Medicine, Faculty of
Medicine, Department of
DSpace
UBCV
Taki, Alissa
Master of Science - MSc
University of British Columbia
Spinal cord injury (SCI) disrupts supraspinal input to sympathetic pathways that control the heart, leading to altered cardiovascular function. While advances have been made in understanding the impact of SCI on cardiovascular function, its effects on sympathetic ganglia remains largely unclear. The objectives of this study were: 1) to examine the role of the superior cervical ganglia (SCG) and stellate ganglia (StG) in controlling cardiovascular function in rats with and without chronic complete transection SCI at the T3 level (T3-SCI), and 2) to assess anatomical plasticity of the ganglia post-injury. 12 male Wistar rats (6 naïve, 6 T3-SCI at 4 weeks post-injury) underwent surgical instrumentation and parasympathetic blockade for assessment of sympathetic control of cardiovascular function. Through left-ventricular and femoral-arterial pressure catheters, resting cardiovascular function was assessed sequentially at baseline, following superior cervical ganglionectomy (SCGX), and after stellate ganglionectomy (StGX). Excised ganglia were used for immunohistochemical analysis to identify cardiac-projecting neurons in the SCG and StG through markers of neuropeptide Y and calbindin-D28k. Tyrosine hydroxylase was also used as a marker to quantify cell activity in the ganglia. T3-SCI rats demonstrated significantly lower cardiac and hemodynamic indices compared to naïve rats. SCGX did not alter these indices in either group, whereas StGX led to a significant decline in cardiovascular function in naïve rats. In T3-SCI animals, responses to StGX were blunted. Comparisons between naïve animals after StGX and T3-SCI animals at baseline (i.e. intact ganglia) revealed similar cardiovascular function. Furthermore, we found that cardiac-projecting neuronal cell bodies were significantly larger in the SCG and StG of T3-SCI animals. In summary, our findings demonstrate that sympathetic pathways through the StG that modulate the heart are effectively disrupted in chronic T3-SCI, leading to reduced resting cardiovascular function and remodelling of the cardiac-projecting postganglionic neurons post-injury.
THE IMPACT OF SPINAL CORD INJURY ON SYMPATHETIC GANGLIONIC CONTROL OF THE HEART by Alissa Taki B.Sc., The University of British Columbia, 2022 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) November 2024 © Alissa Taki, 2024 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: The Impact of Spinal Cord Injury on Sympathetic Ganglionic Control of the Heart Submitted by Alissa Taki in partial fulfilment of the requirements for the degree of Master of Science in Experimental Medicine Examining Committee: Christopher R West, Associate Professor, Department of Cellular and Physiological Sciences, UBC Supervisor Matt S Ramer, Associate Professor, Department of Zoology, UBC Supervisory Committee Member Stephen J Lewis, Professor, Department of Pharmacology, Case Western Reserve University Supervisory Committee Member Brett J Hilton, Assistant Professor, Department of Cellular and Physiological Sciences, UBC Additional Examiner iii Abstract Spinal cord injury (SCI) disrupts supraspinal input to sympathetic pathways that control the heart, leading to altered cardiovascular function. While advances have been made in understanding the impact of SCI on cardiovascular function, its effects on sympathetic ganglia remains largely unclear. The objectives of this study were: 1) to examine the role of the superior cervical ganglia (SCG) and stellate ganglia (StG) in controlling cardiovascular function in rats with and without chronic complete transection SCI at the T3 level (T3-SCI), and 2) to assess anatomical plasticity of the ganglia post-injury. 12 male Wistar rats (6 naïve, 6 T3-SCI at 4 weeks post-injury) underwent surgical instrumentation and parasympathetic blockade for assessment of sympathetic control of cardiovascular function. Through left-ventricular and femoral-arterial pressure catheters, resting cardiovascular function was assessed sequentially at baseline, following superior cervical ganglionectomy (SCGX), and after stellate ganglionectomy (StGX). Excised ganglia were used for immunohistochemical analysis to identify cardiac-projecting neurons in the SCG and StG through markers of neuropeptide Y and calbindin-D28k. Tyrosine hydroxylase was also used as a marker to quantify cell activity in the ganglia. T3-SCI rats demonstrated significantly lower cardiac and hemodynamic indices compared to naïve rats. SCGX did not alter these indices in either group, whereas StGX led to a significant decline in cardiovascular function in naïve rats. In T3-SCI animals, responses to StGX were blunted. Comparisons between naïve animals after StGX and T3-SCI animals at baseline (i.e. intact ganglia) revealed similar cardiovascular function. Furthermore, we found that cardiac-projecting neuronal cell bodies were significantly larger in the SCG and StG of T3-SCI animals. In summary, our findings demonstrate that iv sympathetic pathways through the StG that modulate the heart are effectively disrupted in chronic T3-SCI, leading to reduced resting cardiovascular function and remodelling of the cardiac-projecting postganglionic neurons post-injury. v Lay Summary When the spinal cord is injured, this affects how it can communicate with organs systems, leading to a variety of dysfunctions. Specifically, spinal cord injury disrupts how the sympathetic nervous system controls the heart, leading to decreased heart function. This study aimed to examine the impact of spinal cord injury on sympathetic ganglia, which are clusters of neurons that help transmit signals from the spinal cord to target organs. By comparing heart function in rats with and without spinal cord injury, before and after removing sympathetic ganglia, we found that injury at mid-back level disrupts the pathways connected to the stellate ganglia, a key hub for sympathetic control of the heart. As a result, rats displayed reduced heart function and physical changes to the sympathetic ganglia. To our knowledge, this is the first study to investigate the effects of spinal cord injury on sympathetic ganglia responsible for regulating heart function. vi Preface All animal experiments conducted for this thesis were approved by the University of British Columba Animal Care Committee (ACC-A22-0289) and strictly followed guidelines outlined by the Canadian Council for Animal Care. I was the lead investigator on this project. Research questions, study design, surgical assistance, implementation of the anatomical study, and data analysis were conducted by myself. Transection surgeries were conducted by Erin Erskine and all surgical procedures in the in vivo study were performed by Liisa Wainman. Dr. Christopher R. West was the supervisory author for this project and was involved in project conceptualization and development. Dr. Stephen J. Lewis and Dr. Matt S. Ramer provided consultation for study design of the in vivo and anatomical studies, as well as additional expertise throughout the project. Hands-on training for ganglionectomy protocols and programming for recursive translation analysis were also provided by Dr. Ramer. These data have been presented in a poster presentation at an international conference (Taki A, Wainman L, Erskine E, Ramer MS & West CR. Investigating the Influence of Spinal Cord Injury on Sympathetic Ganglionic Control of the Heart. Poster presented at: American Spinal Injury Association Annual Scientific Meeting. May 2024; San Juan, Puerto Rico) and an internal UBC conference (Taki A, Wainman L, Erskine E, Ramer MS & West CR. Beyond the Cord: The Impact of High-Thoracic Spinal Cord Injury on Sympathetic Ganglionic Control of the Heart. Poster presented at: ICORD Annual Research Meeting. March 2024). An oral presentation of this work was also given at an vii internal UBC conference (Taki A, Wainman L, Erskine E, Ramer MS & West CR. Beyond the Cord: The Impact of High-Thoracic Spinal Cord Injury on Sympathetic Ganglionic Control of the Heart. UBCO Interdisciplinary Student Health Conference. March 2024). None of this work has been published in peer-reviewed journals. viii Table of Contents Abstract ......................................................................................................................... iii Lay Summary ................................................................................................................. v Preface .......................................................................................................................... vi Table of Contents ....................................................................................................... viii List of Tables ................................................................................................................ xi List of Figures ............................................................................................................. xii List of Abbreviations .................................................................................................. xiii Acknowledgments....................................................................................................... xv Dedication ................................................................................................................... xvi 1. Introduction ......................................................................................................... 1 1.1 Spinal cord injury ............................................................................................. 2 1.1.1 Epidemiology and mechanism of traumatic SCI ........................................ 2 1.1.2 The evolving SCI research landscape ....................................................... 3 1.2 Neural control of the cardiovascular system................................................. 4 1.2.1 Afferent and central pathways ................................................................... 5 1.2.2 Parasympathetic regulation ....................................................................... 6 1.2.3 Sympathetic regulation .............................................................................. 7 1.3 Cardiovascular dysfunction following high-thoracic injury ....................... 13 1.3.1 Cardiac-specific dysregulation ................................................................. 14 1.3.2 Hemodynamic challenges ........................................................................ 14 1.3.3 Experimental models of injury .................................................................. 15 2. Purpose and Objectives .................................................................................... 19 3. Methods .............................................................................................................. 21 3.1 Experimental design overview ...................................................................... 21 3.1.1 In vivo physiology study ........................................................................... 23 3.1.2 Anatomical study ..................................................................................... 23 3.2 Animals ........................................................................................................... 23 3.3 Complete T3 transection surgery ................................................................. 24 ix 3.4 Post-surgical care .......................................................................................... 25 3.5 Ganglionectomy protocols ............................................................................ 26 3.5.1 Bilateral superior cervical ganglionectomy ............................................... 26 3.5.2 Bilateral stellate ganglionectomy ............................................................. 27 3.6 In vivo protocol .............................................................................................. 28 3.6.1 Anaesthesia ............................................................................................. 28 3.6.2 Surgical instrumentation .......................................................................... 29 3.6.3 Parasympathetic blockade ....................................................................... 30 3.6.4 Data analysis ........................................................................................... 31 3.6.5 Euthanasia ............................................................................................... 31 3.7 Tissue collection ............................................................................................ 31 3.8 Immunohistochemistry .................................................................................. 32 3.8.1 Histological targets .................................................................................. 32 3.8.2 Staining procedure ................................................................................... 33 3.8.3 Imaging .................................................................................................... 33 3.8.4 Cell size and density analyses ................................................................. 34 3.9 Statistical analysis ......................................................................................... 35 3.9.1 In vivo outcomes ...................................................................................... 35 3.9.2 Anatomical outcomes............................................................................... 35 4. Results ............................................................................................................... 36 4.1 In vivo physiology .......................................................................................... 36 4.1.1 Effects of T3-SCI on resting cardiovascular function ............................... 36 4.1.2 Cardiovascular function after ganglionectomy ......................................... 38 4.1.3 Comparative effects of sympathetic denervation and T3-SCI on cardiovascular function ........................................................................................... 42 4.2 Neural atrophy and plasticity after ganglionectomy ................................... 42 4.2.1 Postganglionic signalling .......................................................................... 42 4.2.2 Cardiac-projecting neurons ...................................................................... 45 5. Discussion ......................................................................................................... 47 5.1 In vivo physiology of sympathetic denervation .......................................... 47 5.1.1 Validation of the T3-SCI model ................................................................ 47 x 5.1.2 Superior cervical ganglionectomy has no direct effects on cardiovascular function ................................................................................................................. 48 5.1.3 Stellate ganglionectomy reduces resting cardiovascular function ............ 49 5.1.4 Stellate ganglionectomy mimics the left-ventricular function of T3-SCI ... 52 5.2 Neural plasticity in paravertebral ganglia after SCI .................................... 52 5.2.1 Cardiac-projecting ganglia undergo plasticity in SCI ................................ 52 5.3 Relevance and Future Directions ................................................................. 54 5.3.1 Implications .............................................................................................. 54 5.3.2 Recording volume-dependent indices and challenges to cardiovascular sympathetic regulation ............................................................................................ 54 5.3.3 Sex-related differences in cardiac sympathetic modulation ..................... 54 5.3.4 Whole-mount staining of ganglia after chronic injury ............................... 55 5.3.5 The sympathetic chain as a therapeutic target ........................................ 55 5.4 Considerations ............................................................................................... 56 5.4.1 Strengths ................................................................................................. 56 5.4.2 Limitations ................................................................................................ 56 6. Conclusion ......................................................................................................... 59 References ................................................................................................................... 60 xi List of Tables Table 3.1. Primary and secondary antibody solutions................................................... 32 Table 4.1. Baseline cardiac and arterial pressure recordings, pre- and post-atropine, in naïve and T3-SCI. ......................................................................................................... 37 Table 4.2. Resting cardiac and hemodynamic indices at baseline and steps of denervation. .................................................................................................................. 39 xii List of Figures Figure 1.1. Schematic of the autonomic nervous system and its relevant cardiovascular targets. ............................................................................................................................ 5 Figure 3.1. Overview of the in vivo and anatomical study designs ............................... 22 Figure 3.2. Photo of the isolated right SCG. ................................................................. 27 Figure 3.3. Photo of the isolated right StG .................................................................... 28 Figure 4.1. Resting hemodynamic indices at baseline and after step-wise denervation of the superior cervical ganglion (SCGX) and stellate ganglion (StGX)......................... 40 Figure 4.2. Resting cardiac indices at baseline and after step-wise denervation of the SCGX and StGX. .......................................................................................................... 41 Figure 4.3. Immunohistochemical analysis of tyrosine hydroxylase (TH) positive postganglionic neurons ................................................................................................. 44 Figure 4.4. Immunohistochemical analysis of cardiac-projecting postganglionic neurons in naïve (n=5) and T3-SCI (n=5) rodents ....................................................................... 46 xiii List of Abbreviations ANS: autonomic nervous system ANOVA: analysis of variance AV: atrioventricular calbindin: calbindin-D28k CO: cardiac output DAPI: 4’,6-diamidino-2-phenylindole DBP: diastolic blood pressure dP/dtmax: maximal rate of rise of left-ventricular pressure waveform HR: heart rate K-S: Kolmogorov-Smirnov LV: left ventricle M2: muscarinic 2 MAP: mean arterial pressure NPY: neuropeptide Y Pmax: maximum LV pressure PBS: phosphate buffered-saline PBS-Tx-Azd: phosphate buffered-saline, Triton-X, sodium azide PNS: parasympathetic nervous system SA: sinoatrial SBP: systolic blood pressure SCG: superior cervical ganglion/ganglia SCGX: superior cervical ganglionectomy xiv SCI: spinal cord injury SNS: sympathetic nervous system SPNs: sympathetic preganglionic neurons StG: stellate ganglion/ganglia StGX: stellate ganglionectomy T3-SCI: T3 complete spinal cord transection TH: tyrosine hydroxylase xv Acknowledgments First, I would like to extend my deepest gratitude to my supervisor Dr. Christopher R. West, who has been instrumental to this work. Thank you for your exceptional mentorship, guidance, and providing me the opportunity to work with great researchers. A huge thank you to everyone in the West lab for their immense support and time spent helping me in this endeavour. A special thanks to Erin Erskine, for all the work you do. I would also like to thank Dr. Christine Voss for being my first mentor and for all the opportunities I have been provided because of you. I would like to thank Dr. Stephen J. Lewis and Dr. Matt S. Ramer for their valuable insights and support, especially in their work to help get this project off the ground. And lastly, but certainly not least, I would like to express my immeasurable gratitude, respect, and admiration for Liisa Wainman. Your endless encouragement and willingness to spend far too many hours in a windowless room with me have been the source of my success, and will continue to guide every success I may find hereafter. xvi Dedication For my family, and my love. 1 1. Introduction Spinal cord injury (SCI) is a complex and life-altering condition, characterized by the multi-faceted damage to vital neural pathways in the spinal cord. In 2019, it was estimated that there are over 30,000 individuals living with traumatic SCI in Canada.1 The incidence and age at injury continues to increase as the numbers and average age of the overall population increase. Along with the increase in prevalence, healthcare costs of SCI are also anticipated to increase by 37% by 2032, with the current lifetime costs estimated to range from $1-3 million CAD per person sustaining an SCI.2,3 Among these costs are interventions related to cardiovascular decline. In the general population, the sympathetic nervous system (SNS) is commonly recognized as the 'fight-or-flight' system, increasing the rate and force of cardiac contraction when activated in response to stressors. However, it is important to recognize that the SNS also works in continuous balance with the parasympathetic nervous system (PNS), fine-tuning heart and vascular functions to maintain homeostasis. When SCI damages the pathways regulating cardiac function, this can cause a considerable impact to normal functioning. Numerous researchers, including our laboratory, have focused their efforts on strengthening or repairing damaged neural pathways within the spinal cord as a method to improve cardiovascular function following SCI. Such a focus, however, has resulted in the field to overlook how changes within the autonomic ganglia may also impact cardiovascular function post-SCI. This section outlines the current field of SCI, its impact 2 on the cardiovascular system, as well as the autonomic nervous system (ANS) and the sympathetic ganglia that control it. 1.1 Spinal cord injury 1.1.1 Epidemiology and mechanism of traumatic SCI Traumatic SCI is defined as injury to the spinal cord resulting from a physical or mechanical trauma.4 Traumatic SCI can occur from many circumstances, and the most common causes vary by age.1 For instance, the most common cause of traumatic SCI in the 65+ age group stem from falls, typically occuring in the home, causing SCI mostly at the cervical level. For individuals aged 20-45, the most common injuries are car accidents or sports injuries that lead to thoracic SCI. Most often, the spinal cord is not directly damaged upon injury, but rather the mechanical force against the vertebrae that overcomes its structural integrity will cause compression or contusion to the cord and its surrounding vasculature.5 Compounding the devastation caused by external injury, the body exacerbates the damage through what is termed as a secondary injury to the spinal cord.4 Local tissue and vasculature damage sustained from initial injury triggers a cascade of detrimental effects involving ischaemia, inflammation, apoptotic cell death, and formation of scar tissue in the injury site that hinders regenerative attempts.4,6–8 These sequelae are also capable of spreading across to neighboring segments of the cord and further complicates capturing the full extent of neural pathway damage resulting from the injury.7,9 Additionally, trauma to the cord may cause the body to go into a state of spinal shock, a transient state 3 of suppressed somatic function below the level of injury.10 These overwhelming events following primary injury can cause permanent damage, serving as a prelude to the challenges that individuals with SCI may face for the rest of their lives.11 1.1.2 The evolving SCI research landscape Secondary health conditions are a commonality in individuals living with chronic SCI. A diverse range of conditions have been widely reported, such as respiratory disorders, neuropathic pain, and muscle spasticity.12,13 Cardiovascular disorders that are caused by disrupted autonomic control are often underreported, as it has largely been eclipsed by the focus on assessing motor and sensory impairments.9,14,15 Only around the turn of the century has there been a shift, with the growing literature recommending clinicians to focus on assessment/management of cardiovascular deficits.15,16 In the early 2000s, a committee was established by the American Spinal Injury Association and International Spinal Cord Injury Organization with the aim to create guidelines for classifications and assessment methods for multi-system dysfunctions caused by autonomic impairment.15 The guidelines conceived, titled ‘International Standards to document Autonomic Function following SCI (ISAFSCI)’, were informative on highlighting cardiovascular phenomena such as orthostatic tolerance and autonomic dysreflexia that are caused by disrupted autonomic function.17 Today, identifying the pathophysiology of cardiovascular health decline for individuals with SCI has become a focus for our laboratory and others.18–20 4 1.2 Neural control of the cardiovascular system Regulation of the cardiovascular system is a complex balancing act made up of many components for ensuring the body's survival. The ANS necessitates rapid and continuous responses to external stimuli to maintain adequate blood flow, which is vital for the delivery of oxygen and nutrients to tissues and organs. Neural mechanisms play a crucial role in controlling the cardiovascular system. Central pathways interact with peripheral connections of the ANS to coordinate rapid signalling onto the heart and vasculature, for appropriate and timely responses to its environment.21 An overview of the neural pathways that control the cardiovascular system is illustrated in Figure 1.1 below. 5 Figure 1.1. Schematic of the autonomic nervous system and its relevant cardiovascular targets. 1.2.1 Afferent and central pathways The ANS transmits signals from central nervous system pathways as response to peripheral afferent signals or higher-order processes in the brain.22 Predominant cardiovascular afferent signals come from baroreflex and chemoreflex activation, where their sensory information influences central autonomic processes. Situated in the aortic and carotid bodies, arterial baroreceptors and peripheral chemoreceptors act to detect changes in the bloodstream. As specialized mechanoreceptors, baroreceptors sense 6 distortions in vessel walls elicited by pulse pressure and act to resist fluctuations outside the normal range of blood pressure, and initiate responses that will activate sympatho-inhibitory, or excitatory, pathways in the brainstem.23 In contrast, activated chemoreceptors increase sympathetic outflow in response to changes in chemical composition of the blood by constant monitoring of oxygen and pH levels.24 Afferent pathways converge at the nucleus of the solitary tract in the medulla oblongata and project to surrounding regions for input-specific neural integration.25 Within these regions, appropriate signals are generated and sent from the brainstem to efferent pathways in the spinal cord or cranial nerves.26 Central pathways for communicating with the sympathetic arm of the ANS involve signalling from a host of different sites, such as the rostral ventrolateral region in the medulla oblongata.25 Axons of these nuclei exit the brainstem into the spinal cord, traversing many levels to synapse with neurons known as sympathetic preganglionic neurons (SPNs), marking the transition to ANS pathways.27 The central pathways of the parasympathetic arm of the ANS involve vagal motor neurons located in the dorsal motor nucleus of the vagus and the nucleus ambiguous, which project through the cranial nerve X (vagus) to directly synapse with cardiac ganglia.28 1.2.2 Parasympathetic regulation The PNS promotes energy conservation during resting conditions by attenuating the function of various organ systems, including the cardiovascular system. Vagal motor neurons in the brainstem synapse with postganglionic neurons in the intrinsic cardiac ganglia to innervate the sinoatrial (SA) and atrioventricular (AV) nodes, as well as the 7 atria and ventricles.29 Notably, there is evidence of dense cholinergic fibers innervating all chambers of the heart, however, the precise role of these fibers in controlling ventricular function has not been elucidated.30,31 Through cholinergic signalling, acetylcholine activates muscarinic 2 (M2) receptors to reduce the activity of cardiomyocytes and slow heart rate.32 This attenuation has negative chronotropic effects, which lower firing rate at the SA node, and negative dromotropic effects, which reduce conduction speeds at the AV node. Parasympathetic control of the heart is tonically active but can be easily offset by sympathetic input, shifting the balance of cardiovascular regulation to an excitatory state.33 1.2.3 Sympathetic regulation 1.2.3.1 Sympathetic control of the cardiovascular system Wallin & Charkoudian describe the SNS as the ‘ultimate integrator of systems physiology’, highlighting its pervasive role across all aspects of cardiovascular control.34 Sympathetic outflow to the cardiovascular system exerts excitatory effects, innervating both the heart (to elevate cardiac performance) and the vasculature (to enhance blood flow throughout the body). Cardiac performance, which refers to the heart's ability to pump blood, is measured by cardiac output (CO), defined as the volume of blood the heart is capable of pumping per minute.35 The heart contains rich sympathetic innervation across the entire myocardium. Sympathetically-mediated activation of β-adrenergic receptors can cause a cascade of excitatory effects across the heart.32 Sympathetic excitation can increase heart rate through increasing the speed of depolarization at the SA node (positive chronotropy), 8 while also increasing conduction velocity at the AV node (positive dromotropy). Additionally, sympathetic innervation has positive inotropic effects by enhancing myocardial contractility, the ability of myocardium to develop force and generate velocity.35 These effects allow the heart to pump more blood by increasing the volume pumped per beat and at a faster rate, consequently increasing CO.22 Peripheral blood vessels also receive extensive neural control from the SNS. Within the muscular tunica media layer of vessels, α-adrenergic receptors respond to postganglionic adrenergic signalling by inducing vasoconstriction.32 This constriction increases vascular resistance, thereby raising peripheral blood pressure to increase blood flow. Individual vascular beds are selectively controlled, allowing for reflexive sympathetic activation to control blood flow in specific regions.36 This selective activation allows for dynamic adjustments of peripheral resistance in response to metabolic demand throughout the body.32 Unlike sympathetic control of the heart, sympathetic vasoconstriction can also be tonically active in large vascular beds in order to maintain arterial blood pressure for adequate perfusion.34 1.2.3.2 Sympathetic preganglionic neurons Central pathways in the brainstem connect to descending sympathetic neurons signalling to SPNs in the spinal cord. The cell bodies of SPNs reside within the intermediolateral region, located in the lateral horn of gray matter in T1-L2 segments of the cord.23 Axons of SPNs leave the spinal cord through ventral roots to enter spinal nerves. SPNs communicate with postganglionic nerves via cholinergic signalling, which then communicate with end-organ systems through adrenergic synapses. Sympathetic input to the heart and upper body vasculature lies between the T1-4 segments of the 9 spinal cord in both humans and rats, while outflow to the lower body vasculature is within T5-L2 segments.37–39 Axons of SPNs can travel in three directions: 1) synapse with postganglionic neurons in a sympathetic ganglion of the same vertebral level, 2) traverse the sympathetic chain to synapse with a ganglion farther away, or 3) synapse with a prevertebral ganglion.40 1.2.3.3 Sympathetic postganglionic neurons Sympathetic ganglia are clusters of postganglionic neuronal cell bodies that reside near the spinal cord to modulate and transmit sympathetic signals from SPNs to target effectors.41 Sympathetic postganglionic neurons possess long, unmyelinated axons to travel long distances for widespread distribution, in contrast to the short axons of parasympathetic postganglionic neurons that reside within their target for local regulation.42 Sympathetic ganglia can be classified into two types: paravertebral ganglia and prevertebral ganglia.41,43 Paravertebral ganglia form the bilateral sympathetic chain that runs parallel to the spinal cord, while prevertebral ganglia form ‘lobes’ that lie ventral to the spinal cord, surrounding major nodes of the abdominal aorta. The sympathetic chain is generally comprised of 23 pairs of ganglia, along with one unpaired coccygeal ganglion; however, there is considerable genetic diversity in the structure of each ganglion, in which fusions between multiple ganglia can occur.44,45 1.2.3.4 Sympathetic ganglia as modulators Traditionally, sympathetic ganglia were regarded as simple relay centers in a monosynaptic pathway. However, this perspective has gradually evolved.46 The 10 identification of multiple neurotransmitters within a ganglion, as observed by Klimaschewski et al., supports the concept of multi-synaptic pathways running through a single ganglion.47 Their work highlighted the presence of a variety of neurotransmitters within the cervical ganglia, suggesting a complex chemical coding of ganglionic transmission to their targets. This has been further supported in the field by a continuation of identifying complex pathways in the sympathetic chain, where a single ganglion can receive converging inputs from various segments of the spinal cord and a single postganglionic neuron can receive input from up to three SPNs.38,48 These pathways allow for a coordination of signals in each ganglion, to facilitate the route to their respective destinations. Recent advancements in observing signal strength and firing patterns have also allowed revision of the traditional perspective. For instance, Klassen et al. utilized microneurography to study the paravertebral ganglia that innervate skeletal muscle circulation. Their findings demonstrated that these ganglia influence the firing patterns of sympathetic transmission, highlighting their active role in sympathetic modulation.49 1.2.3.5 Cardiac-projecting sympathetic ganglia The extrinsic cardiac SNS is composed of the peripheral connections that leave the spinal cord and synapse with the intrinsic cardiac system.50 This system is recognized for its high complexity, with no clear consensus among researchers regarding the pathways that facilitate innervation to the heart.51 Deciphering the functionality of postganglionic innervation has been inconsistent in literature. While multiple paravertebral ganglia have been identified as having physical connections to the heart, establishing their direct role in cardiac sympathetic control remains difficult to prove. 11 The superior cervical ganglia (SCG) reside at the top of the sympathetic chain at the C2/3 segment of the spinal cord.52 The SCG receive innervation from the SPNs originating from the T1-4 spinal levels and project postganglionic axons to numerous structures within the body, such as cerebral and coronary arteries, carotid bodies, the pineal gland, and skin.53,54 Kawashima and others have shown in dissections across multiple species that the SCG possess anatomical connections to the anterior side of the myocardium, however, their functionality remains unclear.44,55,56 Recently, a systematic review from Chen and colleagues investigated the current literature on innervation of the heart by the SCG.51 Their findings support an indirect involvement in cardiac function and presence of innervation across all studied species through in vivo, in vitro, and morphological studies. This indirect involvement pertained to the changes observed in the morphology of the SCG in diseases states, such as post-myocardial infarction, diabetes mellitus, and pulmonary hypertension, which contributed to increased resting HR, blood pressure, and other cardiac-related indices. Only one study by Brandys et al., conducted in dogs, directly stimulated the SCG to measure changes in cardiac indices and found no meaningful contributions.57 Together, these outcomes may suggest the SCG contribute very modestly to cardiac sympathetic control, but that their influence becomes heightened in disease states that involve overactivation of the SNS. How the SCG may function in a state of reduced sympathetic input in SCI is unknown, as this has not yet been explored. Located at the C7/T1 spinal segments, the stellate ganglia (StG) is the fusion of the inferior cervical ganglion and the first thoracic ganglion (or rarely, the fusion of the middle cervical ganglion and the inferior cervical ganglion).52 Cardiac SPNs projecting to the StG 12 reside between T1-5, with the highest input around the T2 level. The StG is considered the primary sympathetic ganglion for cardiac control, as demonstrated by functional and anatomical studies confirming its widespread innervation across the heart.58–60 More specifically, the left stellate ganglion is commonly regarded as having a greater degree of cardiac sympathetic innervation. Alongside cardiac innervation, the StG supplies sympathetic innervation to the head, neck, and upper extremities.45 More recent studies have explored the complexities of the StG. Wu et al. conducted a series of experiments in dogs to manipulate the StG and observed bilateral asymmetry in its control over the heart.61 Stimulation of either ganglion side showed increased systolic blood pressure, while stimulation of the right side showed dominance in increasing heart rate. Following this, Clyburn et al. has shown complex modulation of sympathetic signalling in the StG, where in vitro patch clamp recordings identified collateral pathways within the ganglia.62 These inter-postganglionic connections suggest cardiac innervation is guided by graded response patterns that are specifically controlled by the organizational system within the StG, much like firing patterns observed by Klassen et al. in cervical ganglia innervating skeletal muscle circulation.49 Postganglionic neurons projecting to the heart have been shown to have a variety of neurochemical classes, as described by Anderson and further explored by Richardson et al. through immunohistochemistry and retrograde tracing experiments in rodents.59,63 Injections of Fast Blue into the heart retrogradely labeled cardiac-projecting postganglionic neurons within the StG. Subsequently, these neurons were co-stained with two distinct markers of cardiac transmission. Norepinephrine is the primary neurotransmitter released for adrenergic signalling, but another neuropeptide observed 13 to be co-transmitted in cardiovascular-specific sympathetic transmission is neuropeptide Y (NPY). NPY along with calbindin-D28k (referred simply as calbindin), an intracellular calcium-binding protein, were found to create four unique neurochemical types with traceable axons projecting through cardiac nerves to the myocardium. These were classified as reactive to both NPY and calbindin, reactive to only one, or reactive to neither. Of these four, almost half of the neural population projecting to the heart were reactive to both NPY and calbindin. 1.3 Cardiovascular dysfunction following high-thoracic injury Cardiovascular disease risk has been shown to nearly triple in individuals with SCI and is a leading cause of mortality for this population.64 Thus, managing cardiovascular decline has become primary focus in the treatment of secondary health conditions brought on by SCI. Injuries above the T6 level of the spinal cord can result in partial or complete loss of medullary sympathetic control to the heart. This is due to the loss of supraspinal input to SPNs or direct damage to these cells, disrupting the sympathetic pathways controlling cardiovascular function.65 Described below, this dysregulation can also cause cardiovascular phenomena such as orthostatic hypotension and autonomic dysreflexia. Our laboratory and others have demonstrated this significant impairment in animal models and clinical populations with chronic cervical or high-thoracic SCI. Notably, a recent cohort study by Eun Yoo et al. showed an increased risk of heart failure, arrhythmias, and hypotension occuring at all levels of injury for those living with chronic SCI.66 14 1.3.1 Cardiac-specific dysregulation Central to cardiovascular dysfunction following SCI are the physiological and structural changes induced in the heart. The body of literature documenting these alterations and their underlying mechanisms continues to expand, including research conducted by our laboratory. Williams et al. conducted a meta-analysis on cardiac function in individuals with chronic SCI, showing that across 22 studies, this population had smaller left ventricle (LV) volumes and altered systolic function, resulting in reduced stroke volume and CO.67 Reduced cardiac sympathetic tone caused by loss of supraspinal input has been shown to cause myocardial atrophy, which is reflected in a reduced average LV mass in the SCI population.9,65 Additionally, low total blood volume and venous return contributes to myocardial unloading, the progression of reduced workload in the heart that leads to atrophy and weakens myocardial strength over time. Among the variety of cardiovascular complications, bradycardia and reflex bradycardia are prominent in the acute stage of SCI.18 This is primarily attributed to the loss of sympathetic tone and unopposed vagal activity. The intact vagal tone leads to recurrent bradycardiac episodes, further exacerbating the risk of asystolic events and cardiac arrest. 1.3.2 Hemodynamic challenges Concerns surrounding blood pressure management arise immediately after initial injury and continue to persist in chronic high-level SCI. Reduced cardiac function, in tandem with low total blood volume and reduced sympathetic vasomotor tone, decreases resting systemic blood pressures.67 In addition, orthostatic hypotension is a common 15 occurrence, defined as an event of a large drop in arterial blood pressure and bradycardia when moving from supine to an upright position.39 Conversely, individuals may experience an event of a dramatic increase in blood pressure in association with arrhythmia, termed as autonomic dysreflexia. This potentially life-threatening phenomenon is brought on by a noxious, or non-noxious, stimuli below the level of injury that activates spinal reflexes to increase blood pressure. 1.3.3 Experimental models of injury Along with studying motor and sensory deficits, experimental models of SCI have been utilized to study autonomic dysfunction. Both small and large animal models are employed to characterize various aspects of SCI. In particular, rats have been viewed as an ideal translational model for cardiovascular physiology.37,68,69 In addition to these animals demonstrating similar cardiovascular function as humans, they also exhibit analogous declines in function post-SCI.70 1.3.3.1 In vivo assessments Animal models of SCI offer the benefit of real-time monitoring of changes induced by injury. Established in vivo methods of recording cardiovascular function have been adopted by our laboratory to analyze such changes.37,70 A foundational technique in these studies is the surgical implantation of pressure-volume catheters into the LV coupled with arterial pressure catheterization.71 This approach allows for continuous monitoring of cardiac function and hemodynamics, measuring indices such as stroke volume, CO, and arterial pressure. In addition to measuring cardiovascular function under resting conditions, sympathetic function can be assessed through tests such as controlled 16 hemorrhage and simulated orthostatic hypotension.72,73 These tests elicit maximal sympathetic excitation by activating afferent baroreflex pathways in response to low blood pressure, to provide a more comprehensive understanding of the efferent sympathetic pathways disrupted by SCI. In summary, these methods aid in our advancement of understanding pathophysiology of SCI and advance development of targeted therapeutic interventions. 1.3.3.2 Transection models Complete transection of the spinal cord involves surgical severance of the spinal cord to ensure specific pathways are disrupted. Transection models are frequently employed to assess axonal regeneration and functionality post-injury; however, their ability to disconnect all pathways through the cord also provides a reliable framework for studying basic anatomy and physiology in systems of interest.69,74 Most recently, our laboratory was able to utilize a T3 transection (T3-SCI) rat model to demonstrate in vivo that severing specific bulbospinal sympathetic pathways causes immediate and persistent cardiac dysfunction.65 These findings were reflective of the outcomes observed in a human population of SCI, further validating the use of rodent models in translational SCI research. 1.3.3.3 Cervical vs. high-thoracic SCI models While some researchers have trialed cervical SCI models to focus on cardiovascular deficits, the immense care and complications of such a severe injury makes this model less desirable.75 The use of complete transection at the cervical level is heavily scrutinized from an ethical standpoint due to the high welfare burden and researchers are recommended to consider other options.75 As an alternative, it is 17 favoured by our laboratory and others alike to utilize high-thoracic models (T3 and above) for a balance between accuracy and ethical consideration.37,75,76 High-thoracic models are less prone to complications, preserve upper limb function, and reduce the burden of care on both the laboratory animals and the researchers. The use of high-thoracic SCI models has recently been challenged by Lujan & Di Carlo for its inadequacy to simulate realistic cardiovascular deficits, in comparison to cervical SCI models.77 This theory relies on the idea that preservation at the T1 level allows for intact descending input to the StG, thereby allowing increased sympathetic activation to occur instead. Through in vivo assessments of resting and sympatho-excitatory states, they reported that rats with a chronic T3-SCI were tachycardic with enhanced cardiac sympathetic tone in comparison to naïve rats, similar to results from studies that assessed animal models with mid-thoracic SCI. Conversely, our research team and others have demonstrated that chronic high-thoracic transection and contusion animal models display profound cardiovascular deficits, regardless of T1 preservation.37,70,73,78 In vivo assessments of cardiovascular function produced by our laboratory have shown significant decreases in resting cardiac and peripheral blood pressures, as well as reduced LV contractility in animals with T3-SCI. During sympatho-excitatory challenges, these animals demonstrate reduced sympathetic tone manifested through blunted changes to CO, maximal LV pressure, and peripheral blood pressure. As mentioned above, this T3-SCI rodent model has also been validated to demonstrate reduced LV function and volume that are translational to the clinical manifestations reported in a human population with chronic cervical SCI.65 18 Aside from previous evidence reporting on the cardiovascular deficits observed from high-thoracic SCI models, Lujan & Di Carlo’s analysis does not examine neural plasticity in the ganglia to confirm hyperactivation of postganglionic sympathetic pathways to the heart is occuring.77 In fact, to our knowledge, no studies have focused on the effects of SCI on the paravertebral ganglia that regulate the cardiovascular system. Despite signficant advances in SCI research, there is still uncertainty in the basic mapping of the sympathetic pathways that control the heart and the changes that occur post-injury. Understanding these pathways is crucial for development of effective interventions to mitigate autonomic dysfunction following SCI. 19 2. Purpose and Objectives Paravertebral sympathetic ganglia play a critical role in regulating cardiovascular function. Despite the extensive historical research on the structure and function of these ganglia, there is, to our knowledge, no research identifying the changes that occur in these cardiac-innervating ganglia after SCI. Cervical or high-thoracic SCI impairs descending input to the sympathetic preganglionic neurons controlling the heart, which is purported to impair cardiac function post-injury. The work detailed here could further our understanding of structures that control the heart, as well as guide interventional research for modulation of ganglia, as a way to compensate for the loss of central pathways brought on by SCI. The aims of this study were to: 1. Validate the impairment of cardiovascular function in a chronic model of SCI at the T3 level. 2. Investigate the role of the SCG and StG in modulating sympathetic transmission to the heart following T3-SCI. 3. Demonstrate that the impaired cardiovascular function observed in T3-SCI is attributable to the loss of supraspinal input to the cardiac-projecting paravertebral ganglia. 4. Identify anatomical plasticity in the ganglia following T3-SCI. Hypothesis 1: In rats, T3-SCI will impair cardiovascular function by disrupting sympathetic supraspinal control of the heart. 20 Hypothesis 2: Bilateral denervation of the paravertebral ganglia innervating the heart will reduce resting cardiovascular function in animals with intact spinal cords, while animals with a T3-SCI will not respond to denervation due to the prior loss in sympathetic tone. Hypothesis 3: As a result of the prior loss in sympathetic transmission, animals with a T3-SCI and intact ganglia will mimic animals with intact spinal cord that have undergone bilateral denervation of paravertebral ganglia. Hypothesis 4: T3-SCI will induce plasticity in the sympathetic postganglionic neurons innervating the heart as a result of reduced activity in the absence of supraspinal input. 21 3. Methods Ethics approval was granted by the UBC Animal Care Committee (ACC-A22-0289). All experimental protocols were conducted under strict accordance with the Canadian Council on Animal Care. 3.1 Experimental design overview The presented study was conducted in two parts to investigate both the physiological and anatomical changes that occur in the chronic stage (4 weeks) of complete spinal cord transection at the T3 level compared to naïve (i.e. uninjured) controls. Complete T3-SCI and post-operative care were followed from protocols detailed from previous studies in our laboratory.65 An in vivo experiment was conducted to investigate the acute effect of step-wise sympathetic denervation of the heart via removal of the SCG and the StG in naïve (n=6) and spinal cord-injured (n=6) rats. Using left-ventricular and femoral-arterial pressure catheters, resting cardiac and hemodynamic function was assessed sequentially at baseline, following SCG removal, and after StG removal. These ganglia were then stained for various targets to identify anatomical changes in rats with chronic T3-SCI. An overview of the study design can be found in Figure 3.1. 22 Figure 3.1. Overview of the in vivo and anatomical study designs. Rats in the T3-SCI group underwent complete T3 transection and were survived for 4 weeks prior to the in vivo study. Excised superior cervical ganglia (SCG) and stellate ganglia (StG) were used for the anatomical study. 23 3.1.1 In vivo physiology study 12 male Wistar rats were assigned evenly to two groups. Six rats received a complete spinal cord transection at the T3 level and were survived to a chronic SCI timepoint at 4 weeks post-injury, while six remained uninjured. All rats were housed in our facility under the same conditions and duration. Both groups underwent in vivo assessment for cardiac and hemodynamic function via direct cardiac and arterial catheterization. Rats then underwent bilateral SCG ganglionectomy (SCGX), and bilateral StG ganglionectomy (StGX) to measure the effect of incremental sympathetic denervation of cardiac-projecting ganglia. 3.1.2 Anatomical study The excised SCG and StG from naïve (n=5) and T3-SCI (n=5) animals were prepared for histological examination. Two immunofluorescent staining paradigms were used to assess cellular composition. A co-stain was used identify cardiac-projecting neurons in the SCG and StG, based on the presence/absence of NPY and calbindin.59 Tyrosine hydroxylase (TH; a rate-limiting enzyme in catecholamine biosynthesis) was used as a neural proxy for signalling activity, alongside 4’,6-diamidino-2-phenylindole (DAPI) to stain nuclei.79 3.2 Animals Male Wistar rats were selected as the model organism for this study. The Wistar strain has been used extensively within spinal cord injury research and in our 24 laboratory.37,65,70 Their anatomy has been well defined and translates effectively to the human cardiovascular system. Male rats were selected as studies conducted in our laboratory have largely used only males for SCI survival studies but does not reflect on a greater significance to choose either sex. All rats were received at the UBC Okanagan Animal Facility at 10 weeks of age. Rats arrived at the facility one week prior to the experimental start point for acclimation of their environment, handling practices, diet, and cage mates. 3.3 Complete T3 transection surgery Rats allocated to the T3-SCI group underwent a complete spinal cord transection at the T3 level at 11-12 weeks old. For each of the three days prior to surgery, injections of 10 mg/kg of Baytril (fluoroquinolone antibiotic) were given subcutaneously. Rats were anaesthetized first under chamber induction at 5% isoflurane and 95% O2, then switched over to 1.5-2% isoflurane via inhalation through a nose cone. Body temperature was maintained using a heating pad placed underneath and monitored through a rectal thermometer. Pre-surgical care included lubricating the eyes, hair removal on the dorsal side of the trunk region, and disinfection of the surgical area through three washes of chlorhexidine scrub and 70% isopropyl alcohol rinse. Additionally, subcutaneous injections of 5 ml of lactated Ringer’s solution, 10 mg/kg of Baytril (antibiotic) and 0.05 mg/kg of buprenorphine (analgesia) were administered before surgical protocol began. 25 A dorsal midline incision was made across the cervical to mid-thoracic region and internal structures were bluntly dissected to expose the spinal column. The thoracic region was raised by placing an empty syringe under the chest to angle the T2 and T3 vertebrae downwards and expose the spinal cord. The dura was cut in between the T2 and T3 vertebrae using micro-scissors. A complete transection at the T3 level was performed using micro-scissors and a vacuum suction was used to remove the spinal tissue and debris. Gelfoam was placed in the excised space as a hemostatic and to act as a barrier between the two ends of the lesion. The muscular and cutaneous layers were then sutured. An additional dose of 5 ml of lactated Ringer’s solution was administered before animals were taken off anaesthesia and placed in an incubator for one hour to recover. 3.4 Post-surgical care Once returned to their home cages, rats were given pellets, hydrogel, and fruit on the bottom of the cage for ease of access. Rats were closely monitored four times a day for one week, assessing for health and surgical complications. Each morning, rats were weighed and compared to pre-surgical weight to monitor changes. Weight supplements were given to rats displaying a decrease in 25% from their original weight. For the first three days following surgery, 10 mg/kg of Baytril and 2 mg/kg of Metacam (a non-steroidal anti-inflammatory drug) were administered once in the morning, as well as 0.02 mg/kg of buprenorphine given twice a day, all subcutaneously. Injection sites were alternated to avoid re-injection into the same site. 5 ml of lactated 26 Ringer’s solution was given twice daily for hydration throughout the first week. Appearance, behaviour, and pain were graded. The dosage of buprenorphine was increased to 0.05 mg/kg in animals displaying signs of pain.75 As this injury results temporary loss in bladder control, animals required manual expression to assess and empty contents of the bladder four times a day until spontaneous micturition returned. Once this was confirmed, routine monitoring was performed twice a day until experimental end point. 3.5 Ganglionectomy protocols 3.5.1 Bilateral superior cervical ganglionectomy The SCGX procedure was performed according to a well-established protocol by Savastano and colleagues.80 During surgical instrumentation for intubation and left-ventricular catheterization, a midline incision inferior to the mandible was made into the skin and superficial muscles, followed by blunt dissection of the sternohyoid muscles. The incision was retracted to expose the mandibular glands. The carotid bifurcations were landmarked through a triangular shape made by the internal and external carotid arteries, with the ganglion located in the middle of the triangle, deep to the arteries. The external carotid artery was ligated with suture and moved laterally to isolate the SCG. A photo of the isolated SCG is shown in Figure 3.2. When it was time for removal, the ganglion was excised by cutting the sympathetic chain proximally and distally to the ganglion. Both dissections were completed prior to baseline measures, but not excised. The incisions were hydrated with lactated Ringer’s solution and retractors were removed. 27 Figure 3.2. Photo of the isolated right SCG, view from the ventral side of the neck with head positioned rostral to the ganglia (i.e. top of the image). The external carotid artery (ECA) is pulled laterally and held with sutures to isolate the SCG further. The black arrow is directed at the ganglion. Dotted lines indicated where incision were made to excise the ganglion. 3.5.2 Bilateral stellate ganglionectomy The StGX protocol was designed and validated by our laboratory prior to the study, based on a procedure by Scherschel et al.81 A midline skin incision was made, caudal to the clavicles and extended to the xiphoid process of the sternum. Fascia around the perimeter of the incision was removed to widen the area. The pectoralis major, minor and scalenus muscles were crushed with haemostats and bisected. An incision into the intercostal muscles between the first and second rib was made. A retractor was placed between the ribs to widen the incision. The StG was located laterally to the sternum, just shallow to the second rib. Landmarks to locate the StG included its superior-lateral position relative to the thymus and a vertebral artery that runs across the StG. This artery was displaced carefully to isolate the ganglia. The sympathetic trunk running on either side of the ganglia was isolated and sutures were placed on either side to aid in isolation ECA 28 as depicted in Figure 3.3. The incisions were hydrated with lactated Ringer’s solution and retractors were removed. Figure 3.3. Photo of the isolated right StG, view from the ventral side of the neck with head positioned rostral to the ganglia. The second rib has been removed in this animal to better visualize the StG. The black arrow is directed at the ganglion. Dotted lines indicated where incision was made to excise the ganglion. 3.6 In vivo protocol 3.6.1 Anaesthesia Rats were first anaesthetized under chamber induction at 5% isoflurane and 95% O2 then maintained at 1.5-2% isoflurane via inhalation through nose cone, adjusting the dosage in response to toe-pinch and palpebral reflexes. After instrumentation, animals were switched over to intravenous urethane provided through a venous fluid line inserted into the left jugular vein. An initial bolus of 1.6 mg/kg urethane diluted in physiological 29 saline at an infusion rate of 6ml/hr was given and additional doses of 0.1-0.4 mg/kg were delivered throughout protocol as needed. 3.6.2 Surgical instrumentation After loss of palpebral reflex and decreased frequency of breathing under chamber induction, rats were placed supine on top of a heating pad on the operating table, and a thermometer was inserted into the rectum to maintain body temperature at 37°C. A nose mask connected to a SomnoSuite Anaesthesia System was fitted over the nose and inspired isoflurane at 1.5-2% was administered. Once the rats were confirmed to be under a surgical plane of anaesthesia confirmed by absence of the toe-pinch and palpebral reflexes, surgery was commenced. A midline incision at the mandible extending to the manubrium was made and the sternohyoid muscle was bluntly dissected. The underlying fascia of the sternomastoid muscles were dissected to locate the left jugular vein. The vein was ligated cranially with a suture, to constrict blood flow, and pierced with a needle to advance PE50 tubing into the lumen of the vessel. Incisions were made along both inguinal ligaments and retracted. A fluid line attached to a stop-cock system was inserted into the right femoral artery for blood gas analysis. The right common carotid artery was isolated and ligated while a pressure catheter was advanced into the LV. A pressure catheter was placed into the left femoral artery using the same approach and advanced into the abdominal aorta. 30 Once all tubing were securely attached, the jugular venous fluid line was connected to an infusion pump and animals were converted from isoflurane to intravenous urethane. An initial bolus of 1.6 mg/kg urethane diluted in physiological saline was given at an infusion rate of 6 ml/hr and additional doses of 0.1-0.4 mg/kg were delivered prior to the start of baseline measures as needed. The trachea and right common carotid artery were carefully isolated. A tracheostomy was performed with an endotracheal tube advanced into the trachea and connected to a small-animal ventilator for mechanical ventilation at 100% O2. Tidal volume and breathing frequency were set based on animal body mass derived to ensure appropriate arterial gas concentrations during anaesthesia.71 Both the SCG and StG were isolated prior to baseline measurements, following protocols described in 3.5.1 and 3.5.2. 3.6.3 Parasympathetic blockade In order to isolate sympathetic transmission to the heart, atropine was administered prior to baseline recordings. Atropine functions as an M2 receptor antagonist, thereby impeding cholinergic signalling to the heart.82 Rats received a bolus dose of 2 mg/kg atropine methyl bromide diluted in physiological saline at an infusion rate of 6 ml/hr, followed by a constant rate of infusion of 0.5 mg/kg at 1 ml/hr throughout the study. Sufficient parasympathetic blockade was validated by a substantial increase in heart rate that was sustained after 20 minutes. 31 3.6.4 Data analysis A one-minute representative recording of baseline cardiovascular indices was taken after stabilization of the parasympathetic blockade. Cardiovascular indices of interest included the maximal rate of rise of left-ventricular pressure waveform (dP/dtmax; mmHg/s) and maximum LV pressure (Pmax; mmHg) from the left-ventricular pressure catheter, as well as mean arterial pressure (MAP; mmHg), heart rate (HR; BPM), diastolic, and systolic blood pressure (DBP, SBP; mmHg) from the left femoral arterial pressure catheter. Resting cardiac and hemodynamic assessments were repeated for both SCGX and StGX conditions, where one-minute representative recordings were taken 30 minutes after ganglia removal. 3.6.5 Euthanasia At experimental or humane endpoint, rats were given an overdose of intravenous chloral hydrate diluted in saline. Upon cessation of vitals, rats were exsanguinated through the diaphragm as a secondary method to ensure death. 3.7 Tissue collection The SCG and StG excised during the in vivo protocol were immediately placed in 10% paraformaldehyde to clear the tissue of blood. After 24 hours, ganglia were placed into 20% sucrose solution for 2 days for cryoprotection. The ganglia were then placed in CryoMatrix™ embedding solution within foil casings and frozen at -80 °C, where they were stored until the staining protocol. 32 3.8 Immunohistochemistry 3.8.1 Histological targets In the first staining paradigm, immunofluorescent staining was used to identify active cardiac projecting neurons in the SCG and StG, based on the presence of NPY and calbindin.59 For the second paradigm, TH was used as a neural proxy to measure adrenergic signalling activity, alongside DAPI in ProLong™ Gold antifade mounting medium to stain nuclei. Primary and secondary antibodies are listed in Table 3.1 below. All antibody solutions were diluted in a solution of phosphate buffered-saline, Triton-X, and sodium azide (PBS-Tx-Azd). Table 3.1. Primary and secondary antibody solutions. Histological Target Species Dilution Source Primary Calbindin-D28K Rabbit 1:500 Invitrogen, PA5-85669, Waltham, MA, USA Neuropeptide Y Goat 1:500 Novus Biologicals, NBP1-46535SS, Toronto, ON, Canada Tyrosine Hydroxylase Sheep 1:200 MilliporeSigma, AB1542, Oakville, ON, Canada Secondary/Fluor Rabbit IgG-Dylight 650 Donkey 1:800 Novus Biologicals, NBP1-726960, Toronto, ON, Canada Goat IgG-Alexa 488 Donkey 1:1000 Jackson ImmunoResearch, AB2340428, West Grove, PA, USA Sheep IgG-Cy3 Donkey 1:200 Jackson ImmunoResearch, AB2340727, West Grove, PA, USA 33 3.8.2 Staining procedure Ganglia were cut longitudinally at 10 μm thickness using a cryostat. Sections were mounted on silane-coated slides, with eight per slide, and left to dry for 20-minutes before placing back into -80 °C storage. One slide per co-stain for each ganglion was chosen to be stained. Prior to staining, slides were taken out of storage and dried for one hour. A perimeter was drawn around the tissue using a hydrophobic barrier pen to keep the solutions from falling off the slides during incubation. All wash and incubation steps were taken place on a shaker plate at a low speed. Slides were placed in Coplin staining jars and washed with phosphate buffered-saline (PBS) in three 10-minute intervals, then moved onto a staining tray for incubation with a blocking solution of 10% Normal Donkey Serum in PBS-Tx-Azd for one hour. Slides were then incubated overnight with 300 μl of primary antibody solution per slide. The staining tray cover was placed on top to keep in moisture. The following morning, slides were washed with PBS in three 15-minute intervals, then incubated for two hours with 300 μl of secondary antibody solution per slide, keeping the tray cover on as to not expose the immunofluorescent tags to light. Another wash was done with 0.1 M Phosphate buffer solution in three 10-minute intervals. Slides were air-dried, then cover-slipped with ProLongTM Gold antifade mounting medium with DAPI (without DAPI for NPY/calbindin stain) and left to set for 24 hours prior to imaging. 3.8.3 Imaging Immunofluorescence imaging was conducted using a Zeiss Axio Imager M2 microscope at 20x magnification and processed with the ZEN 2 Blue program. A preview 34 of all slides was generated using Alexa488 fluorescence. Prior to final imaging, each fluorescence channel was observed under live camera to adjust exposure and contrast. Three sections for each co-stain, all at least 40 μm apart, were randomly chosen for analysis of each ganglion. 3.8.4 Cell size and density analyses Analyses of images were performed in ImageJ Fiji and Ilastik for their respective functions. Machine learning algorithms were created in Ilastik for automated object classification based on the detection of cell shape and fluorescence of target markers.83 Overall cell activity was assessed through a neural proxy with the presence of TH. Cardiac-projecting neurons were characterized by colocalization of NPY and calbindin. A particle map was generated for each section, then exported to ImageJ Fiji to measure neuronal profile Feret diameter. Bilateral differences in both the SCG and StG were assessed prior to merging pairs together. Comparisons of cell diameter sizes were conducted through size-frequency analysis using recursive translation, in which neuronal profile diameters were mathematically reconstructed from the initial populations.84 Diameters were categorized into 4 μm bins. The proportional frequency and cumulative sum of proportions from cell diameter categories were calculated and visualized in their respective plots. Concurrently with assessing TH+ cell size, overall area of TH+ fluorescence was analyzed. Threshold analysis in ImageJ Fiji was conducted, in which images were converted to 8-bit and set to a threshold only including pixels with intensity values for the TH+ stain. In each section, perimeters were traced and total section area was calculated 35 to generate a percentage of area of TH+ fluorescence (Area % = (area of TH+ fluorescence / total section area) * 100). 3.9 Statistical analysis 3.9.1 In vivo outcomes In vivo data acquired from Labchart 8 was exported and transferred to R Studio for statistical analysis. The Shapiro-Wilks test for normality was conducted for all cardiovascular indices of interest, separated by each group (naïve, T3-SCI) and step (baseline, SCGX, StGX). A two-way repeated measures analysis of variance (ANOVA) was conducted, alongside Bonferroni corrections for pairwise comparisons to identify within-, between-, and across- group differences. In addition, the absolute change after ganglionectomy from baseline was compared using Independent Student’s t-tests, when specified. All tabulated data are presented as mean ± standard error of the mean with statistical significance set at p < 0.05. 3.9.2 Anatomical outcomes Independent Student’s t-tests were employed to compare percentage of area fluorescent for the TH+ label between naïve and T3-SCI for both ganglia. Kolmogorov-Smirnov (K-S) tests were conducted to compare cumulative frequency of cell sizes. Significance was determined if the value of D was greater than the critical value (1.36 / √𝑁 , where N is sample size). All tabulated data are presented as mean ± standard deviation with statistical significance set at p < 0.05. 36 4. Results 4.1 In vivo physiology 4.1.1 Effects of T3-SCI on resting cardiovascular function Baseline cardiovascular function from the left-ventricular and femoral arterial catheters pre- and post-infusion of atropine are reported in Table 4.1. Parasympathetic blockade was induced through atropine infusion with a dosage that was validated prior to the study. HR significantly increased following atropine infusion in uninjured animals, confirming the inhibition of parasympathetic transmission to the heart (p<0.05). Conversely, atropine infusion in the T3-SCI animals did not significantly increase HR (p>0.05). A main effect of group was observed across all pressure-derived indices. T3-SCI animals before atropine infusion exhibited lower dP/dtmax, Pmax, and SBP, and MAP (all p<0.05). Following atropine infusion, the T3-SCI group retained reductions in all above pressure-derived indices, along with significantly lower DBP (all p<0.05). 37 Table 4.2. Baseline cardiac and arterial pressure recordings, pre- and post-atropine, in naïve and T3-SCI. Naïve T3-SCI p-Value Pre-Atropine Post-Atropine Pre-Atropine Post-Atropine Step Group Int. Cardiac Data dP/dtmax (mmHg/s) 7238 ± 548 8610 ± 557* 4931 ± 358 4786 ± 390 0.01 <0.001 0.02 Pmax (mmHg) 124 ± 2.9 127 ± 4.3 97.6 ± 4.7 95.7 ± 4 0.8 0.007 0.2 Hemodynamic Data SBP (mmHg) 128 ± 5.4 131 ± 2.9 93.8 ± 5.3 89.3 ± 3.8 0.8 <0.001 0.2 DBP (mmHg) 79.2 ± 6.8 87.2 ± 5.4 62.1 ± 4.5 60.3 ± 3.1 0.2 0.006 0.03 MAP (mmHg) 100. ± 6.6 106. ± 4.8 75.5 ± 4.8 72.6 ± 3.5 0.6 <0.001 0.07 HR (BPM) 328 ± 14 416 ± 9.7** 345 ± 16 374 ± 21 <0.001 0.7 0.004 Values are means ± standard error (se) for naïve (n=6) and T3-SCI (n=6) groups. The last column contains p-values for across steps, between groups, and the interaction between steps and groups, extracted from two-way repeated measures ANOVAs. Signficant post hoc pairwise within-group comparisons are indicated as *p<0.05 and **p<0.001. 38 4.1.2 Cardiovascular function after ganglionectomy Cardiac and hemodynamics indices are reported in Table 4.2, with outcomes illustrated in Figure 4.1. As detailed in the study outline in Figure 3.1, both groups underwent a step-wise denervation of the cardiac-projecting paravertebral ganglia. We compared the changes in cardiovascular indices from baseline, following SCGX, and following StGX. Individual two-way repeated measures ANOVAs were conducted for each parameter to assess effects of step and group variables. Main effects for step and group were observed across all pressure-derived indices (all p<0.05). Only a main effect for step was seen for HR. Additionally, there was an interaction effect between step and group for dP/dtmax, SBP, DPB, and MAP (all p<0.05). Post-hoc comparisons revealed that following StGX, dP/dtmax and SBP significantly decreased in naïve animals (both p<0.05), while no significant reductions were observed in T3-SCI animals (dP/dtmax p=0.3; SBP p=0.1). DBP and MAP were reduced in both naïve and T3-SCI groups following StGX (all p<0.05); however, significant interaction effects between step and group (both p<0.05) indicate that the reductions in these indices were of greater magnitude in the naïve group. Pmax was also observed to significantly reduce following StGX in both naïve and T3-SCI groups, with a greater reduction observed in the naïve group when comparing the absolute change from baseline (p<0.05).39 Table 4.3. Resting cardiac and hemodynamic indices at baseline and steps of denervation. Naïve T3-SCI p-Value Baseline SCGX StGX Baseline SCGX StGX Step Group Int. Cardiac Data dP/dtmax (mmHg/s) 8610 ± 557 9060 ± 381 6354 ± 400** 4786 ± 390 5306 ± 545 3839 ± 277 <0.001 <0.001 0.007 Pmax (mmHg) 127 ± 4.3 128 ± 3.1 107 ± 3.7** 95.7 ± 4 96.2 ± 5.0 83.1 ± 2.4* <0.001 <0.001 0.08 Hemodynamic Data SBP (mmHg) 131 ± 2.9 130 ± 3.8 103 ± 4.6** 89.3 ± 3.8 90.4 ± 3.7 78.2 ± 1.9 <0.001 <0.001 0.003 DBP (mmHg) 87.2 ± 5.4 88.4 ± 5.3 65.6 ± 4.8* 60.3 ± 3.1 55.6 ± 3.2 43.9 ± 2.9* <0.001 <0.001 0.04 MAP (mmHg) 106 ± 4.8 106 ± 4.8 82.1 ± 5** 72.6 ± 3.5 69.5 ± 3.5 56.6 ± 2.7* <0.001 <0.001 0.04 HR (BPM) 416 ± 9.7 430 ± 5.8 372 ± 8.5 374 ± 21 400 ± 19 359 ± 10 <0.001 0.09 0.4 Values are means ± se for naïve (n=6) and T3-SCI (n=6) groups. The last column contains p-values for across steps, between groups, and the interaction between steps and groups, extracted from two-way repeated measures ANOVAs. Signficant post hoc pairwise within-group comparisons are indicated as *p<0.05 and **p<0.001.40 Figure 4.1. Resting hemodynamic indices at baseline and after step-wise denervation of the superior cervical ganglion (SCGX) and stellate ganglion (StGX) for naïve (n=6) and T3-SCI (n=6) groups. A. Systolic blood pressure (SBP). B. Diastolic blood pressure (DBP). D. Mean arterial pressure (MAP). C. Heart Rate (HR). In the main plot, solid colours represent means ± se of the naïve and T3-SCI groups, with transparent colours for individual animals. Within-group comparisons of baseline vs. StGX are indicated in the main plot by *p<0.05 and **p<0.001. The inset plot represents means ± se of indices at baseline and after StGX. Between-group comparisons (naïve baseline vs. T3-SCI baseline) and across-group comparisons (naïve StGX vs T3-SCI baseline) are indicated in the inset plot by †p<0.05 and ǂp<0.001. 41 Figure 4.2. Resting cardiac indices at baseline and after step-wise denervation of the SCGX and StGX for naïve (n=6) and T3-SCI (n=6) groups. A. Maximal rate of rise of left-ventricular pressure waveform (dP/dtmax). B. Maximum left-ventricular pressure (Pmax). In the main plot, solid colours represent means ± se of the naïve and T3-SCI groups, with transparent colours for individual animals. Within-group comparisons of baseline vs. StGX are indicated in the main plot by *p<0.05 and **p<0.001. The inset plot represents means ± se of indices at baseline and after StGX. Between-group comparisons (naïve baseline vs. T3-SCI baseline) and across-group comparisons (naïve StGX vs T3-SCI baseline) are indicated in the inset plot by †p<0.05 and ǂp<0.001. 42 4.1.3 Comparative effects of sympathetic denervation and T3-SCI on cardiovascular function A direct comparison of cardiac and hemodynamic parameters between naïve animals post-StGX and T3-SCI animals at baseline revealed notable similarities. Both groups exhibited comparable dP/dtmax, Pmax, SBP, DPB, and MAP (all p>0.05), indicating that baseline cardiovascular function in animals with T3-SCI mimicked cardiovascular function in animals with sympathetic denervation of cardiac-projecting ganglia. 4.2 Neural atrophy and plasticity after ganglionectomy 4.2.1 Postganglionic signalling As a neural proxy, TH was used to measure overall signalling activity in the ganglia, measuring cell size in tandem with overall density of TH+ fluorescence. In Figure 4.2A, representative images of TH+ neuronal profiles in the SCG and StG are shown. The TH+ neuronal profiles were classified in 4 μm bins and plotted as proportional frequencies on a histogram plot in Figure 4.2B. A cumulative sum of proportions was also plotted in Figure 4.2C. Data from each ganglion pair (right and left sides) were combined for analyses. Separate K-S tests for the SCG and StG were conducted to identify differences in the naïve vs. T3-SCI neuronal profile cumulative frequencies. Comparing naïve vs. T3-SCI SCG neuronal profile distributions indicated no differences, suggesting that the naïve and T3-SCI populations had comparable size distributions (D=0.05 < critical value (0.07), p>0.05). Furthermore, comparisons of the naïve vs. T3-SCI StG distributions also showed 43 no significant differences in the size distributions of the naïve and T3-SCI populations (D=0.05 < critical value (0.07), p>0.05). The overall density of TH was assessed using threshold analyses. Percentage of area fluorescent for the TH+ label was measured per section of each ganglion. Independent Student’s t-tests were conducted to compare area fluorescence between naïve and T3-SCI animals for the SCG and StG. No signficant differences were found in TH density between naïve and T3-SCI groups for either the SCG (naïve 68.8 ± 3% vs. T3-SCI 70.9 ± 2%, p=0.7), or the StG (naïve 77.1 ± 3% vs. T3-SCI 73.4 ± 3%, p=0.4).44 Figure 4.3.Immunohistochemical analysis of tyrosine hydroxylase (TH) positive postganglionic neurons in naïve (n=4) and T3-SCI (n=4) rodents A. Representative images of ganglion sections for TH. B. Proportional frequencies of neuronal profile diameters (μm) categorized by 4μm bins. C. Cumulative sum of the proportions of neuronal profile diameters. 45 4.2.2 Cardiac-projecting neurons The same process of neuronal profile analyses described above in 4.2.1 was applied for NPY+/CD+ cells. The K-S test for the SCG indicated a statistically significant difference between the distributions of naïve vs. T3-SCI neuronal populations (D=0.1 > critical value (0.07), p<0.05). This suggests that the samples are drawn from different populations, in which the T3-SCI group had a greater frequency of larger diameter categories. For the StG, the K-S test revealed a significant difference in distribution of the naïve vs. T3-SCI neuronal profiles (D=0.1 > critical value (0.07), p<0.05), in which the T3-SCI population also had a higher frequency of larger diameter categories. 46 Figure 4.4. Immunohistochemical analysis of cardiac-projecting postganglionic neurons in naïve (n=5) and T3-SCI (n=5) rodents. A. Representative images of ganglion sections co-stained for NPY and calbindin. B. Proportional frequencies of neuronal profile diameters (μm) categorized by 4μm bins. C. Cumulative sum of the proportions of neuronal profile diameters. 5. Discussion In this thesis, we aimed to validate our chronic model of T3-SCI for studying cardiovascular deficits resulting from SCI and to demonstrate that these deficits are attributable to loss of supraspinal input to cardiac-projecting paravertebral ganglia. Our in vivo and anatomical studies yielded several significant outcomes: Firstly, our rodent model of T3-SCI exhibited marked attenuation in resting cardiovascular function in comparison to naïve rats. Secondly, we found no functional effect of SCG removal, however, ganglionectomy of the StG was found to lower cardiac and hemodynamic pressure-derived indices in naïve rodents. In T3-SCI animals, some of these indices were also observed to reduce following StG, albeit at a significantly lower magnitude. Comparisons between naïve animals with their StG removed and spinal cord-injured animals at baseline with intact ganglia were found to be remarkably similar. Finally, we found evidence of enlarged cell bodies of cardiac-projecting neurons occurring in both ganglia post-SCI. 5.1 In vivo physiology of sympathetic denervation 5.1.1 Validation of the T3-SCI model Recent studies from our laboratory have shown that resting cardiac and hemodynamic function are reduced in our T3-SCI contusive and transection models, reflective of cardiovascular impairments seen in the clinical population at high-level injuries.9,65,67,70 Our study mirrors those findings, in which all cardiac and hemodynamic 48 pressure-derived indices recorded in our T3-SCI group were diminished, even after parasympathetic innervation was blocked. As previously reported, HR remained unchanged in the chronic T3-SCI model. Notably, rats with a T3-SCI did not respond to the parasympathetic blockade to the same degree as the naïve group, suggesting that parasympathetic withdrawal may be occurring to balance the reduced sympathetic tone.85 5.1.2 Superior cervical ganglionectomy has no direct effects on cardiovascular function Resting cardiac and hemodynamic function did not appear to change after SCGX in naïve or T3-SCI animals. Prior retrograde tracing and anatomical studies have validated that the SCG exhibits physical connections to the myocardium across many species, but have not elaborated on the function of these pathways.44,55,86 Specifically, retrograde tracing from the heart to paravertebral ganglia has shown that the SCG contributes to sympathetic innervation on the anterior side of the myocardium in a small area, in comparison to the StG which densely innervates across both anterior and posterior aspects.55 Additionally, the SCG is known to possess innervation to the coronary arteries.44 Whilst the StG is widely accepted to functionally regulate the LV, the potential role of the SCG has been heavily debated.51,55 A systematic review by Chen et al. highlighted that functional studies provided indirect evidence that the SCG can modulate cardiac function, mostly by reporting altered structure of the SCG in chronic disease states which are associated with altered sympathetic signalling, such as post-myocardial infarction.51 The change in SCG structure 49 in states of altered sympathetic function suggest that there are compensatory mechanisms that upregulate signalling through the SCG to increase sympatho-excitation to the heart. It is, however, noteworthy that the authors did not report any studies that examined cardiovascular function immediately following SCGX, or after a loss in supraspinal input. To our knowledge, this current study is the first to record cardiovascular function in real-time after SCGX in rodents. As we found no differences in any cardiovascular indices following SCGX in healthy and T3-SCI rodents, this implies that the SCG play no role in regulating short-term cardiac function. There were also no changes in cardiovascular function amongst spinal cord-injured rodents after SCGX, suggesting that no functional remodeling occurred within the SCG to compensate for the loss of sympathetic innervation, despite seeing morphological changes to its cardiac-projecting neurons. 5.1.3 Stellate ganglionectomy reduces resting cardiovascular function As we observed no changes in cardiac indices following SCGX, it is probable that the intact StG alone is sufficient to drive sympathetic output. This assumption is in agreement with previous studies that have demonstrated cardiac modulation by the StG. Specifically, electrical stimulation of the StG has been demonstrated to increase both cardiac contractility and blood pressure, whereas removal of the StG has the opposite effect, decreasing these parameters.60,61,87,88 After StG removal in naïve animals in our study, all cardiac and hemodynamic pressure-derived indices were diminished. Indicated by the main effect on cardiac measures dP/dtmax and Pmax, the observed reduction in LV systolic function can be 50 attributed to the loss of input to the LV from cardiac sympathetic postganglionic neurons originating in the StG, a finding consistent with reports by Buckley et al. and others.60,60,87,88 In anaesthetized pigs, Buckley et al. demonstrated decentralized cardiac sympathetic control following partial denervation of the StG.88 Stimulation of the StG with projections to the myocardium elicits positive inotropic effects, enhancing LV contractile force.61,89 Decreases in cardiac pressures seen following StG removal indicate a disruption in key sympathetic pathways responsible for maintaining the heart's contractility through SNS contributions. Peripheral blood pressures SBP, DBP, and MAP were also seen to reduce following StGX in naïve animals. Similarly, Wu and colleagues found a marked decrease in blood pressure after removing the StG in dogs.61 This finding implies that, since major sympathetic pathways leading to peripheral vasculature remained intact, this decrease in blood pressure is likely due to a reduction in CO. Blood pressure is a product of peripheral resistance and CO; by severing the connections of StG to the heart, the cardiac arm contributing to blood pressure is impeded. The loss of sympathetically-mediated inotropic effects on the heart results in a reduction in CO. This would consequently result in decreased blood flow, leading to a sustained drop in blood pressure.90 Arguably, sympathetic responses in peripheral vasculature may compensate for this loss through vasoconstriction to restore homeostasis, however, it is possible that this response would not be sufficient to maintain resting blood pressure against the reduced function of the heart. Following StGX in spinal cord-injured animals, there was a reduction in Pmax, DBP, and MAP; however, these changes were not as pronounced as the reductions in cardiac 51 function observed in the naïve group after StGX. Additionally, dP/dtmax and SBP remained unchanged in the T3-SCI group. Reductions in select indices imply that there may be some sympathetic signals are still reaching the StG in our T3-SCI group. This could be from spared SPNs projecting from the T1 segment or plasticity of pathways from the sub-lesional region.19 Nevertheless, our findings strongly suggest that T3-SCI effectively disrupts sympathetic pathways through the StG that is required to maintain normal resting cardiovascular function. One parameter of note that remained unchanged in both groups after StGX was HR. Previous studies have implicated outflow from the StG in control of eliciting positive chronotropic effects on the heart.55,60,91 In a recent study by Rajendran et al., innervation of the SA node by the StG was identified through retrograde tracing and optogenetic stimulation of the StG that elicited increases in HR.91 Despite a positive dose-response shown in HR with increased stimulation strength, the change between resting and sympatho-excited states showed no significant increases to HR, even at the highest stimulation strength. Moreover, studies by Wu et al. and Ajijola et al. have demonstrated statistically insignificant increases in HR accompanied by significantly increased blood pressure during StG stimulation in dogs and humans, respectively.61,92 In addition, Wu et al. reported no changes to HR following StGX in their animals. The statistical insignificance of a StG-mediated HR response alongside significant changes in blood pressure are reflected in our findings as well. The naïve group displayed an 8.4% decrease in HR after StGX that showed no statistical significance, in comparison to the T3-SCI group which showed a 1.4% decrease in HR. 52 5.1.4 Stellate ganglionectomy mimics the left-ventricular function of T3-SCI Our laboratory and others have previously suggested that T3-SCI reduces sympathetic control of the LV, which in turn reduces pressure-generating capacity of the heart. Results from the present study directly support this postulate as all cardiac and hemodynamic pressures were reduced to a greater degree following StGX in naïve animals, in comparison to the smaller change demonstrated in T3-SCI animals. In fact, these indices in the naïve group following StGX demonstrated no statistical difference to those obtained in the T3-SCI group at their baseline. This suggests that SCI-induced loss of supraspinal input effectively attenuates cardiac sympathetic control through the StG. 5.2 Neural plasticity in paravertebral ganglia after SCI 5.2.1 Cardiac-projecting ganglia undergo plasticity in SCI We hypothesized that postganglionic neurons projecting to the heart would undergo structural changes due to the loss of efferent signalling to the SCG and StG. When comparing neuronal profile diameter sizes, the T3-SCI group showed a higher frequency of larger NPY+/CD+ neuronal profile diameters in both ganglia, indicating potential somatic enlargement occuring post-injury. In the literature, there appears to be no consensus on the mechanism and function of somatic enlargement in the paravertebral ganglia. Outside of SCI, structural changes to the StG following cardiovascular events has been documented. Ajijola et al. observed increased cell body sizes in neurons of the StG from patients post-myocardial infarction and non-ischemic cardiomyopathy.93 They propose two distinct explanations for cell growth: in cases of 53 myocardial infarction, axonal damage to postganglionic neurons triggers neurotrophic signalling, leading to the release of trophic factors that promote cell size expansion and repair damaged connections; whereas in cardiomyopathy, chronic sympathetic hyperactivation results in increased neuronal size, which may blunt transmission efficiency. This latter explanation is supported by Singh et al. and Peruzzi et al., whom explain that increased cell body size in the StG causes lower excitability to account for sympathetic hyperactivation in the heart.94,95 Moreover, rationale proposed by Fioretto et al. suggests that the enlargement of postganglionic neuronal soma supports neural plasticity in response to the loss of efferent signalling to the ganglia.96 Their stereological study of unilateral SCG ganglionectomy in sheep revealed that the remaining ganglion responded to the disruption in efferent signalling by enlarging the neuronal cell bodies. They suggest that this is a compensatory mechanism to enhance sensitivity of remaining postganglionic neurons and increase the strength of their output. This compensatory mechanism may also account for our findings, as the ganglia could be enhancing sensitivity in the pathways that continue to receive innervation from the spinal cord after T3-SCI. Despite the increase in cell size observed in both ganglia following SCI, there were no effective functional outcomes, as evidenced by the low resting cardiovascular function in the T3-SCI group and their minimal responses after the removal of the SCG or StG. 54 5.3 Relevance and Future Directions 5.3.1 Implications Our findings contribute the overall understanding of structures that control cardiovascular function and the neurological consequences of SCI. This is the first study to examine the impact of SCI on paravertebral ganglia that innervate the heart, providing novel insights to this field. Additionally, these outcomes also validate our laboratory’s T3-SCI model as a sufficient pre-clinical model for studying cardiovascular deficits in SCI. 5.3.2 Recording volume-dependent indices and challenges to cardiovascular sympathetic regulation Our in vivo physiology study measured resting cardiovascular indices through left-ventricular and femoral arterial pressure catheters. Volume-based indices were not evaluated due to limited resources. Future research that pairs cardiac volume indices with our assessments of load-dependent pressure indices would provide a fuller picture into the physiology of ganglionic control of the heart. Challenges to elicit maximal sympatho-excitation such as orthostatic hypotension would also add to the picture, assessing the maximal of cardiovascular sympathetic function following sympathetic ganglionectomy in uninjured and SCI conditions. 5.3.3 Sex-related differences in cardiac sympathetic modulation In a recent study by Jia et al, findings from a national community health survey indicated that females with SCI had a lower prevalence of cardiovascular diseases compared to males with SCI.97 Sex-related differences in cardiovascular function have been documented in the literature, pertaining to structure of the heart as well as 55 autonomic control.98,99 In a study by Dart et al., they observed a difference in resting cardiac sympathetic activity. It stands to reason that repeating our study in female rats to assess sex-related differences in sympathetic ganglionic modulation of the heart would be warranted, as this has not been previously explored. 5.3.4 Whole-mount staining of ganglia after chronic injury As this study utilized a representative postganglionic population distribution to analyze differences, it would be beneficial to conduct morphological analyses in whole-mount staining. Described by Rajendran et al., whole-mount staining of the stellate would allow for 3D visualization of cardiac innervation projected from the paravertebral ganglia.91 This would also allow for volume-based assessments of the ganglia, to identify whole-structure changes that may occur following SCI. 5.3.5 The sympathetic chain as a therapeutic target Within the SCI field, spinal cord stimulation has become a prominent topic of interest for improving a variety of motor and autonomic functions. Recent studies have recommended methods such as epidural stimulation as a possible intervention to improve cardiovascular dysfunction.100–102 The mechanisms of long-term cardiovascular improvement following electrical stimulation of the spinal cord has not yet been elucidated; however, one of several theories suggests that the restoration of sympathetically-induced vasoconstriction causes activation of sensory afferents, leading to a re-conditioning of the baroreflex.103,104 Furthermore, sympathetic activation could also be working to improve vascular tone and correct maladaptive remodelling of the cardiovascular system. 56 Under the same reasoning, electrical stimulation of the sympathetic chain may be a possible therapeutic target for activating sympathetic pathways directly to the heart and re-condition autonomic regulation. Targeting the StG would provide means to bypass unsalvageable cardiac-projecting efferent pathways in the spinal cord and elicit sympathetic-activation of the cardiovascular system. This approach could serve as an alternative method to improve vascular tone, while also mitigating SCI-induced systolic dysfunction and cardiomyocyte atrophy. 5.4 Considerations 5.4.1 Strengths The use of an in vivo model allowed us to assess pathophysiology of SCI through an invasive approach that would not be feasible with human participants. This methodology provides a controlled environment where variables can be precisely manipulated and observed, in real-time. In tandem with the functional outcomes, the detailed anatomical assessments of the ganglia strengthen the conclusions of this thesis. Through this dual approach, we provide a comprehensive understanding of the impact of SCI on the cardiac-projecting paravertebral ganglia. 5.4.2 Limitations Limitations in this thesis include the surgical preparation of our in vivo study. The use of urethane anaesthesia, as well as the requirement to open the chest to excise the StG, reduce the validity of studying real-world cardiovascular physiology of our animals. However, the outcomes we measure are based on the differences between groups, or 57 across steps, under the same conditions. This approach allows for comparisons to be made without compromising the validity of our findings. Additionally, the order of ganglionectomies was not randomized as the SCG were always removed first. As a result, SCG function was never isolated. This was due to the innervation pathways of the paravertebral ganglia, where preganglionic input to the SCG required axons to travel up the sympathetic chain in order to reach it. Therefore, removing the StG first would denervate the SCG as well. Another limitation in this study was the inability to completely remove the StG in a few animals, due to the malformation of ribcages observed in the T3-SCI rodents. In some instances, the StG was near impossible to remove without damaging the ribcage or surrounding vasculature. Therefore, lidocaine was locally administered onto the ganglia to block transmission instead, and excised for histological preparation post-mortem. Using a pharmacological block on the ganglia was validated prior to the study, in which is was shown to demonstrate the same effects as excision of the ganglia in a healthy animal. Lastly, a limitation of this study was in regards to conclusions made about cell morphology without reference to the overall cell population in each ganglion. Proportional frequency was compared through the population of cells that were positive for the markers indicative of cell activity, not including neurons that may be inactive or perhaps not positive for the select markers. One of our markers of interest was TH, a rate-limiting enzyme used in catecholamine synthesis. This was used specifically to identify postganglionic adrenergic signalling occuring in a ganglion, however, without a pan-neuronal stain, this introduces bias in making assumptions about entire cell populations within the ganglion. Nevertheless, comparing the naïve and T3-SCI populations offers valuable insights into 58 the anatomical pathology associated with SCI. Further research is needed to characterize overall cell populations and investigate accurate comparisons made for the topography of neurons in rodent SCG and StG. 59 6. Conclusion The aim of this thesis was to identify the effects of high-thoracic SCI on ganglionic control of the heart. Functional and anatomical assessments of the influence of the SCG and StG on cardiac and hemodynamic function in a T3-SCI model of injury were assessed. We report decreased resting cardiac and hemodynamic function following stellate, but not superior cervical, ganglionectomy in naïve rats. This negative effect to cardiovascular function was minimal in T3-SCI rats, validating that supraspinal descending input to the heart is severely impacted after high-thoracic SCI. In our anatomical study, the morphology of cardiac-projecting postganglionic neurons and overall signalling activity were explored. It was found that cardiac-projecting neurons of both ganglia display enlarged cell body sizes in chronic SCI, while density of TH+ cells in the ganglia remained unchanged. To date, this study is the first to investigate cardiac-projecting ganglia following SCI. 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Thesis/Dissertation
eng
Experimental Medicine
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
West, Christopher R.
The impact of spinal cord injury on sympathetic ganglionic control of the heart
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