Ligation of left anterior descending coronary artery: The pericardium was removed via blunt dissection to allow for access to the left anterior descending (LAD) coronary artery. The LAD coronary artery was located descending from the left coronary artery (or in cases where the left coronary artery is not visible, directly from the left aortic sinus) through the interventricular groove towards the apex of the heart (69). A 6/0

Prolene suture (Ethicon Inc., USA) was threaded around the LAD coronary artery by passing the thread from the base of the pulmonary conus, beneath the left atrium, passing next to the right wall of the left atrial appendage (70). The suture was loosely tied in a triple knot. To induce an infarct, the ligatures were tightened and tied again to prevent loosening. Successful ligation resulted in discolouration of the left ventricle (Figure 2.7).

Figure 2.7. Successful ligation of the left anterior descending (LAD) coronary artery. Suture is tied around the LAD coronary artery. Discolouration can be seen near the apex of the heart (light blue arrow).

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Cardiac sympathetic nerve isolation: Using a cotton swab and fine tooth tweezers, fat and connective tissue at the region of the 1st and 2nd ribs were meticulously cleared to expose the stellate ganglion and cardiac sympathetic nerve (CSN) (Figure 2.8). The CSN was identified and was carefully isolated with minimal stretching and contact to prevent nerve damage. Once isolated, a fine ligature (6/0 silk USP,

Resorba, Germany) was threaded under the nerve using fine curved forceps and tied at the region of the nerve most visibly distal to the stellate ganglion. The region of the nerve distal to the ligature and was cut with micro-scissors and carefully placed on a platinum recording electrode. To prevent desiccation of the nerve, the nerve was enveloped with paraffin oil.

Figure 2.8. Image of the left cardiac sympathetic nerve branching from the stellate ganglion.

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Recording of sympathetic activity: Sympathetic activity was recorded 10 minutes before and 4 hours after the induction of a MI.

Via bio-amplifier (BMA-200, AC/DC Bioamplifier, USA), noise interference was filtered and raw sympathetic activity was amplified. To confirm the presence of sympathetic activity, a speaker connected to the bio-amplifier allowed for audible detection of spikes of activity, characterised by irregular beats. The altered signal from the bio-amplifier was connected to an oscilloscope, allowing for visible real-time identification of sympathetic excitation potentials.

An amplitude discriminator (WD-2 Time-Amplitude Window Discriminator, Dagan Corp.,

Minneapolis, MN) was used to distinguish between noise interference and genuine sympathetic activity on the oscilloscope.

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2.3. Experimental Protocol

Rats were randomly assigned into one of three groups, 1) Sham, 2) MI, or 3) MI + ATB. All rats underwent surgical preparation to allow for electrophysiological and haemodynamic data collection. Once cSNA stabilised, baseline data was recorded 10 minutes prior to coronary occlusion (MI and MI + ATB group only), and was continuously recorded for four hours after

(Figure 2.9). The MI + ATB group were centrally administered with 10µL of ATB (10µg/mL) one hour after the induction of an MI. At the end of the four hour recording, rats were terminated with 0.5mL of KCl. The brain was injected with 5µL of Evans blue for confirmation of administration in ATB treated rats. Hearts subjected to MI were extracted and stained for infarct size analysis.

Figure 2.9. Experimental protocol timeline. After surgery, baseline recordings of haemodynamic and electrophysiological data were collected from all three groups (Sham, MI, MI + ATB), to which 10 minutes later at ‘0 hours’, MIs (MI; myocardial infarction) were induced in the MI and MI + ATB group. ATB (atosiban) was administered 1 hour later in the MI + ATB group. At 4 hours, rats which survived for the entire protocol were terminated. Hearts from the MI and MI + ATB group were removed and stained for infarct size analysis.

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2.4. Data collection and statistical analysis

Equipment set-up: Before initiation of the recording protocol, ABP was recorded via a deltran pressure transducer

(Utah Medical Products Inc., Utah, USA), sampled at 400Hz and amplified via bridge amplifier

(MacLab, AD Instruments). The amplified signal was then sent to an 8-channel PowerLab data acquisition system (PowerLab 8/35, AD Instrument Pty Ltd, Australia) connected to a

Macintosh desktop running ‘LabChart Pro’ recording software (AD Instrument Pty Ltd,

Australia 1986-213). Heart rate (HR) was then generated from the ABP channel via computed input from Labchart.

The channel recording for cSNA was calibrated against a 100µV signal produced by the bio- amplifier (BMA-200. AC/DC Bioamplifier, USA). This 100µV signal was used for two-point calibration (0µV and 100µV), allowing for accurate assessment of sympathetic activity. This raw sympathetic signal was then subsequently used to generate an integrated cSNA signal. The actual value of the integrated signal was determined by subtracting the background noise value at the end of the recording protocol.

Statistical analysis Data was collected 5 minutes before (-5) and during coronary occlusion (0) and at time points

5, 10, 20, 30, 60, 75, 90, 120, 180 and 240 minutes post infarct which was consequently organised on Excel (Microsoft, 2010). Arrhythmic episodes were calculated in hourly intervals

(0-1, 1-2, 2-3 and 3-4). Data collected from cSNA was normalised to percentage changes from baseline using the equation !"#$%&"'!()*+$,"'$. Means and SEMs were generated using excel )*+$,"'$ for individual timepoints in all groups for cSNA, ABP, HR and arrhythmias which were then analysed on Prism (GraphPad software 7.0, San Diego, CA, USA). Via one-way analysis of variance (ANOVA) tests, differences in timepoints were identified. Statistically significant

26 differences were then compared to baseline via Dunnett’s post hoc test and between timepoints via Bonferroni post hoc test. Individual time points between sham and MI, as well as MI and

MI + ATB were compared via 2-way ANOVA, with significant differences analysed via

Bonferroni post hoc test. Differences in infarct size was analysed via unpaired t-test. Kaplan-

Meier survival analysis was conducted to identify the survival curves between all three groups.

A P value of ≤0.05 was considered statistically significant.

2.5. Infarct size analysis

2,3,5 Triphenyl tetrazolium chloride staining: At the completion of the SNA recording procedure, rats were euthanised with 0.5mL of KCl.

Once sympathetic activity was no longer detected, the background noise value was obtained for subtraction from the integrated sympathetic signal. Hearts were immediately extracted and submerged with ice-cold phosphate buffered saline (1% PBS) solution. To flush interior of the heart, a cannula attached to a PBS filled 10mL syringe was inserted into the aorta and secured using a piece of thread (4/0 silk USP, Resorba, Germany) and then slowly infused into the heart.

Once the effluent no longer contained traces of blood, the heart was perfused with 50mL of

37˚C 2,3,5-triphenyl tetrazolium chloride (TTC) (Sigma-Aldrich®, St Louis, MO) solution via perfusion pump for 15-20 minutes at 0.5mL/gm/min (Figure 2.10).

After the TTC perfusion, hearts were placed in a freezer (-20 degrees) for approximately 20 minutes. Hearts were then placed in a rodent heart matrix (Zivic Instruments, Pittsburgh, PA) and sliced transversely into uniform 3mm sections. The hearts were sandwiched between two

Perspex panels and tightened with screws to ensure a uniform thickness of 2mm in all slices before being photographed using a microscope camera (Leica IC 80HD, Germany).

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Figure 2.10. Staining of heart with 2,3,5-triphenyl tetrazolium chloride (TTC). (A) Heart is perfused with TTC. Infarcted areas are stained white while non-infarcted areas are stained red. (B) transverse cross-section of the heart. Infarcted areas are identified (dotted green area) and compared with the overall area of the left ventricle (purple line).

Quantification of infarct size: Quantification of infarct size was conducted using ImageJ software (version 1.53a, NIH). The total areas of the left ventricle (including atrioventricular septum), ventricular lumen and infarcted zone were calculated. The area of the ventricular lumen was subtracted from the area of the left ventricle to determine the actual left ventricular area. Then, the proportion of area of the infarcted zone was compared with the actual left ventricular area to determine infarct size.

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3. RESULTS

3.1. Baseline data

Before the induction of a MI, baseline electrophysiological and haemodynamic data were collected and quantified (Table 3.1). No significant differences were observed pre-MI at baseline between the sham, MI and MI + ATB groups in all of the variables.

Table 3.1. Baseline electrophysiological and haemodynamic parameters. Data is presented as mean ± SEM. Data for absolute integrated cardiac sympathetic nerve activity (cSNA), mean arterial blood pressure (MABP), heart rate (HR) and total body weight was collected from sham (n = 6), MI (n = 7) and MI + ATB (n = 6) groups. The P-value was calculated from all 3 groups via one-way ANOVA. There were no significant difference between any of the groups for each variable. Sham MI MI + ATB P value (n = 6) (n = 7) (n = 6) Integrated cSNA (µV.s) 4.52 ± 1.03 4.23 ± 0.98 2.02 ± 0.71 0.1472

MABP (mmHg) 72 ± 6 87 ± 5 77 ± 3 0.1134

HR (bpm) 403 ± 12 372 ± 21 376 ± 11 0.4773

Total body weight (g) 398 ± 15 407 ± 15 437 ± 11 0.2276

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3.2. Mortality

Of the 10 rats in the MI group, five rats died during the four hour recording protocol, exhibiting a 50% survival rate at 240 minutes (Figure 3.1) Intriguingly, all six MI + ATB rats survived for the entire duration of the experimental protocol (100% survival).

Further analysis of electrophysiological and haemodynamic changes in the MI group were analysed using data from seven rats, including those that died before four hours, by using the data collected until their time point of death. Two rats died in less than 30 minutes, which was attributed to sudden cardiac death due to cardiac arrythmias. Rats that died from blood loss or hypotension were conclusively excluded from this experiment.

Figure 3.1. Kaplan-meier survival curves depicting survival rates of sham (n=6), MI (n=10) and MI + ATB (n=6) groups over the duration of 4 hours. MI + ATB and sham exhibited no change in survival rate (100% survival) in comparison to the 50% survival rate of the MI group at 4 hours. Dotted line represents time of ATB injection in the MI + ATB group.

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3.3. Arrhythmic episodes

Arrhythmic episodes associated with acute MI were identified and quantified into hourly intervals to observe changes in incidence over time. Arrhythmias were exhibited in MI and MI

+ ATB groups upon induction of an acute MI and were identified via irregular changes in blood pressure, seen as a succession of ectopic beats which occur between sets of normal cardiac activity (Figure 3.2). Arrhythmias were most commonly seen in the first hour post-infarct, with a significant decrease from 29 ± 7 to 1 ± 1 episodes after the first hour in the MI group (n

= 7, P < 0.0001; Figure 3.3). The MI + ATB group also exhibited a significant decrease from

35 ± 12 in the first hour to 3 ± 2 episodes in the 1st to 2nd hour (n = 6, P = 0.0073). In both groups, the incidence of arrhythmias in the 2nd to 3rd hour continued to decrease, however were not significantly different to that of the 1st to 2nd hour. No arrhythmias were detected in the 3rd to 4th hour. No significant differences were found between MI and MI + ATB groups.

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Figure 3.2. Typical Labchart trace depicting arrhythmic episodes between periods of normal cardiac beats following acute myocardial infarction. Arrhythmic episodes are visualised as irregular fluctuations of blood pressure (blue boxes). MABP; mean arterial blood pressure, HR; Heart rate, Raw SNA; Raw sympathetic nerve activity, IntSNA; integrated sympathetic nerve activity.

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Figure 3.3. Arrhythmic episodes in hourly intervals. Arrhythmias in the MI group significantly reduced after the 1st hour (n = 7, one-way ANOVA with Dunnett’s post hoc test; ****P < 0.0001). Similarly, the MI + ATB group also exhibited a significant decrease in arrhythmias after the 1st hour (n = 6, **P = 0.0073). No significant differences were observed between groups.

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3.4. Infarct size analysis

The proportion of necrotic tissue created as a result of the MI (i.e. infarct size) was identified via TTC staining, to which the area of infarcted tissue was quantified to determine the severity of the infarct (Figure 3.4). Animals in the MI group that did not survive past 30 minutes upon onset of a MI were excluded from infarct size analysis as TTC staining could only accurately detect necrotic damage after ~30 minutes of ischemia.

The MI + ATB group had a mean infarct size of 36 ± 4% (n = 6) which was significantly larger compared to the MI group which had a mean infarct size of 23 ± 1% (n = 7). Infarct sizes between groups was found to be significantly different (P = 0.0051).

Figure 3.4. Infarct sizes of MI and MI + ATB groups. Infarct size is expressed in percentages as a proportion of the total left ventricle. MI + ATB (n = 6) was found to have a significantly larger infarct size (36 ± 4%) compared to that of the MI group (23 ± 1%, n = 7, unpaired t-test; **P = 0.0051)

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3.5. CSNA response to acute myocardial infarction and atosiban

Sham cSNA remained stable and similar to baseline for all 4 hours with no significant changes between time points.

In the MI group, cSNA began to increase at 30 minutes post infarct, but exhibited a significant increase at 180 minutes (49 ± 14%, P = 0.0032) and 240 minutes (79 ± 21%, P =0.0001) when compared to baseline (Figure 3.5). When compared to the sham group at similar time points, a significant difference between groups was observed at 120 minutes (P = 0.0417), 180 minutes

(P = 0.0069), and at 240 minutes (P < 0.0001).

Similar to the MI group, the MI + ATB group displayed significantly elevated cSNA levels at

180 minutes (61 ± 21%, P = 0.0497) and 240 minutes (90 ± 35%, P = 0.0007) when compared to baseline. When comparing percentage increases in cSNA between MI and MI + ATB, no significant differences were identified. Overall, the MI + ATB group exhibited a increase in cSNA similar to that of an untreated MI.

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Figure 3.5. Percentage change in cardiac sympathetic activity over time following acute MI or sham. MI rats were either untreated (MI; n = 7) or treated with ATB (MI + ATB; n = 6). The MI group shows a significant increase from baseline at 180 minutes (**P = 0.0032) and at 240 minutes (****P = 0.0001). When compared to sham (n=6), significant differences between MI and sham were observed at 120 minutes († P = 0.0417), 180 minutes († P = 0.0069), and at 240 minutes († P < 0.0001). MI + ATB exhibits a significant increase from baseline at 180 minutes (61 ± 21%, *P = 0.0497) and 240 minutes (90 ± 35%, ***P = 0.0007). Dotted line indicates time of ATB (atosiban) administration in the MI + ATB group. * indicates significant difference from baseline (one-way ANOVA with Dunnett’s post-hoc test; P < 0.05). † indicates significant difference to sham (2-way ANOVA with Bonferroni’s post-hoc test; P < 0.05).

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3.6. Haemodynamic responses to acute MI and atosiban

As expected, the haemodynamic data for the sham group remained stable for all four hours of recording with no significant changes over time. No significant changes in mean arterial blood pressure (MABP) was observed between groups (Figure 3.6a). Heart rate remained stable in all three groups for the entire four hour protocol with no significant differences observed between groups (Figure 3.6b).

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Figure 3.6. Changes in haemodynamic parameters following acute myocardial infarction or sham. (A) Graph depicts mean arterial blood pressure (MABP) between groups. No significant differences were observed between sham (n = 6), MI (n = 7), and MI + ATB (n = 6). (B) Comparison of heart rate (HR) parameters between groups. No significant changes were found between groups and between time points. Dotted line indicates time of ATB (atosiban) administration in the MI + ATB group.

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4. DISCUSSION

This study aimed to assess the efficacy of ATB on elevated cSNA one hour following the onset of an acute myocardial infarction. Direct electrophysiological nerve recordings showed an expected increase in cSNA following coronary occlusion that consistently elevated over time.

Administration of ATB one hour after coronary occlusion did not exhibit a reduction in cSNA and instead continued to rise over time, following a similar progression to that of an untreated

MI. However, due to differences in infarct size between groups, it was not possible to comprehensively establish whether the lack of response in cSNA to ATB was due to a decrease in efficacy in ATB due to delayed administration, or as a result of the MI group expressing cSNA levels that are lower than what is usually observed in literature. Haemodynamic factors such as MABP and HR remained largely stable throughout the recording protocol.

4.1. Mortality

As the leading cause of death globally, a significant factor contributing to the high mortality rate associated with acute MI is the elevation in cSNA, which is known to drive the generation of potentially-fatal arrhythmias shortly following the onset of an infarct (1). In this study, rats in the MI group displayed a 50% mortality rate in the four hours following MI induction, which concurs with that previously reported in literature (7, 40, 71). Intriguingly, in MI rats that were treated with the oxytocin receptor blocker, ATB, there was 100% survival in the four hours post MI.

Identification of infarct size has long been employed as a prognostic determinant of outcomes associated with acute MI, with infarct size correlating to the extent of impaired left ventricular function and mortality (71-74). In this study, the infarct size of the MI group (23 ± 1%) was considerably lower than the ~40% infarct size consistently reported in previous related studies

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(7, 40, 72). Moreover there is little observed variation in haemodynamic parameters and left ventricular function compared to a normal functioning heart if the infarct size is found to be less than 30% (71). Hence, given the significant difference in infarct size between untreated

MI rats (23%) and MI+ATB rats (36 ± 4%) in our current study, it is not possible to make definitive and conclusive comparisons regarding the effect of delayed ATB administration on the magnitude of sympathetic activation. In recognition of this weakness, however, I do note that the MI + ATB group still exhibited a 100% survival rate despite the larger infarct size and associated mortality risk compared to the untreated MI group.

The mechanisms underpinning arrhythmogenesis is multifactorial, of which increased cSNA is known to strongly influence the risk of sudden cardiac death (2-4). Despite the obvious difference in infarct size between MI groups, Arrhythmias were observed, mostly within the first hour, in both MI and MI + ATB groups with similar incidence rates. All recorded deaths were attributed to arrhythmia-induced sudden cardiac failure.

4.2. Sympathetic responses to MI

Upon onset of an acute MI, elevation of cSNA occurs within an hour post-infarct, with the rise in cSNA reaching maximal levels within 2-3 hours (39). This elevation in cSNA has been found to be sustained over time for as long as 6 months after the MI (75). As mentioned previously, cSNA is closely associated with arrhythmogenesis and sudden cardiac death, emphasising the detrimental effects associated with the pathological stimulation of cSNA.

Here, this study reported a 78 ± 21% increase in cSNA following acute MI, which is considerably lower than that reported in previous studies conducted on anaesthetised rat models which exhibit increases in cSNA ranging from 110%-190% (7, 40). The reason for this moderate overall increase in cSNA can possibly be attributed to the smaller infarct size of the

40

MI group, which was lacking the capacity to trigger a large increase in cSNA. Remarkably, there is no study that has directly correlated infarct size with the magnitude of sympathetic activation and, thus, remains an area of research that warrants further investigation.

Additionally, the location of OT expressing neurons in regions of sympathetic regulation, and its stimulation of cSNA increase in response to acute MI remains an area of active research in further understanding the mechanisms involved in MI-associated cSNA as well as the potential treatment strategies for cSNA regulation.

4.3. The effect of Atosiban on elevated cSNA

ATB is a competitive OTR antagonist with a promising safety profile commonly used in treatment for women experiencing pre-term labour (60, 62). Although ATB has been found to have no side effects on the cardiovascular system in pregnant women (58), information regarding the effects of ATB on acute MI remain scarce.

Based on information from a previous study which discovered that immediate administration of ATB effectively prevented the increase in cSNA following a MI (7), in this study I hypothesised that ‘delayed’ ATB treatment one hour after the onset of an acute MI would effectively reduce sympathetic activation. However, this study was unable to show any decrease in cSNA upon administration of ATB as the elevation of cSNA followed that of the untreated MI group, therefore indicating that ATB administration one hour after the onset of an acute MI does not reduce elevated cSNA levels back to pre-infarct levels.

Interestingly, although the infarct size for the MI + ATB group (36 ± 4%) was similar to that of previous reports, the overall increase in cSNA at four hours was 90 ± 35% which is less than expected based on the severity of the infarct which would usually exhibit a larger overall increase ranging from 110 – 190% if untreated (7, 40). Possible implications of this finding

41 could be that the actual effect of ATB is underestimated due to differences in infarct size when compared with the MI group in this study, or that ATB suppresses the overall increase in cSNA, causing the elevated cSNA level to be similar to that of a weaker infarct. However, further testing is required to confirm this statement.

Another possible confounding factor regarding the impact of delayed administration of ATB on cSNA relates to the pharmacodynamics of ATB. As a competitive antagonist, ATB targets the same OTR binding site as OT (76). Given the one hour delay in ATB administration, it is possible that the amount of OT already bound to OTRs in the RVLM, the site of sympathetic origin, may be more saturated than if ATB were administered immediately post-MI. This would therefore reduce the number of available receptors for ATB to bind to in order to cause an inhibitory effect. This possibly suggests that the efficacy of ATB is very much time-reliant, and a delay of one hour may be considered too late to cause any significant changes in cSNA.

4.4. Study limitations

Irregular infarct sizes between study groups

Despite the fact that all experimental groups were randomised, the infarct sizes of untreated

MI group in this study were inexplicably but significantly less severe compared to that of the

MI + ATB group. The probability that all rats which were randomly assigned to the MI group would have smaller infarcts is remote, but not impossible. Differences in the severity of infarct has been shown to alter electrophysiological and haemodynamic responses, which weakens the comparative analysis between study groups (71). This has led to difficulty in accurately determining the effect of delayed ATB administration on elevated cSNA. In order to obtain similar infarct sizes between groups, additional experiments should be conducted on a larger sample size, while focusing on refinement of the LAD coronary occlusion technique.

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Administration of atosiban

In order to recreate the clinical application as a therapeutic drug, the use of retosiban was initially suggested due to its ability to cross the blood brain barrier (BBB) and its intravenous administration method. However, I was unable to acquire retosiban due to its sudden unavailability and instead selected ATB as a substitute. ATB exhibits mechanisms of action similar to that of retosiban and is a suitable substitute for this concept-based study, but is unable to be administered intravenously due to its inability to bypass the BBB. In order to rectify this,

ICV surgery was required to allow for direct administration to the brain. As this method of central administration is non-specific, it is not possible to confirm that ATB did not affect other oxytocinergic areas of the central nervous system. Furthermore, ATB is not wholly specific to

OT as compared to retosiban, as it can bind to vasopressin receptors (VPR) as well (77).

Therefore, if ATB is unable to bind to OTRs, there is a possibility that ATB may bind to VPRs instead. Vasopressin is also associated with inhibitory sympathetic regulation (78, 79), which may have potentially introduced confounding variables due to the possibility of vasopressin inhibition negatively impacting the sympathetic response by preventing sympathetic inhibition.

To address this issue, immunohistochemical staining can be used to identify the specific neural pathways affected by ATB.

Lack of Sham group treated with ATB

Due to time constraints, the 4th study group ‘Sham + ATB’ was not conducted as part of this study. Although this omission may not greatly affect the overall outcome of this study, it reduces the ability for meaningful comparison of results between control and treatment groups.

To amend this, future experimentation should be conducted with inclusion of the 4th study group.

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Direct electrophysiological recordings

Direct sympathetic nerve recordings have been traditionally used as a reliable approach to obtain information regarding changes in SNA in anaesthetized animals from specific sympathetic nerves (80). Maintenance of an intact signal for 4 hours required careful nerve placement, insulation and constant supervision to prevent nerve displacement or distortion of the sympathetic signal from fluid accumulation in the chest cavity. The isolation of the cardiac sympathetic nerve was a demanding procedure due to the high risk of nerve damage, thus leading to a limited sample size for this study. A steep learning curve was required in order to perfect surgical techniques and to minimise operation times. For this reason, a large number of rats were excluded from this study based on the fact that the cardiac sympathetic nerve was irreparably damaged before the recording protocol had even begun. Despite the difficulty, successfully isolated cardiac sympathetic nerves provided an accurate representation of the changes occurring in cSNA over time in response to MI and drug treatment which was able to be easily compared to previous studies.

Animal models

The use of murine models have long been used as a means to investigate acute MI and its associated mechanisms due to their cardiovascular similarity to humans and easy accessibility, maintenance, surgical handling and genetic manipulation (81). Although there are anatomical and physiological differences between human and rodent, there are many advantages to a using a rat model. It is possible to obtain data from a variety of sympathetic nerves in a rodent body via direct nerve recordings, while data collection of sympathetic activity in humans is mostly limited to indirect recordings which reduces the accuracy of sympathetic representation (82,

83). Discrepancies between species is to be expected no matter how physiologically and anatomically similar the two groups are, but the use of rodent models have been proven

44 throughout the years to be one of the most appropriate models to use in analysing acute MI

(81).

Effect of anaesthesia The use of anaesthetics is a common limitation due to its potential confounders in cSNA and cardiovascular regulation. Commonly used anaesthetics such as barbiturates have been shown to depress cardiovascular function and its response to hypothalamic input (84). To circumvent these issues, urethane was used in this study to induce surgical anaesthesia. Urethane is commonly used as the anaesthetic of choice in animal studies due to it having little to no impact on the autonomic and cardiovascular system and its ability to maintain anaesthesia over long periods of time, with the physiological parameters being similar to that of an unanaesthetised animal (85, 86).

It was necessary for the administration of anaesthetics due to the highly invasive surgical procedures required in this study. As urethane was used, the confounding factors associated with changes in autonomic and cardiac regulation was controlled and consistent within all groups. Furthermore, consistent doses of urethane was used in each rat, therefore reducing any potential confounders associated with drug administration.

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4.5. Future directions

The findings from this study gives rise to several factors that would be in need of further investigation. A study identifying the efficacy of ATB over time and therefore the optimal window of time needed for effective treatment would allow us to determine the applicability of ATB as a potential primary response strategy for acute MI.

Another aspect to examine is the long term benefits/outcomes that could be potentially associated with ATB/retosiban treatment, by assessing changes in cSNA, haemodynamics and

OT levels four weeks after an acute MI.

Tracking the time course of OT-neuronal activation using fibre photometry during an acute MI would also provide further insight into whether neuronal OT activation arises during, or in response to an infarct which may shift the perspective of OT-related treatment.

In this study, male Sprague-Dawley rats were used for experimentation. This was done to minimise confounders caused by gender differences and the associated hormonal differences.

Nonetheless, it is important to determine if ATB’s effect on cardiovascular regulation in females is similar to that of males, or if there are any other undiscovered effects to other organs in the body that are regulated by OT when used as a treatment for MI, due to ATB’s initial purpose as a pre-term labour therapeutic.

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4.6. Conclusions

To date, medical advancements in developing therapeutic strategies have been successful in reducing the mortality risk associated with acute MI. However, primary response strategies targeting the increase in cSNA remain an area of active research. This study aimed to explore the applicability of ATB as a potential therapeutic drug for acute MI by investigating the efficacy of ATB administration one hour post-infarct via in vivo electrophysiological recordings of cSNA in a MI rat model. This study was unable to show any significant differences in cSNA elevation between the MI and MI + ATB group. Although infarct sizes between groups were significantly different, which would affect the validity of comparison between groups, the MI + ATB group did not exhibit any decrease in cSNA upon administration of ATB at one hour post infarct. One could potentially argue that although ATB was not reduced to pre-infarct levels, there is a possibility that ATB may weaken the increase in CSNA over time, however further investigation is required to fully elucidate the therapeutic potential of ATB. In summary, administration of ATB one hour after a MI did not reduce elevated cSNA back to pre-infarct levels.

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REFERENCES

1. Brunner-La Rocca H, Esler M, Jennings G, Kaye D. Effect of cardiac sympathetic nervous activity on mode of death in congestive heart failure. Eur Heart J. 2001;22(13):1136-43. 2. Ramchandra R, Hood SG, Denton DA, Woods RL, McKinley MJ, McAllen RM, et al. Basis for the preferential activation of cardiac sympathetic nerve activity in heart failure. Proceedings of the National Academy of Sciences. 2009;106(3):924-8. 3. Hjalmarson Å, Gilpin EA, Kjekshus J, Schieman G, Nicod P, Henning H, et al. Influence of heart rate on mortality after acute myocardial infarction. The American journal of cardiology. 1990;65(9):547-53. 4. Kolettis TM, Kontonika M, Barka E, Daskalopoulos EP, Baltogiannis GG, Tourmousoglou C, et al. Central Sympathetic Activation and Arrhythmogenesis during Acute Myocardial Infarction: Modulating Effects of Endothelin-B Receptors. Front Cardiovasc Med. 2015;2:6. 5. Jung C, Wernly B, Bjursell M, Wiseman J, Admyre T, Wikström J, et al. Cardiac-Specific Overexpression of Oxytocin Receptor Leads to Cardiomyopathy in Mice. J Card Fail. 2018;24(7):470-8. 6. Morris M, Callahan MF, Li P, Lucion AB. Central oxytocin mediates stressinduced tachycardia. J Neuroendocrinol. 1995;7(6):455-9. 7. Roy RK, Augustine RA, Brown CH, Schwenke DO. Activation of oxytocin neurons in the paraventricular nucleus drives cardiac sympathetic nerve activation following myocardial infarction in rats. Communications biology. 2018;1(1):1-11. 8. Yu R, Wang F, Yin J, Shi Y, Wang Y, Wen S, et al. Expression of oxytocin receptor in the rat superior cervical ganglion after myocardial infarction. Int J Clin Exp Pathol. 2018;11(2):739-47. 9. Joyner RW, Van Capelle F. Propagation through electrically coupled cells. How a small SA node drives a large atrium. Biophys J. 1986;50(6):1157-64. 10. Portaluppi F, Vergnani L, Manfredini R, Fersini C. Endocrine mechanisms of blood pressure rhythms. Ann N Y Acad Sci. 1996;783(1):113-31. 11. Conte MR. Gender differences in the neurohumoral control of the cardiovascular system. Ital Heart J. 2003;4:367-70. 12. Gabella G. Autonomic nervous system. eLS. 2012. 13. Akselrod S, Gordon D, Madwed JB, Snidman NC, Shannon DC, Cohen RJ. Hemodynamic regulation: investigation by spectral analysis. American Journal of Physiology-Heart and Circulatory Physiology. 1985;249(4):H867-H75. 14. Jansen AS, Van Nguyen X, Karpitskiy V, Mettenleiter TC, Loewy AD. Central command neurons of the sympathetic nervous system: basis of the fight-or-flight response. Science. 1995;270(5236):644-6. 15. Wang W-Z, Gao L, Pan Y-X, Zucker IH, Wang W. AT1 receptors in the nucleus tractus solitarii mediate the interaction between the baroreflex and the cardiac sympathetic afferent reflex in anesthetized rats. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 2007;292(3):R1137-R45. 16. Wang W-Z, Gao L, Wang H-J, Zucker IH, Wang W. Interaction between cardiac sympathetic afferent reflex and chemoreflex is mediated by the NTS AT1 receptors in heart failure. American Journal of Physiology-Heart and Circulatory Physiology. 2008;295(3):H1216-H26. 17. Gao J, Zhang F, Sun H-J, Liu T-Y, Ding L, Kang Y-M, et al. Transneuronal tracing of central autonomic regions involved in cardiac sympathetic afferent reflex in rats. J Neurol Sci. 2014;342(1- 2):45-51. 18. Coote J, Yang Z, Pyner S, Deering J. Control of sympathetic outflows by the hypothalamic paraventricular nucleus. Clin Exp Pharmacol Physiol. 1998;25(6):461-3. 19. Duan YC, Xu B, Shi Z, Gao J, Zhang SJ, Wang W, et al. Nucleus of solitary tract mediates cardiac sympathetic afferent reflex in rats. Pflugers Arch. 2009;459(1):1-9.

48

20. Badoer E. Hypothalamic paraventricular nucleus and cardiovascular regulation. Clin Exp Pharmacol Physiol. 2001;28(1-2):95-9. 21. Lee SK, Ryu PD, Lee SY. Differential distributions of neuropeptides in hypothalamic paraventricular nucleus neurons projecting to the rostral ventrolateral medulla in the rat. Neurosci Lett. 2013;556:160-5. 22. Mo N, Wallis DI, Watson A. Properties of putative cardiac and non-cardiac neurones in the rat stellate ganglion. J Auton Nerv Syst. 1994;47(1-2):7-22. 23. Siuda ER, McCall JG, Al-Hasani R, Shin G, Park SI, Schmidt MJ, et al. Optodynamic simulation of β-adrenergic receptor signalling. Nature communications. 2015;6(1):1-13. 24. Ungerer M, Böhm M, Elce J, Erdmann E, Lohse M. Altered expression of beta-adrenergic receptor kinase and beta 1-adrenergic receptors in the failing human heart. Circulation. 1993;87(2):454-63. 25. Moniotte S, Kobzik L, Feron O, Trochu J-Nl, Gauthier C, Balligand J-L. Upregulation of β3- adrenoceptors and altered contractile response to inotropic amines in human failing myocardium. Circulation. 2001;103(12):1649-55. 26. Freeling JL, Li Y. Age-related attenuation of parasympathetic control of the heart in mice. Int J Physiol Pathophysiol Pharmacol. 2015;7(3):126-35. 27. Cheng Z, Powley TL. Nucleus ambiguus projections to cardiac ganglia of rat atria: an anterograde tracing study. J Comp Neurol. 2000;424(4):588-606. 28. Rajendran PS, Chui RW, Ajijola OA, Vaseghi M, Armour JA, Ardell JL, et al. Neural control of cardiac function in health and disease. Atlas of Cardiac Innervation: Springer; 2017. p. 13-35. 29. Guyton AC. The relationship of cardiac output and arterial pressure control. Circulation. 1981;64(6):1079-88. 30. Roth GA, Abate D, Abate KH, Abay SM, Abbafati C, Abbasi N, et al. Global, regional, and national age-sex-specific mortality for 282 causes of death in 195 countries and territories, 1980– 2017: a systematic analysis for the Global Burden of Disease Study 2017. The Lancet. 2018;392(10159):1736-88. 31. Goldstein JA, Demetriou D, Grines CL, Pica M, Shoukfeh M, O'Neill WW. Multiple complex coronary plaques in patients with acute myocardial infarction. N Engl J Med. 2000;343(13):915-22. 32. DeWood MA, Spores J, Notske R, Mouser LT, Burroughs R, Golden MS, et al. Prevalence of total coronary occlusion during the early hours of transmural myocardial infarction. N Engl J Med. 1980;303(16):897-902. 33. Falk E. Plaque rupture with severe pre-existing stenosis precipitating coronary thrombosis. Characteristics of coronary atherosclerotic plaques underlying fatal occlusive thrombi. Heart. 1983;50(2):127-34. 34. Thygesen K, Alpert JS, White HD, TASK FORCE MEMBERS: Chairpersons: Kristian Thygesen JSA, Harvey D. White *, Biomarker Group: Allan S. Jaffe C, Fred S. Apple , Marcello Galvani , Hugo A. Katus , L. Kristin Newby , Jan Ravkilde, ECG Group: Bernard Chaitman C-o, Peter M. Clemmensen , Mikael Dellborg , Hanoch Hod , Pekka Porela, et al. Universal definition of myocardial infarction. Circulation. 2007;116(22):2634-53. 35. Shahzad S, Hasan A, Faizy AF, Mateen S, Fatima N, Moin S. Elevated DNA Damage, Oxidative Stress, and Impaired Response Defense System Inflicted in Patients With Myocardial Infarction. Clin Appl Thromb Hemost. 2018;24(5):780-9. 36. Wang X, Guo Z, Ding Z, Mehta JL. Inflammation, Autophagy, and Apoptosis After Myocardial Infarction. J Am Heart Assoc. 2018;7(9). 37. Paradies G, Paradies V, Ruggiero FM, Petrosillo G. Mitochondrial bioenergetics and cardiolipin alterations in myocardial ischemia-reperfusion injury: implications for pharmacological cardioprotection. Am J Physiol Heart Circ Physiol. 2018;315(5):H1341-h52. 38. Malliani A, Schwartz PJ, Zanchetti A. A sympathetic reflex elicited by experimental coronary occlusion. Am J Physiol. 1969;217(3):703-9. 39. Jardine DL, Charles CJ, Ashton RK, Bennett SI, Whitehead M, Frampton CM, et al. Increased cardiac sympathetic nerve activity following acute myocardial infarction in a sheep model. J Physiol. 2005;565(Pt 1):325-33.

49

40. Schwenke DO, Tokudome T, Kishimoto I, Horio T, Shirai M, Cragg PA, et al. Early ghrelin treatment after myocardial infarction prevents an increase in cardiac sympathetic tone and reduces mortality. Endocrinology. 2008;149(10):5172-6. 41. Ramchandra R, Hood SG, Xing D, Lambert GW, May CN. Mechanisms underlying the increased cardiac norepinephrine spillover in heart failure. Am J Physiol Heart Circ Physiol. 2018;315(2):H340-h7. 42. Lown B, Verrier RL. Neural activity and ventricular fibrillation. N Engl J Med. 1976;294(21):1165-70. 43. Fishbein MC, Maclean D, Maroko PR. The histopathologic evolution of myocardial infarction. Chest. 1978;73(6):843-9. 44. Weisman HF, Bush DE, Mannisi JA, Weisfeldt ML, Healy B. Cellular mechanisms of myocardial infarct expansion. Circulation. 1988;78(1):186-201. 45. Rajendran PS, Nakamura K, Ajijola OA, Vaseghi M, Armour JA, Ardell JL, et al. Myocardial infarction induces structural and functional remodelling of the intrinsic cardiac nervous system. J Physiol. 2016;594(2):321-41. 46. Hwang D, Lee JM, Kim HK, Choi KH, Rhee TM, Park J, et al. Prognostic Impact of β- Blocker Dose After Acute Myocardial Infarction. Circ J. 2019;83(2):410-7. 47. Bradford WD, Chen J, Krumholz HM. Under-utilisation of b-blockers after acute myocardial infarction. Pharmacoeconomics. 1999;15(3):257-68. 48. Domburg RT, Hendriks JM, Kamp O, Smits P, Melle M, Schenkeveld L, et al. Three life years gained after reperfusion therapy in acute myocardial infarction: 25-30 years after a randomized controlled trial. Eur J Prev Cardiol. 2012;19(6):1316-23. 49. Jahan R, Saver JL, Schwamm LH, Fonarow GC, Liang L, Matsouaka RA, et al. Association between time to treatment with endovascular reperfusion therapy and outcomes in patients with acute ischemic stroke treated in clinical practice. JAMA. 2019;322(3):252-63. 50. Petty MA, Lang RE, Unger T, Ganten D. The cardiovascular effects of oxytocin in conscious male rats. Eur J Pharmacol. 1985;112(2):203-10. 51. Michelini LC, Marcelo MC, Amico J, Morris M. Oxytocinergic regulation of cardiovascular function: studies in oxytocin-deficient mice. American Journal of Physiology-Heart and Circulatory Physiology. 2003;284(6):H2269-H76. 52. Uvnäs Moberg K, Handlin L, Kendall-Tackett K, Petersson M. Oxytocin is a principal hormone that exerts part of its effects by active fragments. Med Hypotheses. 2019;133:109394. 53. Sawchenko P, Brown E, Chan R, Ericsson A, Li H-Y, Roland B, et al. The paraventricular nucleus of the hypothalamus and the functional neuroanatomy of visceromotor responses to stress. Prog Brain Res. 107: Elsevier; 1996. p. 201-22. 54. Pyner S. Neurochemistry of the paraventricular nucleus of the hypothalamus: implications for cardiovascular regulation. J Chem Neuroanat. 2009;38(3):197-208. 55. Yang Z, Han D, Coote JH. Cardiac sympatho-excitatory action of PVN-spinal oxytocin neurones. Autonomic neuroscience : basic & clinical. 2009;147(1-2):80-5. 56. Hosoya Y, Matsukawa M, Okado N, Sugiura Y, Kohno K. Oxytocinergic innervation to the upper thoracic sympathetic preganglionic neurons in the rat. Exp Brain Res. 1995;107(1):9-16. 57. Inoue T, Kimura T, Azuma C, Inazawa J, Takemura M, Kikuchi T, et al. Structural organization of the human oxytocin receptor gene. J Biol Chem. 1994;269(51):32451-6. 58. Reversi A, Rimoldi V, Marrocco T, Cassoni P, Bussolati G, Parenti M, et al. The oxytocin receptor antagonist atosiban inhibits cell growth via a “biased agonist” mechanism. J Biol Chem. 2005;280(16):16311-8. 59. Wsol A, Kasarello K, Kuch M, Gala K, Cudnoch-Jedrzejewska A. Increased Activity of the Intracardiac Oxytocinergic System in the Development of Postinfarction Heart Failure. BioMed Research International. 2016;2016. 60. Chan J, Cabrol D, Ingemarsson I, Marsal K, Moutquin J-M, Fisk NM. Pragmatic comparison of β2-agonist side effects within the Worldwide Atosiban versus Beta Agonists study. European Journal of Obstetrics & Gynecology and Reproductive Biology. 2006;128(1-2):135-41. 61. Grzesiak M, Gaj Z, Kocyłowski R, Suliburska J, Oszukowski P, Horzelski W, et al. Oxidative Stress in Women Treated with Atosiban for Impending Preterm Birth. Oxid Med Cell Longev. 2018;2018:3919106.

50

62. Weissman A, Tobia RS, Burke YZ, Maxymovski O, Drugan A. The effects of oxytocin and atosiban on the modulation of heart rate in pregnant women. The Journal of Maternal-Fetal & Neonatal Medicine. 2017;30(3):329-33. 63. Nishioka T, Anselmo-Franci JA, Li P, Callahan MF, Morris M. Stress increases oxytocin release within the hypothalamic paraventricular nucleus. Brain Res. 1998;781(1-2):57-61. 64. Callahan MF, Thore CR, Sundberg DK, Gruber KA, O'Steen K, Morris M. Excitotoxin paraventricular nucleus lesions: stress and endocrine reactivity and oxytocin mRNA levels. Brain Res. 1992;597(1):8-15. 65. Swanson LW, McKellar S. The distribution of oxytocin- and neurophysin-stained fibers in the spinal cord of the rat and monkey. J Comp Neurol. 1979;188(1):87-106. 66. Adams JW, Sakata Y, Davis MG, Sah VP, Wang Y, Liggett SB, et al. Enhanced Gαq signaling: a common pathway mediates cardiac hypertrophy and apoptotic heart failure. Proceedings of the National Academy of Sciences. 1998;95(17):10140-5. 67. D'Angelo DD, Sakata Y, Lorenz JN, Boivin GP, Walsh RA, Liggett SB, et al. Transgenic Galphaq overexpression induces cardiac contractile failure in mice. Proc Natl Acad Sci U S A. 1997;94(15):8121-6. 68. Paxinos G, Watson C. The rat brain in stereotaxic coordinates: hard cover edition: Elsevier; 2006. 69. Iaizzo PA. Handbook of cardiac anatomy, physiology, and devices: Springer Science & Business Media; 2009. 70. Samsamshariat SA, Samsamshariat ZA, Movahed MR. A novel method for safe and accurate left anterior descending coronary artery ligation for research in rats. Cardiovasc Revasc Med. 2005;6(3):121-3. 71. Pfeffer MA, Pfeffer JM, Fishbein MC, Fletcher PJ, Spadaro J, Kloner RA, et al. Myocardial infarct size and ventricular function in rats. Circ Res. 1979;44(4):503-12. 72. Weisman HF, Bush DE, Mannisi JA, Bulkley BH. Global cardiac remodeling after acute myocardial infarction: a study in the rat model. J Am Coll Cardiol. 1985;5(6):1355-62. 73. Anversa P, Beghi C, Kikkawa Y, Olivetti G. Myocardial infarction in rats. Infarct size, myocyte hypertrophy, and capillary growth. Circ Res. 1986;58(1):26-37. 74. Sobel BE, BRESNAHAN GF, SHELL WE, YODER RD. Estimation of infarct size in man and its relation to prognosis. Circulation. 1972;46(4):640-8. 75. Graham LN, Smith PA, Stoker JB, Mackintosh AF, Mary DA. Time course of sympathetic neural hyperactivity after uncomplicated acute myocardial infarction. Circulation. 2002;106(7):793-7. 76. Ślusarz MJ, Ślusarz R, Meadows R, Trojnar J, Ciarkowski J. Molecular dynamics of complexes of atosiban with neurohypophyseal receptors in the fully hydrated phospholipid bilayer. QSAR & Combinatorial Science. 2004;23(7):536-45. 77. Åkerlund M, Bossmar T, Brouard R, Kostrzewska A, Laudanski T, Lemancewicz A, et al. Receptor binding of oxytocin and vasopressin antagonists and inhibitory effects on isolated myometrium from preterm and term pregnant women. BJOG. 1999;106(10):1047-53. 78. Suzuki S, Takeshita A, Imaizumi T, Hirooka Y, Yoshida M, Ando S-i, et al. Central nervous system mechanisms involved in inhibition of renal sympathetic nerve activity induced by arginine vasopressin. Circ Res. 1989;65(5):1390-9. 79. Malpas SC, Coote JH. Role of vasopressin in sympathetic response to paraventricular nucleus stimulation in anesthetized rats. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 1994;266(1):R228-R36. 80. Stocker SD, Muntzel MS. Recording sympathetic nerve activity chronically in rats: surgery techniques, assessment of nerve activity, and quantification. American Journal of Physiology-Heart and Circulatory Physiology. 2013;305(10):H1407-H16. 81. Bryda EC. The Mighty Mouse: the impact of rodents on advances in biomedical research. Mo Med. 2013;110(3):207. 82. Moretti J-L, Burke SL, Evans RG, Lambert GW, Head GA. Enhanced responses to ganglion blockade do not reflect sympathetic nervous system contribution to angiotensin II-induced hypertension. J Hypertens. 2009;27(9):1838-48.

51

83. Diedrich A, Jordan J, Tank J, Shannon JR, Robertson R, Luft FC, et al. The sympathetic nervous system in hypertension: assessment by blood pressure variability and ganglionic blockade. J Hypertens. 2003;21(9):1677-86. 84. Peiss CN, Manning JW. Effects of sodium pentobarbital on electrical and reflex activation of the cardiovascular system. Circ Res. 1964;14(3):228-35. 85. Hara K, Harris RA. The anesthetic mechanism of urethane: the effects on neurotransmitter- gated ion channels. Anesth Analg. 2002;94(2):313-8. 86. Maggi C, Meli A. Suitability of urethane anesthesia for physiopharmacological investigations in various systems. Part 2: Cardiovascular system. Experientia. 1986;42(3):292-7.

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