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Cardiovascular and metabolic function after thoracic spinal cord injury Inskip, Jessica Ann 2010

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  CARDIOVASCULAR AND METABOLIC FUNCTION AFTER THORACIC SPINAL CORD INJURY   by   Jessica Ann Inskip   B.Sc., The University of British Columbia, 2006     A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF   MASTER OF SCIENCE   in   The Faculty of Graduate Studies   (Zoology)     THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   April 2010     © Jessica Ann Inskip, 2010   ii ABSTRACT Spinal cord injury (SCI) has the potential to disrupt autonomic pathways in the spinal cord leading to a range of autonomic dysfunctions.  The cardiovascular (CV) and metabolic sequelae can restrict the lives of individuals with SCI and contribute to the deterioration of their cardiometabolic health. Here I investigated the whole-body CV and metabolic ramifications of experimental SCI in rats.  Complete thoracic SCI was performed at two different levels in order to determine whether these outcomes demonstrated a level dependence.  High-(T3) and low- (T10) thoracic SCI both result in flaccid hindlimb paralysis, but have different effects on the level of supraspinal autonomic control.  CV and metabolic function were assessed at several times post-injury to investigate changes over time. Animals with acute high-thoracic SCI displayed resting hypotension that resolved with time post-injury.  However, their capacity to control blood pressure (BP) in response to physiological stimuli remained deficient; animals with high-thoracic SCI displayed pronounced orthostatic hypotension (OH) and severe episodes of sensory stimulation- induced hypertension known as autonomic dysreflexia (AD).  The resting BP and heart rate of animals with low-thoracic SCI, and their ability to respond to orthostatic stress, was indistinguishable from sham controls. Lipid metabolism was also disordered by SCI in a level-dependent pattern.  Animals with high-thoracic SCI carried increased white adipose tissue and had higher circulating triacylglycerol levels compared to animals with low-thoracic SCI and sham controls. However, there was no difference in the distribution of cholesterol-carrying lipoproteins.   iii Carbohydrate metabolism in animals with SCI did not support the diabetic profile suggested by the lipid results.  Overall, animals with SCI were more sensitive to glucose and insulin than sham-injured animals.  The pronounced ketone response to fasting in animals with high-thoracic SCI suggests that there are diverse effects on substrate metabolism. This work introduces simple tests that can be performed to investigate several important and understudied autonomic outcomes of SCI.  The results reveal the importance of the intact autonomic nervous system in regulating CV and metabolic function.  The disparity between motor and autonomic function encourages modifying our current conventions so that we stratify subjects by their autonomic injury level and their motor deficits.   iv  TABLE OF CONTENTS  Abstract .........................................................................................................................ii Table of Contents ......................................................................................................... iv List of Tables ................................................................................................................vi List of Figures..............................................................................................................vii List of Abbreviations..................................................................................................viii Acknowledgements....................................................................................................... ix Dedication...................................................................................................................... x Co-Authorship Statement ............................................................................................xi 1 Introduction............................................................................................................. 1 1.1 Introduction................................................................................................... 1 1.2 General anatomy of the autonomic nervous system ....................................... 2 1.3 The autonomic nervous system and baroreflex cardiovascular control ........... 4 1.4 Cardiovascular sequelae of SCI..................................................................... 6 1.4.1 Neurogenic shock.............................................................................. 8 1.4.2 Orthostatic hypotension..................................................................... 9 1.4.3 Autonomic dysreflexia .................................................................... 11 1.4.4 Cardiovascular circadian rhythms.................................................... 14 1.5 Energy metabolism...................................................................................... 15 1.5.1 Glucose metabolism and the autonomic nervous system.................. 15 1.5.2 Lipid metabolism and the autonomic nervous system ...................... 16 1.5.3 Circadian metabolic rhythms........................................................... 18 1.6 Metabolic sequelae of SCI........................................................................... 19 1.6.1 Body composition ........................................................................... 20 1.6.2 Energy balance................................................................................ 21 1.6.3 Substrate metabolism ...................................................................... 22 1.6.4 Circadian metabolic rhythms following SCI.................................... 23 1.7 Blood sugar control following SCI .............................................................. 24 1.8 Experimental objectives .............................................................................. 26 1.9 References .................................................................................................. 28 2 Cardiovascular responses to orthostatic stress, colorectal distension and sexual stimulation in rats with spinal cord injury................................................................. 44 2.1 Introduction................................................................................................. 44 2.2 Materials and methods................................................................................. 48 2.2.1 Surgeries......................................................................................... 48 2.2.2 Physiological testing ....................................................................... 50 2.2.3 Statistical analysis ........................................................................... 52 2.3 Results ........................................................................................................ 53 2.4 Discussion................................................................................................... 72 2.5 References .................................................................................................. 79 3 Cardiometabolic risk factors in experimental spinal cord injury ....................... 84 3.1 Introduction................................................................................................. 84 3.2 Materials and methods................................................................................. 87 3.2.1 Surgery and animal care.................................................................. 87   v 3.2.2 Magnetic resonance imaging ........................................................... 90 3.2.3 MRI post-processing and image analysis......................................... 91 3.2.4 Visceral white adipose tissue dissection .......................................... 91 3.2.5 Blood lipid profiling........................................................................ 92 3.2.6 Statistical analysis ........................................................................... 94 3.3 Results ........................................................................................................ 95 3.4 Discussion................................................................................................. 109 3.5 References ................................................................................................ 116 4 Energy expenditure, circadian rhythms, and carbohydrate metabolism after thoracic Spinal cord injury....................................................................................... 123 4.1 Introduction............................................................................................... 123 4.2 Materials and methods............................................................................... 125 4.2.1 Surgery and animal care................................................................ 126 4.2.2 Telemetry...................................................................................... 127 4.2.3 Housing ........................................................................................ 129 4.2.4 Calorie consumption ..................................................................... 129 4.2.5 Blood sampling ............................................................................. 130 4.2.6 Glucose tolerance test.................................................................... 130 4.2.7 Insulin tolerance test ..................................................................... 131 4.2.8 Circadian glucose.......................................................................... 131 4.2.9 Data analysis................................................................................. 131 4.3 Results ...................................................................................................... 132 4.3.1 Calorie consumption and body weight........................................... 132 4.3.2 Core temperature and circadian temperature rhythms .................... 132 4.3.3 Circadian activity patterns............................................................. 137 4.3.4 Total daily activity ........................................................................ 140 4.3.5 Circadian blood glucose ................................................................ 143 4.3.6 Glucose sensitivity ........................................................................ 143 4.3.7 Insulin-induced hypoglycemia....................................................... 148 4.3.8 Fasting and non-fasting blood glucose........................................... 151 4.3.9 Fasting and non-fasting blood ketones........................................... 154 4.4 Discussion................................................................................................. 154 4.5 References ................................................................................................ 163 5 Discussion ............................................................................................................ 167 5.1 Cardiovascular function after experimental SCI ........................................ 168 5.2 Recommendations for future cardiovascular research in experimental SCI 170 5.3 Metabolic function after experimental SCI ................................................ 171 5.4 Recommendations for future metabolic research using this model ............. 175 5.5 Metabolic implications for future experimental SCI research..................... 177 5.6 Insights and limitations of experimental SCI research................................ 178 5.7 Concluding remarks .................................................................................. 182 5.8 References ................................................................................................ 184 Appendix: Animal care certificate............................................................................ 189     vi LIST OF TABLES  Table 2.1 Resting cardiovascular variables were similar across injury levels and time post-injury. ............................................................................................................ 55 Table 2.2 Blood pressure and heart rate responses to colorectal distension were equally pronounced at one month and three months following T3 SCI. .............................. 65 Table 2.3 Sexual stimulation elicited hypertension and tachycardia in animals with T3 SCI........................................................................................................................ 71 Table 3.1 Non-fasting serum lipid concentrations in high-thoracic, low-thoracic and sham-injured rats reveal that rats with high-thoracic SCI have elevated triglyceride levels one month post-injury................................................................................ 105 Table 3.2 Non-fasting blood lipids measured using a home cholesterol test system showed similar results to laboratory quantification. ............................................. 108 Table 4.1 Animals with SCI experienced mild hypoglycemia in response to a 12-hour fast at one month post-injury, but not at three months post-injury............................... 153   vii LIST OF FIGURES  Figure 2.1 Orthostatic hypotension was present after high- but not low-thoracic transection, and was more severe acutely after injury............................................. 57 Figure 2.2 Animals exhibited tachycardia in response to an acute orthostatic stress. ...... 60 Figure 2.3 Colorectal distension elicited hypertension and bradycardia in animals with high-thoracic SCI, while animals with low-thoracic SCI experienced milder hypertension accompanied by tachycardia ............................................................. 62 Figure 2.4 Colorectal distension elicited pronounced hypertension and bradycardia in animals with T3 SCI at one and three months post-injury. ..................................... 67 Figure 2.5 Representative arterial blood pressure recording of an animal with T3 SCI undergoing penile sheath retraction one month post-injury. ................................... 69 Figure 3.1 Rats with high-thoracic SCI and those with low-thoracic SCI recover to their pre-operative weights shortly after injury............................................................... 97 Figure 3.2 Rats with high-thoracic SCI carried increased visceral and subcutaneous fat one month post-injury............................................................................................ 99 Figure 3.3 Visceral white adipose tissue dissection and wet weight measurement was sensitive to changes after SCI. ............................................................................. 102 Figure 4.1 Calorie consumption and body weight throughout the 12-week experiment.134 Figure 4.2 Core temperature was decreased by T3 SCI but was normalized by two weeks post-injury. .......................................................................................................... 136 Figure 4.3 Nocturnal activity patterns were not disrupted by high- or low-thoracic SCI. ............................................................................................................................ 139 Figure 4.4 Average daily activity was reduced by both high- and low-thoracic SCI acutely after injury............................................................................................... 142 Figure 4.5 Circadian blood glucose variations were not disrupted by high- or low-thoracic SCI...................................................................................................................... 145 Figure 4.6 Animals with high- and low-thoracic SCI displayed improved glucose tolerance compared to sham-injured animals........................................................ 147 Figure 4.7 Animals with T3 SCI and animals with T10 SCI displayed rapid insulin- induced hypoglycemia at one and three months post-injury, respectively............. 150 Figure 4.8 Animals with high-thoracic SCI exhibit a greater ketone response to fasting. ............................................................................................................................ 156   viii LIST OF ABBREVIATIONS AD, autonomic dysreflexia ATP, adenosine triphosphate ANS, autonomic nervous system AUC, area under the curve BP, blood pressure BMI, body mass index bpm, beats per minute CHOL, cholesterol CT, computed tomography CRD, colorectal distension CV, cardiovascular CVLM, caudal ventrolateral medulla CVD, cardiovascular disease DAP, diastolic arterial pressure DEXA, dual energy X-ray absorptiometry E, epinephrine GTT, glucose tolerance test HDL-C, high-density lipoprotein cholesterol HR, heart rate HSL, hormone sensitive lipase HUT, head-up tilt IML, intermediolateral ITT, insulin tolerance test KITT, rate constant for the disappearance of glucose LDL-C, low-density lipoprotein cholesterol LPL, lipoprotein lipase MAP, mean arterial pressure MRI, magnetic resonance imaging NE, norepinephrine NEFA, non-esterified fatty acid NTS, nucleus of the solitary tract OH, orthostatic hypotension PNS, parasympathetic nervous system SAP, systolic arterial pressure SCI, spinal cord injury SCN, suprachiasmatic nucleus scWAT, subcutaneous white adipose tissue SEM, standard error of the mean SNS, sympathetic nervous system SPN, sympathetic preganglionic neuron T3, third thoracic level of the spinal cord T10, tenth thoracic level of the spinal cord vWAT, visceral white adipose tissue WAT, white adipose tissue   ix ACKNOWLEDGEMENTS I am much obliged to all of the people that helped make this thesis possible.  First and foremost to my supervisors, Dr. Matt Ramer and Dr. Andrei Krassioukov for demonstrating the curiosity and tenacity necessary of good scientists, for providing guidance where appropriate and for giving me the freedom to flounder a bit on my own. I would also like to thank Dr. Bill Milsom for serving on my committee and for providing helpful comments on my thesis. Thanks also to all the members of the Ramer and Krassioukov labs: especially to Leanne for sharing her brain with me over the past few years, for demonstrating such high standards in writing, teaching and presenting, and driving me to hockey games; to Lesley for leading by example and prodding me along when needed; to Andrew for keeping morale high and sharing his excitement about teaching; to Ward for encouraging experiments and sharing his fascination in science and human nature; and to Byron and Nima for their many hours of thankless animal care and technical assistance. Finally, thanks to Chad for putting up with the inevitable self-doubt and other unbecoming attributes that emerge in stressful times.   x DEDICATION    To my family   xi CO-AUTHORSHIP STATEMENT The work presented in Chapters 2, 3 and 4 were prepared with contributions from members of Dr. Andrei Krassioukov’s laboratory, Dr. Matt Ramer and Dr. Ward Plunet. In Chapter 2, I contributed to the identification and development of the experimental question and design.  Leanne M. Ramer and I performed the surgical procedures and animal care; together we collected the data.  I performed the data analysis and manuscript preparation.  I am grateful to Leanne Ramer, Dr. Krassioukov and Dr. Ramer, who all provided helpful suggestions on the manuscript. The design and development of the work in Chapter 3 was a collaborative effort between Leanne Ramer, Dr. Plunet and I.  Technical assistance in data acquisition using the MRI was provided by Andrew Yung and Dr. Piotr Kozlowski.  Post-processing was also performed by Andrew Yung and Dr. Kozlowski.  Image analysis was performed by John Byron Ramsey, Leanne Ramer and myself.  Dr. Plunet and Leanne Ramer assisted me with data collection.  I performed the data analysis and manuscript preparation in collaboration with Leanne Ramer, Dr. Plunet, Dr. Ramer and Dr. Krassioukov. I designed the experiments presented in Chapter 4 with support and collaboration from Dr. Plunet and Leanne Ramer.  Leanne Ramer and I performed all of the surgical procedures. I performed the data collection with assistance from Dr. Plunet and John Byron Ramsey.  I was responsible for the data analysis and manuscript preparation, with helpful suggestions from both Dr. Plunet and Leanne Ramer.     1 1 INTRODUCTION 1.1 Introduction Spinal cord injury (SCI) carries a significant social cost in health care and a tremendous impact on individual quality of life.  The incidence of traumatic SCI in Canada, which accounts for approximately 81% of all SCIs (Hitzig et al., 2008), is between 42.4 and 52.5 per million (Dryden et al., 2003; Pickett et al., 2006).  While the majority of injuries occur in those under 35 years old, our aging population has created a bimodal distribution, with peaks at both 30 and 60 years old (Dryden et al., 2003; Eng and Miller, 2008; Kattail et al., 2009). Individuals injured today can expect significantly improved acute care and management of secondary complications compared to the past three decades (Strauss et al., 2006). But, while there are far fewer deaths in the acute setting, lifelong health remains a clinical concern; cardiovascular disease (CVD) is common and cardiovascular (CV) mortality is high (Garshick et al., 2005).  CVD strikes earlier in this population and life expectancy is decreased compared to the general population (Strauss et al., 2006). The past few decades have seen a greater appreciation for the diverse range of dysfunctions that can accompany SCI in addition to paralysis (Anderson, 2004; Anderson, 2006; Alexander et al., 2009a; Alexander et al., 2009b).  SCI can disrupt motor, sensory, and autonomic pathways traveling rostrally and caudally within the spinal cord.  Autonomic dysfunctions have a significant impact on quality of life and functional independence and they have been identified as a research priority by individuals with SCI (Anderson, 2004).  However, in general, our animal models are overwhelmingly focused on motor outcomes after SCI (Inskip et al., 2009).  Despite the   2 clinical significance of abnormal CV and metabolic function following SCI, the characteristics and pathophysiology are poorly understood. 1.2 General anatomy of the autonomic nervous system In order to understand the autonomic sequelae of SCI it is important to appreciate the structure and function of the intact autonomic nervous system (ANS).  The ANS maintains the body’s internal homeostasis; a task that it carries out via the regulation of visceral reflexes, cardiac and smooth muscle activity and glandular secretions. Anatomically, with the exception of the adrenal medulla, information is relayed to effector organs via ganglia, resulting in diffuse action compared to the somatic system. The ANS is composed of three branches: the two main branches are the sympathetic nervous system (SNS) and parasympathetic nervous system (PNS), and the third is the enteric nervous system (Langley, 1921).  For the most part, the PNS and SNS innervate the same targets, but the functions that they regulate are often opposite in nature; a stable internal environment is maintained by a balance of activity in both systems. Both the SNS and PNS reach their effector targets by first synapsing in a peripheral ganglion; the first neuron in this chain is termed preganglionic, the second ganglionic. Sympathetic preganglionic neurons (SPNs) reside in the thoracolumbar spinal cord and the majority are found in small clusters in the lateral horn of the grey matter in the intermediolateral (IML) cell column (Henry and Calaresu, 1972; Pyner and Coote, 1994). SPN axons exit the spinal cord via the ventral roots and travel via the white ramus and synapse in sympathetic paravertebral ganglia, which lie close to the cord, or travel further, through paravertebral ganglia, to synapse in prevertebral ganglia (Jänig, 2006). Parasympathetic preganglionic neurons (PPNs) reside in the nucleus ambiguus of the   3 brainstem and in the sacral cord, in the sacral parasympathetic nucleus in the lateral horn (Banrezes et al., 2002).  Unlike SPNs, PPNs synapse in local ganglia, usually in close proximity to their final target (reviewed in Brading, 1999). The autonomic control of the CV system is carried out through its innervation of the heart and blood vessels.  The heart is innervated by sympathetic ganglionic neurons in the upper thoracic paravertebral sympathetic ganglia, whose SPNs originate in the upper thoracic cord (T1-T4) (Strack et al, 1988; reviewed in Jänig, 2006).  Sympathetic ganglionic nerves terminate in both the atria and ventricles of the heart (reviewed in Levick, 2003).  Vagal parasympathetic preganglionic neurons exit the brainstem and innervate local cardiac ganglia in the atrial nodes and atria itself (reviewed in Levick, 2003).  Increased sympathetic activity results in tachycardia, while increased parasympathetic activity results in bradycardia (reviewed in Levick, 2003). Peripheral vasomotor pathways control vascular tone and ultimately total peripheral resistance.  This role is carried out primarily via sympathetic vasoconstrictor pathways and adrenergic innervation. With the exception of erectile tissue and intracranial vessels, for the most part, the PNS does not innervate blood vessels (reviewed in Jänig, 2006). The dense sympathetic adrenergic innervation of the splanchnic vascular bed is particularly important for BP control (Cowen et al., 1982; Rowell, 1990).  The spinal outflow to this vasculature bed originates in T5-L2 (reviewed in Jänig, 2006); injuries above this level often result in disordered CV control. The majority of the abdominal visceral organs are innervated by both branches of the ANS (Yamaguchi, 1992).  The sympathetic ganglionic neurons that innervate the visceral organs originate primarily in the prevertebral celiac ganglion and superior mesenteric   4 ganglion, their preganglionic inputs originating in the lower thoracic cord (T5-T12) (Kiba, 2004; Uyama et al., 2004).  Vagal parasympathetic neurons innervate local ganglia in visceral organs, to the level of the transverse colon, and sacral parasympathetic neurons innervate the distal colon and pelvic organs (reviewed in Jänig, 2006).  The liver and pancreas are both richly innervated by the PNS and SNS (Yamaguchi, 1992).  Unlike most visceral organs, adipose tissue only receives sympathetic innervation (Youngstrom and Bartness, 1995; Giordano et al., 2006).  There, nerve endings are found both in the vasculature and in the parenchyma (Youngstrom and Bartness, 1995). 1.3 The autonomic nervous system and baroreflex cardiovascular control The regulation of BP is arguably one of the most critical functions of the ANS.  It must be able to detect deviations in BP and respond to maintain stable BP (Cowley, 1992). One of the ways that this is achieved is by the reflex regulation of SNS and PNS activity, resulting in altered vasomotor tone and cardiac output (Fisher et al., 2006; Guyenet, 2006). The arterial baroreceptor-mediated reflex (baroreflex) is one of the most important and well-studied means of short-term BP regulation (Dampney et al., 2002).  The successful execution of this reflex requires the coordination of afferent, central and efferent components. The afferent arm of the baroreflex begins with the baroreceptors, stretch-sensitive mechanoreceptors located in the arterial wall of the aortic arch and carotid sinus (Bock and Gorgas, 1976).  Baroreceptors respond to both the degree and speed of arterial pressure change between 50 and 150 mmHg (reviewed in Kirchheim, 1976).  The sensory afferents that contain baroreceptors in the aortic arch have their cell bodies in the nodose ganglion and those from the carotid sinus lie in the petrosal ganglion (Kirchheim, 1976).   5 Their central axons join the vagus or glossopharyngeal nerve, enter the brainstem, and project bilaterally to the nucleus of the solitary tract (NTS) (reviewed in Jänig, 2006). In the brainstem, second-order neurons travel from the NTS to the ipsilateral caudal ventrolateral medulla (CVLM) (Chalmers et al., 1991).  The rostral ventrolateral medulla then integrates signals from both the NTS and the CVLM (Ross et al., 1984) and sends descending projections to vasomotor and cardiomotor SPNs in the IML cell column of the thoracolumbar cord (Amendt et al., 1979). Parasympathetic cardiomotor information is relayed from the NTS to preganglionic vagal neurons in the ipsilateral nucleus ambiguus (reviewed in Jänig, 2006). The vasomotor effects of the baroreflex are carried out by the sympathetic innervation and control of resistance arteries, while the cardiac component of the baroreflex is effected by the reciprocal actions of both branches of the ANS.  The bradycardic response to hypertension is predominated by the increase in vagal activity (Leon et al., 1970; Pickering et al., 1972).  The tachycardic response to hypotension is effected by a combination of withdrawal of vagal tone and increase in sympathetic activity, with the former acting rapidly and the latter more slowly (Chen et al., 1982; Parlow et al., 1995). The rapid vagal baroreflex response is also seen in its reflex effects on stroke volume (Casadei et al., 1992).  Together, the baroreflex mediated autonomic effects on vasomotor tone, HR, and stroke volume effectively regulate short-term changes in BP. The renin-angiotensin-aldosterone system participates in BP maintenance on a longer- term basis, on the order of hours.  In response to reductions in effective circulating blood volume, caused by a reduction in pressure or volume, juxtaglomerular cells of the kidney release renin (Gomez and Lopez, 2009).  Renin increases angiotensin II levels, which   6 causes potent vasoconstriction, facilitates sympathetic transmission and stimulates aldosterone release from the adrenal cortex (He and MacGregor, 2003; Nelson and Cox, 2005).  Circulating aldosterone ultimately results in blood volume expansion (MacGregor et al., 1981).  Together, these effects buffer changes in effective circulating blood volume and maintain BP.  The relationship between the blood volume stimulus and renin response is modulated by the SNS and circulating catecholamines (Kirchheim et al., 1988). 1.4 Cardiovascular sequelae of SCI The profound CV dysfunctions associated with high-level injuries can be among the most frustrating and debilitating sequelae of SCI; from the very beginning, these complications can interfere with functional independence and community reintegration by impeding participation in rehabilitation programs. SNS function is most strongly affected by SCI level due to the location of the SPNs throughout the thoracolumbar cord (Jänig, 2006).  Sympathetic fibers exit the spinal cord throughout the thoracolumbar cord therefore higher injuries leave a greater number of sympathetic nerves without supraspinal control.  The spinal outflow to the splanchnic vasculature is located in spinal cord segments T5-L2 (Strack et al., 1988; Hsieh et al., 2000).  This vasculature bed holds between 20-25% of total blood volume (Rowell, 1990) and is critical for BP control due to its ability to mobilize a large amount of blood volume (Brunner et al., 1988).  The loss of this major element of BP control has consequences on resting BP, maintenance of BP during orthostatic challenge, and control of the reflex vasomotor circuits.   7 The drastic changes in sympathetic vasomotor activity after injury highlight the importance of descending BP regulatory mechanisms.  The loss of this descending control results in low sympathetic vasoconstrictor activity below the injury and thus low resting BP (Stjernberg and Wallin, 1983).  This low, as opposed to absent, sympathetic activity reveals that the local spinal reflex arc remains intact and capable of generating sympathetic tone (reviewed in Guyenet, 2006).  Indeed, in individuals with SCI, stimulation of peripheral somatic sensory afferents below the lesion still initiates some degree of sympathetic activation (Stjernberg and Wallin, 1983; Wallin, 1986; Karlsson et al, 1998).  This can be problematic, as these circuits can be pathologically activated without the important modulation from brainstem (see 1.4.3 Autonomic dysreflexia below). Resting HR is also dependent on the autonomic level of injury.  As vagal HR control is almost always intact following SCI, it is the level of sympathetic cardiac control and resultant balance between that and vagal control that determines HR. Complete cervical SCI typically results in resting bradycardia due to the total loss of sympathetic cardiomotor control (Claydon and Krassioukov, 2006).  On the other hand, mid and low-thoracic SCIs tend to result in resting tachycardia, as the body attempts to compensate for low resting BP, and lower stroke volume (Hjeltnes, 1977; Jacobs et al., 2002), by increasing HR (Claydon and Krassioukov, 2006).  High-thoracic injuries (T1- T4) are hard to characterize as they can disrupt a portion of cardiac sympathetic control, resulting in an atypical HR response (Claydon and Krassioukov, 2008). The location of injury within the cord is another important determinant of CV dysfunction, as most SCIs are anatomically incomplete (Fawcett, 2002).  The descending   8 vasomotor pathways in the human spinal cord lie in the lateral funiculus; damage to this area causes significant CV pathology (Furlan et al., 2003; Krassioukov, 2006). Ultimately, both level and completeness of injury have consequences on resultant CV dysfunction. 1.4.1 Neurogenic shock Acutely after injury, the primary CV concern is supporting patients through the period of neurogenic shock.  Neurogenic shock typically presents as severe hypotension accompanied by reflex bradycardia (Atkinson and Atkinson, 1996; Krassioukov and Claydon, 2006; McMahon et al., 2009).  It is thought to primarily be due to the recent loss of descending medullary basal sympathetic tone to the sympathetic preganglionic vasomotor neurons in the thoracolumbar cord (Calaresu and Yardley, 1988).  This results in reduced systemic vascular resistance, decreased venous return and decreased systemic BP.  Bradycardia is also common during neurogenic shock, especially following high- SCI that disrupts the brainstem control of cardiac sympathetic neurons (Lehmann et al., 1987).  The most common form of medical intervention for neurogenic shock in in the form of fluid resuscitation to induce volume expansion, but it can also include pharmacological pressor therapy (McMahon et al., 2009).  The goal of this therapy is generally to maintain MAP above 85 mmHg (Vale et al., 1997). Neurogenic shock is distinct from spinal shock, the period following SCI when somatic reflexes are absent.  While they can both be present in the acute period, spinal shock tends to resolve earlier than neurogenic shock (Atkinson and Atkinson, 1996; Silver, 2000).  Neurogenic shock has also been documented in experimental animal models (Mayorov et al., 2001), although it is generally less severe and resolves more quickly   9 than in humans (Krassioukov and Claydon, 2006).  Even after the resolution of neurogenic shock, CV function rarely returns to normal; orthostatic hypotension (OH), autonomic dysreflexia (AD) and altered circadian rhythms can persist throughout a person’s lifetime. 1.4.2 Orthostatic hypotension The ability to modulate CV activity to maintain BP when changing body position is crucial to provide adequate blood to the brain.  This is one of the most important goals of the ANS (Goadsby, 2004).  The disruption of the ANS by SCI can compromise this task, manifesting in hypotension in response to a postural change.  Orthostatic hypotension (OH) is defined as a decrease in systolic arterial pressure (SAP) of ≥ 20 mmHg or in diastolic arterial pressure (DAP) of ≥ 10 mmHg within three minutes of an orthostatic challenge (Consensus Committee of the American Autonomic Society and the American Academy of Neurology, 1996). Clinical symptoms often accompany the BP changes, but they are not necessary for the diagnosis of OH.  When they do occur, common symptoms result from poor perfusion of the brain, including light headedness, dizziness, visual disturbances (blurred, tunnel, scotoma, graying/blacking out, colour deficits), and loss of consciousness (Mathias, 1995).  Non-specific symptoms include weakness and fatigue, and muscle aches localized to the neck, shoulders, and lower back (Mathias, 1995). Like most aspects of CV dysfunction, OH is most prevalent in individuals with high-level SCI and most severe in those with complete injuries (Sidorov et al., 2008).  The incidence of OH is particularly high in the acute post-injury period (Sidorov et al., 2008).  At this time, OH associated with sitting is the most common barrier to individuals’ participation in rehabilitation and early wheelchair use (Illman et al., 2000; Tederko et al., 2006).  For   10 some, the severity of OH decreases with time post-injury; for others, the management of OH is a lifelong consideration (Claydon and Krassioukov, 2006; Sidorov et al., 2008). Clinically, orthostatic tolerance is quantified using CV recording during a postural challenge, most commonly in the form of a tilt table, but can also be assessed using a stand-up test or lower body negative pressure.  In neurologically intact subjects, a 45 degree head-up tilt triggers an increase in plasma noradrenaline and renin activity and results in little change in BP (Mathias et al., 1980).  Recently, a modified orthostatic stress test has been developed called the “sit-up test” (Claydon and Krassioukov, 2006). The main advantages of this test are that it is simple and less severe than a tilt-test, which trigger a more modest fall in BP.  As a result, it can be used in the acute phase of SCI and performed using standard hospital beds (Claydon and Krassioukov, 2006). Clinical testing has revealed some of the compensatory responses of the body as it attempts to mitigate the severity of hypotension.  Muscle spasms have been recorded during tilting, whose mechanical effects increase the venous return to the heart and transiently increase BP (Mathias, 1987; Tschakovsky and Sheriff, 2004).  In the partial or total absence of sympathetic vasomotor control, the renin-angiotensin-aldosterone pathway also assumes a more important role in BP control (Mathias and Frankel, 1999). This axis has a number of effects on the CV system that can reduce the severity of OH, including vasoconstriction and increasing plasma volume.  The reliance on this system can be observed during prolonged head-up tilt, where both plasma renin activity and aldosterone increase more rapidly and to a higher degree in tetraplegic subjects than in controls, who increase plasma noradrenaline concurrently (Mathias et al., 1975; Mathias et al., 1980).  Resting plasma renin activity is also higher than normal in individuals with   11 tetraplegia (Mathias et al., 1975), suggesting that mild chronic hypotension can continue to activate this axis over prolonged periods.  All of these studies have informed and improved the monitoring and management of clinical OH.  To date, I have not found any evidence OH being studied in animal models of SCI. 1.4.3 Autonomic dysreflexia SCI perturbs the ability of the ANS to maintain CV homeostasis by disrupting central control of cardiac function and peripheral vasomotor pathways.  It also leaves local spinal sympathetic circuitry distal to the injury to operate independently from supraspinal control.  When the injury is severe, and at a high-thoracic or cervical level, these two changes can combine to produce dangerous, uncontrolled, elevations in BP.  Autonomic dysreflexia (AD), defined as an increase in systolic BP of ≥ 20 mmHg from baseline, is essentially an exaggerated sympathetic reflex of sympathetic vasomotor neurons distal to the injury (Krassioukov et al., 2007).  It is triggered by afferent input below the level of SCI, which reflexively activates peripheral vasoconstriction, culminating in the profound elevation of whole-body BP. The symptoms associated with AD range widely, from mild sweating to severe headaches (Mathias and Frankel, 1999).  Like OH, AD can occur asymptomatically, even when significant changes in BP are experienced (Ekland et al., 2008).  A number of the symptoms, including flushing, sweating, and pounding heart, result from the reflex autonomic responses of the upper body attempting to compensate for the massive sympathetic discharge below the injury level (Guttmann and Whitteridge, 1947; Mathias and Frankel, 1999).  The HR compensation during an episode of AD is often bradycardic. This baroreflex-triggered response, primarily effected by an increase in vagal activity   12 (Gao et al., 2002), is generally insufficient to completely compensate for the sympathetic vasomotor tone, therefore BP remains high (Schmitt et al., 2001b).  On the other hand, no HR change or tachycardia can be observed in severe cervical injuries, whose sympathetic cardiac reflex activity opposes the baroreflex-mediated vagal HR effects (Kewalramani, 1980). AD is tested clinically by monitoring BP during procedures such as routine urethral catheterization (Fagius and Karhuvaara, 1989), cystometric bladder filling (Giannantoni et al., 1998; Igawa et al., 2003), or vibrostimulation for sperm retrieval (Claydon et al., 2006; Ekland et al., 2008).  Each of these activities are potent AD-inducing stimuli for individuals who suffer from AD.  To model these visceral stimuli in animals with SCI, bladder filling or colorectal distension (CRD) is used to trigger AD (Osborn et al., 1990; Krassioukov and Weaver, 1995).  Rodents with severe high-level SCI also experience AD, which is manifested in a CV response similar to the human condition (Krassioukov and Weaver, 1995).  CRD has proven to be a robust trigger of AD.  Since its inception, it has been used to characterize the development of AD over time, identify spinal elements of its pathophysiology and assess severity of damage to autonomic pathways (Krassioukov and Weaver, 1996; Maiorov et al., 1997b; Maiorov et al., 1998; Krenz et al., 1999; Cameron et al., 2006; Hou et al., 2008).  In uninjured rats and humans, visceral distension elicits a small increase in BP, but this is effectively counteracted by the baroreceptor reflex, which returns whole-body BP to resting values (Krassioukov and Weaver, 1995). Like OH, AD generally occurs in individuals with injury at or above the sixth thoracic segment (T6) as these injuries disrupt the supraspinal control of the splanchnic   13 vasculature.  In this population of individuals with cervical or high-thoracic SCI the prevalence of AD is between 50% to 90% (Lindan et al., 1980; Mathias and Frankel, 1983; Mathias and Frankel, 1999).  While AD is most common in individuals with neurologically complete SCI, it can also occur in those with incomplete injuries (Helkowski et al., 2003).  Indeed, neither the presence of AD nor the level of sympathetic dysfunction can be inferred from the degree of motor dysfunction (Curt et al., 1996; Claydon and Krassioukov, 2006).  The association between the presence of AD and loss of sympathetic skin responses (SSR) suggests that the disruption of supraspinal sympathetic control is necessary for the development of AD (Curt et al., 1996). However, the loss of descending control is not sufficient for AD, as AD is not ubiquitous in individuals with sympathetically complete tetraplegia (Curt et al., 1996; Claydon and Krassioukov, 2006).  Additional aberrant changes, such as the expansion of sensory afferent terminals in the spinal cord (Krenz et al., 1999; Cameron et al., 2006) and supersensitivity of the vascular smooth muscle (Teasell et al., 2000; Yeoh et al., 2004) both contribute to the profound sympathetic reflex response to sensory input. AD is typically described and reported in the chronic post-injury period.  However, there have also been cases of “early” AD, occurring in the acute post-injury period in both rats and humans (Osborn et al., 1990; Maiorov et al., 1998; Silver, 2000; Krassioukov et al., 2003).  Early AD is thought to be a result of the acute loss of descending inhibition from supraspinal centres - a distinct etiology from the subacute development of the syndrome. Whatever the etiology, dealing with extreme hypertensive episodes becomes a life-long concern for individuals who develop AD.  Despite this, we know very little about   14 whether the severity of AD changes over time or what long-term effects these episodes of extremely high BP have on resting CV variables. 1.4.4 Cardiovascular circadian rhythms Circadian patterns in autonomic activity correlate with circadian patterns in BP and HR, and are considered to be responsible for these rhythms (Furlan et al., 1990).  A reduction in sympathetic activity and plasma catecholamines during the night are associated with the nocturnal fall in BP, while elevated BP during the day coincides with a rise in sympathetic activity (Linsell et al., 1985; Furlan et al., 1990).  HR follows a similar diurnal rhythm, beginning to rise a few hours prior to waking, and peaking midmorning – a pattern that seems to be driven primarily by the modulation of vagal parasympathetic cardiac activity (Burgess et al., 1997; Scheer et al., 2004).  This rhythm is considered a true endogenous circadian rhythm, driven by the biological clock in the suprachiasmatic nucleus (SCN), as it persists even in unmasking conditions, where light, sleep, physical activity, and feeding are constant and controlled (Kerkhof et al., 1998; Scheer et al., 2001).  BP, on the other hand, does not vary when measured in unmasking conditions, suggesting it is normally entrained by environmental and behavioral cues (Van Dongen et al., 2001). Not surprisingly, circadian BP variation is abolished following complete cervical SCI (Krum et al., 1991; Nitsche et al., 1996; Munakata et al., 1997).  Specifically, there is a failure to increase BP during the day.  However, the HR rhythm is maintained, supporting the principle role of vagal activity in this rhythm generation.  Individuals with mid- and low-thoracic SCI display normal nocturnal dipping of both BP and HR (Nitsche et al., 1996; Munakata et al., 1997).  Circadian CV rhythms are often preserved in incomplete   15 cervical injuries (Nitsche et al., 1996) – though, of course, it is the integrity of the autonomic pathways that is the most important factor in this regard.  These rhythms have also been examined in animal models of SCI.  High-thoracic SCI abolished circadian rhythmicity in BP and HR acutely, but these rhythms were restored by seven days post- injury (Mayorov et al., 2001).  Together, these trends begin to illustrate the level- and time-dependence of CV function following SCI. 1.5 Energy metabolism Energy metabolism is regulated by a combination of hormonal and neural cues.  The ANS plays an important role in controlling the storage and release of both major fuel substrates – glucose and free fatty acids.  Following meals, these substrates are quickly cleared from the circulation and their endogenous release is suppressed (Ahima, 2006). Excess substrates are stored as triglycerides or as glycogen for retrieval during times of need.  This balance is partly mediated by the reciprocal sympathetic and parasympathetic control of the visceral organs. 1.5.1 Glucose metabolism and the autonomic nervous system The ANS plays an important role in modulating glucose levels.  Blood glucose levels are tightly regulated, as both hypo- and hyperglycemia can be damaging to tissues in the body.  Generally speaking, increases in sympathetic activity elevate blood glucose, while increases in vagal activity reduce it.  These effects are carried out by the direct neural control to both the pancreas and liver (Nonogaki, 2000).  In the pancreas, the ANS modulates the release of insulin and glucagon.  Sympathetic activity reduces insulin secretion from pancreatic β-cells and increases glucagon secretion from the α-cells (Kiba, 2004).  Conversely, parasympathetic activity stimulates insulin secretion from β-cells   16 (Yamaguchi, 1992).  Pancreatic hormones are released into the portal vein and travel to the liver, where they modulate glucose release.  There, insulin inhibits glycogenolysis, promotes glycogen synthesis, and suppresses gluconeogenesis (Petersen et al., 1998; Edgerton et al., 2009), while glucagon stimulates glycogenolysis and gluconeogenesis (Chiasson et al., 1975; Magnusson et al., 1995).  Sympathetic activity in the liver promotes glycogen breakdown by increasing the activity of rate limiting enzymes involved in glycogenolysis, thus increasing hepatic glucose output; vagal activity promotes glycogen synthesis by increasing the activity of glycogen synthesis, decreasing plasma glucose in the process (Nonogaki, 2000) Plasma glucose concentrations display a circadian rhythmicity in both rodents and humans – peaking shortly before the beginning of the waking hours (Carroll and Nestel, 1973; Bellinger et al., 1975; La Fleur et al., 1999).  This endogenous rhythm is controlled by the SCN and effected primarily by the level of sympathetic activity to the liver (La Fleur et al., 1999; Kalsbeek et al., 2004; Cailotto et al., 2005).  However, the vagal input must also provide important control, as either sympathectomy or vagotomy abolish this rhythm (Cailotto et al., 2008).  These results reinforce the concept of the requirement for balanced ANS activity and lead to predictions regarding how SCI might affect circadian blood glucose (see 1.8 Experimental objectives below).  Circadian glucose patterns have not been reported in the literature after clinical or experimental SCI. 1.5.2 Lipid metabolism and the autonomic nervous system Adipose tissue exists in both brown and white form, but only the latter is involved in lipid storage (Cinti, 2001).  White adipose tissue (WAT) constitutes the main energy storage depot for the body, in which energy is stored as triacylglycerides (triglycerides) (Cinti,   17 2001).  Unlike glycogen, triglycerides are stored anhydrously and have high energy density, making them well suited to provide large energy reserves (Olsson and Saltin, 1970; Gurr and James, 1971).  Despite these differences, in many ways WAT plays a similar role to the liver; both act as buffers for the daily fluctuations in plasma substrates (Frayn, 2002).  In addition to modulating short-term changes in plasma substrates, WAT is also the long-term repository for energy – excess energy collected over long periods is reflected in the size of WAT depots (Frayn et al., 2003).  During acute and long-term periods of calorie deficit, stored triglycerides can be retrieved from these depots – a task which is primarily achieved by the ANS. The role of the ANS in lipid metabolism is exclusively mediated by the SNS.  The SNS has a direct role in adipocyte metabolism, via its innervation of the parenchyma, and an indirect role by its control of adipose tissue blood flow (Youngstrom and Bartness, 1995; Frayn et al., 2003).  The effects of SNS activity on adipocyte metabolism can be mediated by both α- and β-adrenergic receptors (Klein et al., 1994; Holm, 2003; Collins et al., 2004).  Both norepinephrine (NE) and epinephrine (E) promote lipolysis by activating intracellular hormone-sensitive lipase (HSL) via G-protein signaling, which increases non-esterified fatty acid (NEFA) efflux from adipocytes (Frayn et al., 2003; Holm, 2003).  Conversely, catecholamines reduce lipogenesis by inhibiting the catalytic activity of lipoprotein lipase (LPL) in WAT capillaries (Ramsay, 1996).   LPL’s role is to hydrolyse lipoproteins, converting them into NEFAs and promoting fatty acid storage (Ramsay, 1996).  Lipid metabolism is also modulated indirectly by the catecholaminergic regulation of adipose tissue blood flow (Samra et al., 1996).  Both NE and E increase blood flow, which effects plasma fatty acid levels by providing triglyceride substrate for   18 LPL and promoting NEFA efflux by clearing NEFAs from the local circulation (Kurpad et al., 1995; Samra et al., 1996). As well as regulating the balance between lipolysis and lipogenesis, the SNS is partly responsible for the regulation of the size of WAT depots.  With age, these depots typically grow by a combination of hypertrophy and hyperplasia (Nestel et al., 1969). Sympathetic innervation partly regulates the proliferation of adipocytes and when the sympathetic innervation is interrupted to individual depots adipocytes undergo hyperplasia (Youngstrom and Bartness, 1998).  Despite these roles of the SNS in adipocyte metabolism, we do not know how WAT depots are affected by SCI. 1.5.3 Circadian metabolic rhythms Similar to the CV system, metabolic activity varies significantly throughout the sleep- wake cycle.  Daily fluctuations in energy consumption and expenditure are reflected prominently in animals’ body temperature, physical activity levels and food consumption. These metabolic rhythms are driven predominantly by the circadian clock in the SCN, which is synchronized by light exposure (Meijer and Schwartz, 2003).  On the other hand, modifications in metabolism, such as hypo- or hypercaloric intake, can feed-back and influence the circadian clock and perturb the synchronization between light and metabolism (Challet et al., 1997; Mendoza et al., 2007, 2008).  These interactions severely complicate the ability to establish causal relationships and between circadian rhythmicity and metabolic activity.  Nonetheless, several clinical metabolic disorders, including obesity and diabetes, have been associated with the disruption of daily physiological rhythms such as altered sleep-wake cycles and lipolysis (Aronson, 2001; Bray and Young, 2007; Ruger and Scheer, 2009).  Light-dark shifts repeated at twice-   19 weekly intervals demonstrate that acute circadian disruption can be a powerful obesogenic stimulus in rodents as well (Tsai et al., 2005).  The specific mechanisms for these interactions have yet to be fully resolved; however, it is clear that there is some interaction between circadian disruption and metabolic abnormalities. 1.6 Metabolic sequelae of SCI Individuals with SCI share many of the same concerns about their health and body as those without SCI.  They are interested in attaining and maintaining a healthy weight, developing muscle, improving exercise performance and preventing the development of metabolic disorders. While on-line SCI forums are replete with informal discussions about strategies to address these concerns, evidence-based information is minimal (Wilt et al., 2008).  Research is needed to clarify how metabolism is altered by SCI injury and ultimately to provide viable options for maintaining long-term health. Broadly speaking, we know that SCI results in altered body composition, energy balance, and substrate metabolism (each discussed in detail below). These changes combine to produce an unfavourable metabolic profile; muscle stores are decreased and adipose tissue is easily gained and not easily accessed.  The profound lifestyle changes typically associated with SCI can make it difficult to determine whether metabolic sequelae are the direct result of SCI or secondary to lifestyle changes.  The study of elite athletes with SCI, compared to both non-athletic individuals with SCI and able-bodied controls, provides one strategy to partly dissociate these two factors. Unlike CV dysfunction, for which we have a good idea of the association between injury level and dysfunction (Mathias and Frankel, 1999; Claydon and Krassioukov, 2006), the metabolic sequelae of SCI have not been rigorously documented in relation to SCI level   20 (Wilt et al., 2008); it is unclear whether they are level dependent.  Animal models provide one approach to study this relationship. 1.6.1 Body composition SCI alters body composition by precipitating changes in muscle and fat tissue.  In both humans and rodents, there is a decrease in muscle mass due to disuse atrophy and a shift in myofiber type to predominantly fast-twitch (type II) fibers (Grimby et al., 1976; Lieber et al., 1986; Round et al., 1993; Castro et al., 1999; Gregory et al., 2003; Biering- Sorensen et al., 2009).  A reduction in muscle mass leads to decreased glucose uptake, and this is compounded by decreased insulin mediated glucose uptake in the unused muscle (Seider et al., 1982).  Furthermore, intramuscular adipose tissue, a pathogenic feature that is inversely correlated with insulin sensitivity (Goodpaster et al., 2000), is increased after SCI (Elder et al., 2004).  Finally, the shift to fast-glycolytic muscle fibers results in a muscle type that is more easily fatigued and has reduced adenosine triphosphate (ATP) yield (Biering-Sorensen et al., 2009).  All of these changes likely contribute to the problems in carbohydrate metabolism after SCI (Bauman and Spungen, 1994; Bauman et al., 1999a). Lipid metabolism is also changed as a consequence of SCI.  Grossly, this can be observed in the increased prevalence of overweight (body mass index (BMI) between 25 kg/m2 and 29.9 kg/m2) and obese (BMI≥30 kg/m2) individuals in the SCI population (Gater, 2007; Weaver et al., 2007; Rajan et al., 2008).  However, due to the decreased lean mass described above, individuals with SCI do not necessarily appear overweight, and yet they can still possess a pathological accumulation of adipose tissue (Jones et al., 1998; Gater, 2007).  Despite similar waist circumferences and total adipose tissue, men with SCI carry   21 increased adipose tissue in their abdomen compared to able-bodied controls (Edwards et al., 2008; Maruyama et al., 2008). At the plasma level, some unfavorable cholesterol level changes have been reported, including increased LDL-C, decreased HDL-C and high post-prandial triglyceride levels (Brenes et al., 1986; Bauman et al., 1992; Krum et al., 1992; Bauman et al., 1999b; Nash et al., 2005; Nash and Mendez, 2009).  However, when the literature in the field is considered as a whole, there is inconsistent evidence regarding the direction and degree of all of these changes (Wilt et al., 2008).  Furthermore, the study of active compared to sedentary individuals with SCI reveals that some of these differences, including the decrease in HDL-C, can be reversed with physical activity (Dearwater et al., 1986).  This suggests that the decrease in HDL-C can be attributed to the sedentary life-style often associated with SCI rather than SCI per se.  More research is needed to determine if and how SCI affects the plasma lipid profile directly. 1.6.2 Energy balance One likely contributor to the prevalence of obesity after SCI is a positive energy balance created by a reduction in total energy expenditure that is not offset by energy intake. Total daily energy expenditure is made up of three components: resting energy expenditure (65%), physical activity (25-30%), and the thermic effect of food (5-10%) (Buchholz and Pencharz, 2004).  While the thermic effect of food is similar between those with SCI and able-bodied subjects, both resting energy expenditure and physical activity are lower after SCI (Buchholz et al., 2003a, b).  The decrease in resting energy expenditure can be attributed to the sheer decrease in fat free mass (see 1.6.1 Body composition above) (Buchholz et al., 2003a).  The energy expenditure associated with   22 physical activity is decreased due to the reduced quantity of muscle able to be recruited during activity. Energy intake, on the other hand, does not seem to be altered significantly by SCI. Macronutrient intake in individuals with SCI remains within the acceptable distribution ranges for the able-bodied (Walters et al., 2009); however, the proportion of fat and refined carbohydrates remain high in North Americans with SCI compared to recommended intake (Groah et al., 2009).  This suggests that there is a role for nutritional education and counseling following SCI (Groah et al., 2009).  Overall, a positive energy balance likely represents a notable contributor to metabolic complications following SCI. 1.6.3 Substrate metabolism Substrate metabolism is another aspect of global metabolism that is perturbed by SCI. Fat and carbohydrate are the major substrates that are metabolised to produce ATP (Spriet and Hargreaves, 2006).  The ratio of fuel use can vary widely depending on the activity, intensity, and length of exertion (Bülow, 2004).  At rest, both individuals with SCI and those without use approximately equal fat and carbohydrate for oxidative metabolism (Bülow, 2004; Astorino and Harness, 2009).  During calorie deficits, for example during fasting or sustained physical exercise, able-bodied individuals rely on fat oxidation more than carbohydrate oxidation.  During exercise, the respiratory exchange ratio of SCI individuals reveals a reliance on carbohydrate oxidation – even when exercise intensity is progressively increased (Astorino and Harness, 2009).  Unlike able- bodied individuals, those with SCI seem to depend almost entirely on their glycogen stores (Horowitz, 2006; Astorino and Harness, 2009).  Even endurance trained athletes with SCI are unable to increase their fat oxidation levels during strenuous wheelchair   23 cycling exercise (75% VO2 peak) (Knechtle et al., 2004).  This reduced capacity to mobilize lipids during time of need likely contributes to the increased adipose stores in this population (Edwards et al., 2008).  Furthermore, the reliance on glycogen stores, already depleted due to muscle atrophy, places individuals at risk of hypoglycemic events (Schmitt et al., 2001a). 1.6.4 Circadian metabolic rhythms following SCI Most circadian metabolic rhythms are strongly influenced by the sleep-wake cycle.  Sleep patterns are often disrupted as a result of SCI; people with SCI report difficulty falling asleep, are awakened more often throughout the night, and describe their sleep quality as poor (Biering-Sorensen and Biering-Sorensen, 2001; Norrbrink Budh et al., 2005). Common nighttime complaints, and likely causes for disordered sleep patterns, include obstructive sleep apnea, pain, difficulty voiding, and muscle spasms (Biering-Sorensen and Biering-Sorensen, 2001; Norrbrink Budh et al., 2005).  Rodents with low-thoracic SCI also experience altered sleep patterns compared to sham-injured controls, which manifests as increased wakefulness during the light cycle and more frequent sleep during the dark cycle (Esteves et al., 2007).  The alteration of the sleep-wake cycle likely affects metabolism in both humans and rodents. The disruption of typical melatonin secretion may contribute to disordered sleep-wake cycles.  Melatonin release from the pineal gland is governed by the SCN (Perreau-Lenz et al., 2003) and is synchronized by light (Benstaali et al., 2001).  This circuit is vulnerable to disruption by cervical and high-thoracic SCI as it travels from the hypothalamus via the cervical cord, before exiting the spinal cord at the upper thoracic levels and traveling to the pineal gland via the superior cervical ganglion (Larsen et al., 1998).  Cervical SCI   24 results in abnormally low melatonin levels with no nocturnal increase (Kneisley et al., 1978; Li et al., 1989; Zeitzer et al., 2000).  Given the importance of the sleep-wake cycle and rhythmic release of hormones in the modulation of metabolism, it is possible that altered circadian rhythmicity contributes to the metabolic pathologies that are prevalent in the SCI population (Bauman and Spungen, 1994; Bauman et al., 1999a; Lee et al., 2005; Gater, 2007; Edwards et al., 2008). 1.7 Blood sugar control following SCI Paradoxically, both hypoglycemia and hyperglycemia occur with increased frequently in individuals with SCI (Schmitt et al., 2001a).  The most concerning aspect of impaired blood sugar control is hyperglycemia, due to its association with glucose intolerance, insulin resistance and diabetes (DeFronzo et al., 1989; Fery, 1994).  Fasting hyperglycemia has been documented in up to 23% of individuals with SCI (Duckworth et al., 1983).  Hyperglycemia, triggered by a high-glucose drink, is also more severe in individuals with SCI, and takes longer to restore resting glucose levels (Bauman and Spungen, 1994; Bauman et al., 1999a).  Together these results suggest that people with SCI have some difficulty maintaining glucose homeostasis.  Indeed, the mean insulin increase in response to glucose administration is also higher after SCI, which suggests that insulin resistance is likely present in this population (Duckworth et al., 1980; Bauman and Spungen, 1994).  The development of insulin resistance after SCI has been attributed to altered body composition (Duckworth et al., 1980) and prolonged inactivity (Bauman and Spungen, 1994).  There has not been any research into whether the autonomic control of visceral organs plays an important role in this response.   25 The typical response to acute hypoglycemia involves a number of counter regulatory changes that are partly effected by the SNS and thus could be affected by SCI.  The acute stage of insulin-induced hypoglycemia is counteracted primarily by an increase in adrenal catecholamine and pancreatic glucagon secretions (Yamaguchi, 1992).  Complete SCI disrupts the descending autonomic control to both of these organs, and there is some evidence that SNS dysfunction may contribute to hypoglycemia (Mathias et al., 1979). Individuals with tetraplegia fail to increase plasma adrenaline and noradrenaline during hypoglycemia (Mathias et al., 1979; Corrall and Frier, 1981).  However, normoglycemia is restored notwithstanding, which supports the redundancy between the adrenal and pancreatic counter regulatory responses to acute hypoglycemia.  Indeed, the glucagon response is preserved after high-level SCI, which suggests that the local pancreatic response is sufficient to increase glucagon secretion (Marliss et al., 1973; Palmer et al., 1976; Corrall and Frier, 1981; Havel et al., 1994).  Many of the symptoms typically associated with hypoglycemia, such as sweating, anxiety, hunger, and tachycardia, are triggered by the centrally-mediated catecholamine increase and therefore do not occur after complete high SCI (Mathias and Frankel, 1999; Schmitt et al., 2001a).  It is likely that in the absence of symptoms, mild hypoglycemia is more frequently left to develop into full-blown hypoglycemic events, contributing to the increased incidence in the SCI population. It is not known whether experimental SCI results in resting hyperglycemia, exaggerated response to a glucose load, if insulin-induced hypoglycemia resolves normally, or whether any of these responses are associated with level of SCI.   26 1.8 Experimental objectives The overarching objective of the series of studies included in this thesis was to determine whether thoracic SCI level, and resultant sympathetic dysfunction, contributes to altered whole-body CV and metabolic function. The aim of the first series of experiments (Chapter 2) was to describing the deficits in BP control in relation to SCI level and time post-injury. While AD has been well described, the hypotension associated with orthostatic challenge has never been assessed after experimental SCI.  Therefore, this work completes the CV profile after experimental SCI. Based on the clinical manifestations of OH, I hypothesized that OH would be most severe in high-level thoracic SCI acutely after injury.  I further posited that both OH and AD would remain stable after one month post-injury. The next series of experiments were conducted to determine whether lipid metabolism is altered by SCI (Chapter 3).  This is another system where central SNS activity plays a major role in controlling short- and long-term function.  Here I employed gross measures of adiposity and serum measures of cardiometabolic risk to determine whether these were related to injury level – and consequently to the severity of sympathetic dysfunction. Given the importance of sympathetic activity in mobilizing lipids from adipose tissue, I hypothesized that adipose tissue accumulation would be titrated by injury level - with the greatest accumulation following high-thoracic SCI.  I expected that this increase in adipose tissue would be reflected similarly in serum cholesterol and triglyceride levels. 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J Clin Endocrinol Metab 85:2189-2196.    44 2 CARDIOVASCULAR RESPONSES TO ORTHOSTATIC STRESS, COLORECTAL DISTENSION AND SEXUAL STIMULATION IN RATS WITH SPINAL CORD INJURY1 2.1 Introduction Animals with spinal cord injury (SCI) exhibit cardiovascular (CV) abnormalities and dysfunctions similar to those observed clinically.  In the first week post-injury, animals with high-thoracic SCI experience low resting blood pressure (BP) due to neurogenic shock and loss of circadian rhythms (Mayorov et al., 2001); in the longer term, these animals are prone to autonomic dysreflexia (AD), characterized by episodes of hypertension, which is typically induced experimentally by colon distension (Krassioukov and Weaver, 1995; Leman et al., 2000; Mayorov et al., 2001).  However, there are many other stimuli activities of daily living that can lead to debilitating hypo- or hypertension that have never been tested in experimental animal research. Orthostatic hypotension (OH) is one aspect of CV dysfunction that has received little attention in experimental SCI.  OH is a clinical concern and a frustrating reality that can restrict the activities and functional independence of individuals with SCI throughout their lives (Illman et al., 2000; Claydon and Krassioukov, 2006; Chelvarajah et al., 2009; Jegede et al., 2009).  It is typically defined by a decrease in systolic arterial pressure (SAP) of greater or equal to 20mmHg, or decrease in diastolic arterial pressure (DAP) of 10mmHg or more, within three minutes of being subjected to an orthostatic challenge  1 A version of this chapter will be submitted for publication.  Inskip, J.A., Ramer, L.M., Ramer, M.S. and Krassioukov, A.V. (2010) Cardiovascular responses to orthostatic stress, colorectal distension and sexual stimulation in rats with spinal cord injury.   45 (Consensus Committee of the American Autonomic Society and the American Academy of Neurology, 1996). OH is one of the most common barriers to beginning early rehabilitation (Illman et al., 2000; Tederko et al., 2006); as many as 73.6% of individuals are unable to participate in physiotherapy because their BP falls dramatically with the assumption of a seated posture or transfer to a wheelchair (Illman et al., 2000). There is some reduction in the frequency and severity of OH over time. The incidence decreases dramatically from 7 days to 30 days post-injury; however, 74% of those with motor complete cervical SCI, and 20% of individuals with upper thoracic motor complete SCI continue to suffer from OH one month post-injury (Sidorov et al., 2008).  For a large portion of individuals with high- thoracic or cervical injuries, OH persists in the chronic period following injury and limits the use of standing wheelchairs or frames (Chelvarajah et al., 2009).  More than a year post-injury, 50% of individuals with cervical SCI experienced marked SAP decreases during a “sit-up” test (average decrease -15 mmHg), a passive maneuver in which the subject is moved from a supine to seated position by the raising of the torso and flexion of the knees to a 90 degree angle (Claydon and Krassioukov, 2006).  While there are several pharmacological methods (Freeman, 2008; Krassioukov et al., 2009b), non- pharmacological methods (Elokda et al., 2000; Sampson et al., 2000; Faghri and Yount, 2002; Gillis et al., 2008), and several promising novel targets (Wecht et al., 2005; Wecht et al., 2007; Wecht et al., 2009) to mitigate the symptoms and incidence of OH, OH continues to restrict the daily activities of individuals with SCI. One of the unique features of managing OH in this population is that a majority also alternately deal with crippling bouts of hypertension.  These sudden episodes of high BP   46 triggered by noxious or non-noxious sensory input below the injury level, known as autonomic dysreflexia (AD), add another challenge to the daily lives of many with injuries above T6 (Mathias and Frankel, 1999; Teasell et al., 2000).  The symptoms of AD can cause mild to severe discomfort but untreated episodes have the potential to develop into life-threatening crises, which can culminate in seizures, pulmonary edema, retinal or intracerebral hemorrhage, or even death (Yarkony et al., 1986; Colachis and Fugate, 2002; Pan et al., 2005; Valles et al., 2005; Dolinak and Balraj, 2007; Calder et al., 2009).  While the initiating stimulus may vary between individuals, common triggers include a distended bladder, routine bowel activity, or a tight wheelchair strap.  Acute management involves the removal of the precipitating stimulus, movement into a seated position, and if hypertension is sustained then antihypertensive medications are initiated (Krassioukov et al., 2009a). Sexual activity-induced AD has recently emerged as an important topic in CV management after SCI.  The CV abnormalities associated with sexual stimulation have typically been studied in men undergoing vibrostimulation for sperm retrieval; however, their presence has implications for sexual activity more generally after SCI.  Research conducted during vibrostimulation has informed us about silent and malignant AD (Elliott and Krassioukov, 2006; Ekland et al., 2008).  Like most episodes of AD, those induced by vibrostimulation are typically alleviated by the removal of the triggering stimulus; however, several cases have been described where persistent periods of dysreflexia occurred following sexual activity, which worsened over a one-week period (Elliott and Krassioukov, 2006).  These episodes, termed malignant AD, are troubling because they do not resolve with stimulus removal.  Also of grave concern is silent AD,   47 in which individuals are asymptomatic despite pronounced increases in SAP (Linsenmeyer et al., 1996; Kirshblum et al., 2002; Ekland et al., 2008).  This discrepancy between symptoms and measured BP can make it impossible for individuals to detect when they are experiencing a dysreflexic episode, and so they might fail to initiate treatment.  The potential dangers associated with these episodes makes education and counseling an integral aspect of sexual health and CV management after SCI.  Despite being a critical issue in the clinical management of individuals with SCI, CV responses to sexual stimulation have scarcely been reported in SCI animals (Sansone et al., 1997), and never in male SCI rats. The main goal of this study was to characterize the CV responses to three clinically relevant stimuli known to elicit profound CV changes during routine activities of individuals with SCI.  Orthostatic tolerance, which has not been examined before in experimental SCI, was assessed at one week (acute), one month (subacute) and three months (chronic) after T3 or T10 SCI.  In order to examine the effect of time post-injury on the severity of AD, colorectal distension (CRD)-evoked AD was examined in animals with T3 SCI at one and three months post-injury.  Finally, sexual stimulation was tested as an additional trigger of AD in animals with T3 SCI at one month post-injury. Together these experiments demonstrate the feasibility of adapting novel stimuli to the battery of autonomic assessments currently performed in experimental SCI laboratories. Given that the recovery of autonomic function, particularly bladder, bowel and sexual function, is a priority for individuals with SCI (Anderson, 2004), we propose that the autonomic assessments detailed herein be added to the arsenal of motor, sensory and autonomic tests currently in use after experimental SCI.   48 2.2 Materials and methods All animal procedures were performed in accordance with the guidelines of the Canadian Council for Animal Care and approved by the University of British Columbia Animal Care Committee.  All animals used were male Wistar rats (n=55; 300-400g) 2.2.1 Surgeries Prophylactic antibiotics were administered for three days pre-operatively (enrofloxacin, Baytril; 10 mg/kg, subcutaneous (s.c.), Associated Veterinary Purchasing (AVP), Langley, Canada).  On the day of surgery, rats were anesthetized with ketamine hydrochloride (Vetalar; 70 mg/kg, intraperitoneal (i.p.), University of McGill Animal Resources Centre, Montreal, Canada) and medetomidine hydrochloride (Domitor; 0.5 mg/kg, i.p., AVP).  Buprenorphine (Temgesic; 0.02 mg/kg, s.c., University of McGill Animal Resources Centre, Montreal, Canada) and ketoprofen (Anafen; 5 mg/kg, s.c., AVP) were administered prior to surgery. A complete transection was performed at T3 (n=23) or T10 (n=22), or a sham surgery was performed at T3 (n=10).  The surgical protocols used in our laboratory have recently been described in detail elsewhere (Inskip et al., 2010).  For T3 SCI, a midline incision was made over the T2 process and the overlying muscles were blunt dissected.  The T2 spinous process was removed and the intervertebral gap at T2/3 was cleared of connective tissue. The dura was opened with microscissors and the spinal cord was cut using small scissors. Complete transection was confirmed under the surgical microscope, haemostasis was achieved and gelfoam was inserted at the transection gap. The muscle layer and skin layer were closed with sutures.  The anesthetic was reversed with   49 atipamezole hydrochloride (Antisedan; 50 µL, 5mg/mL, s.c., AVP).  Lactated Ringer’s solution was administered (5mL, s.c.) and animals were kept in a temperature-controlled environment for one hour post-operatively.  The T10 SCI was performed as above, except the incision was performed at the lower thoracic level, a laminectomy was perfomed at T8, and the spinal cord was cut at T10. Animals were closely monitored acutely after injury.  Antibiotics (enrofloxacin; 10 mg/kg, s.c.) and analgesics (Temgesic; 0.02 mg/kg, s.c. and Anafen; 5 mg/kg, s.c.) were continued for three days post-injury.  Urinary bladders were emptied by manual compression four times daily for the first week post-injury, and three times daily after that, until the return of reflex micturition, about 10 days post-injury.  All animals received a high-calorie diet, including fruit, Ensure (Meal replacement, Shopper’s Drug Mart) and TransGel liquid supplement (Charles River Laboratories Inc., Pointe-Claire, Canada).  Rat kibble (Lab Diet 2001, PMI Nutrition International, Jamieson’s Pet Food Dist., Delta, Canada) was provided ad libitum and water was provided using modified low-reaching water bottles.  Animals were housed in modified cages, with a rubber mat on top of the regular bedding, to facilitate locomotion.  Rats were weighed and monitored using other objective criteria every day for the first two weeks after SCI, and every second day thereafter. Carotid artery cannulation was performed at one week, one month or three months after injury.  Animals received ketoprofen (5 mg/kg, s.c.) pre-operatively, and surgery was carried out under isoflurane anesthetic (Aerrane; 5% induction, 1-2% maintenance in 100% O2, AVP).  The left common carotid artery was cannulated with an intra-arterial polyurethane catheter (3 Fr; Instech Laboratories Inc., Plymouth Meeting, PA) and filled   50 with a lock solution (1:10 heparin in 5% dextrose, AVP).  The cannula was tunneled subcutaneously and surfaced from a small incision between the scapulae. 2.2.2 Physiological testing Not all groups of animals were subjected to all aspects of physiological testing at all timepoints.  Sham animals were only subjected to an orthostatic stress at one month post- injury, as we did not expect any changes in their BP control over time.  CRD and sexual stimulation were not performed in sham-injured animals; these stimuli are difficult to perform in these animals because these animals are sensate.  Furthermore, we have shown that they do not experience AD when subjected to CRD (Krassioukov and Weaver, 1995).  The response of animals with T10 SCI to CRD was tested, though sexual stimulation was not. In preparation for physiological recording, rats were handled daily by the same researchers throughout the experiment.  In addition, all rats were habituated to the testing movements and physical environment several times before testing. Animals were acclimated to the testing room for at least one hour prior to testing. At the time of testing, cannulae were flushed with a small amount of heparin (0.01 mL; 1:10 heparin in 5% dextrose) and attached via a 30 cm length of polyethylene tubing (PE-50) to a fluid filled dome mounted on a pressure transducer (SP844, MEMScAP, Norway).  Data was recorded using PowerLab (AD Instuments, Australia) and saved for off-line analysis. Heart rate (HR) was calculated from the interbeat interval. Before any physiological manipulations began, baseline beat-to-beat BP was collected from animals in their home cage for 5 minutes.  Following baseline recording, an acute   51 orthostatic stress was applied by a weight supported 90° head-up tilt for 10 seconds.  This maneuver was performed using a tea towel to provide loose restraint: rats were held on the experimenter’s forearm until baseline BP was achieved at which point the experimenter tilted her forearm, and the rat, to 90 degrees, keeping the abdomen of the rat in the same location during the tilt; this position was held for 10 seconds and then the forearm was returned to a horizontal position, again pivoting around the abdomen of the rat.  Three tilts were performed in each recording session, with a rest period between each tilt to allow BP to return to baseline values. Colorectal distension (CRD) was performed similarly to methods described previously (Krassioukov and Weaver, 1995).  Briefly, the distal tip of a Swan-Ganz catheter (Edwards Lifesciences, Mississauga, Canada) was generously covered in lubricant and inserted 5 cm into the rectum.  The catheter was secured to the proximal tail using tape, being careful not to constrict the tail in the process.  A five-minute rest period was allowed before any further testing to allow BP to return to baseline if it was altered during the process of inserting the rectal catheter.  To begin the period of CRD, the balloon tip of the catheter was slowly filled with 2 mL of air over a period of 10 seconds, using a 3 cc syringe connected to the balloon inflation port.  The balloon distends to the approximate size of several fecal boli, and exerts a pressure of about 35 mmHg (unpublished observations).  The pressure was maintained for a period of 60 seconds at which time the syringe was removed and the pressure alleviated.  A 10-minute recovery period was recorded following CRD. Sexual stimulation was performed by loose restraint of the rat and retraction of the preputial sheath for 30 seconds.  This well-described method was first developed as a   52 standard way to initiate sexual reflexes in order to describe the different stages of the rat sexual response (Hart, 1968). 2.2.3 Statistical analysis Baseline CV variables were averaged over a period of 5 minutes just prior to any experimentation.  For all manipulations, a 30-s period immediately prior to the start of each event (tilt, CRD or sexual stimulation) was used as a baseline reference period. During the tilt maneuver and sexual stimulation, BP and HR were averaged over one- second intervals.  During CRD and throughout the recovery period, BP and HR were averaged over 10-second intervals. All data are expressed as means ± standard error of the mean (SEM).  All data analysis was performed using Prism 5 for Mac OS X (GraphPad Software Inc., San Diego, CA). The CV responses to orthostatic stress and penile sheath retraction were expressed as change in BP relative to the average baseline variables of each animal.  All baseline BP and HR data were compared using two-way analysis of variance (ANOVA), followed by the Bonferroni post-test to conduct pairwise comparisons between groups.  Changes in systolic BP in response to orthostatic challenge were first analysed using a one-sample t- test to determine whether changes were significantly different from zero.  A one-way ANOVA was used to compare BP changes between groups.  A one-sample t-test was used to determine whether HR changes during tilting were significantly different from zero.  A two-sample t-test was used to compare BP and HR responses to CRD at one and three months post-injury.  A one-way repeated measures ANOVA was used to determine how long it took for animals to recover pre-distension systolic BP after CRD.  A one- sample t-test was used to determine whether BP and HR responses to sexual stimulation   53 were significantly different from zero.  Paired t-tests were used to compare baseline CV variables and those during sexual stimulation. 2.3 Results Resting BP and HR values are presented in Table 2.1.  Each variable was compared for the same injury level at different times post-injury, as well as for different injury levels at the same time post-injury.  There was a significant difference in systolic BP between groups (p<0.0001).  Pairwise comparisons revealed that systolic arterial pressure (SAP) was significantly lower in animals with T3 SCI one week, but not one month, after injury compared to sham-injured controls (p<0.05).  SAP was higher in animals with T10 SCI one month post-injury compared to animals with T3 SCI one month post-injury (p<0.0001), T10 SCI one week post-injury (p<0.01) and T10 SCI three months post- injury (p<0.05). The average SAP responses to the acute orthostatic stress (Figure 2.1) are shown at one week (a), one month (b) and three months (c) post-injury.  Animals with T3 SCI responded to tilting with a profound decrease in SAP, while the T10 and sham groups responded with an increase in SAP.  To quantify the hypotension in animals with T3 SCI, BP change was expressed as the maximum decrease in SAP during tilting (Figure 2.1d). The maximum decrease in SAP during the tilt was significantly different from zero in all high-thoracic SCI groups; at one week post-injury (p=0.0011), one month post-injury (p=0.0039) and three months post-injury (p=0.0088).  This decline in BP was more severe at one week than at one month or three months after injury (both p<0.0001).  The   54 Table 2.1 Resting cardiovascular variables were similar across injury levels and time post-injury. Baseline beat-to-beat blood pressure (BP) recording from intra-carotid cannulae averaged over a period of 5 minutes, revealed that there was little difference in resting BP or heart rate (HR) between groups.  Only systolic arterial pressure (SAP) differed between groups: animals with T3 SCI at one week post-injury experienced resting hypotension; at one month post-injury, animals with T10 SCI demonstrated higher SAP than sham controls and animals with T10 SCI at one week or three months.  Generally speaking, the resting cardiovascular variables were similar between groups. Symbols: * denotes significantly different from T3 SCI one month post-injury (p<0.0001); § denotes significantly different from sham-injured animals one month post- injury; † denotes significantly different from T10 SCI one week (p<0.01); ‡ denotes significantly different from T10 SCI three months (p<0.05). Abbreviations: bpm, beats per minute; DAP, diastolic arterial pressure; HR, heart rate; MAP, mean arterial pressure;. SAP, systolic arterial pressure.   55   SAP (mmHg) DAP (mmHg) MAP (mmHg) HR (bpm) 1 month (n=5) 140.0 ± 3.8 118.4 ± 4.0 125.6 ± 3.9 445.6 ± 16.2 Sham 3 months (n=5) 142.0 ± 3.3 113.4 ± 5.7 122.9 ± 4.4 494.9 ± 15.9 1 week (n=5) 115.1 ± 6.3*§ 99.4 ± 5.1 104.7 ± 5.4 502.9 ± 6.6 1 month (n=7) 127.2 ± 4.9 105.2 ± 5.0 112.5 ± 4.9 493.7 ± 11.1 T3 SCI 3 months (n=8) 132.5 ± 3.7 111.61 ± 5.3 118.6 ± 4.6 471.0 ± 9.7 1 week (n=6) 133.3 ± 6.2 110.9 ± 3.1 118.4 ± 3.9 503.8 ± 11.7 1 month (n=5) 159.8 ± 1.8* † ‡  119.8 ± 1.2 133.1 ± 1.2 494.9 ± 15.9 T10 SCI 3 months (n=7) 137.0 ± 3.9 115.1 ± 4.3 122.4 ± 3.4 442.0 ± 9.4      56 Figure 2.1 Orthostatic hypotension was present after high- but not low-thoracic transection, and was more severe acutely after injury. One week post-injury (a) animals with T3 SCI (n=5) showed a pronounced decline in systolic BP in response to a 90-degree tilt, while those with T10 SCI (n=6) showed little BP change during the same maneuver.  One month post-injury (b) animals with T3 SCI (n=5) experienced a modest decline in BP in response to an orthostatic stress, while sham-injured animals (n=5) and animals with T10 SCI (n=5) showed a slight increase in BP.  Three months post-injury (c) animals with T3 SCI (n=5) also experienced a modest decrease in BP and animals with T10 SCI (n=6) again showed a moderate increase in BP over this time.  The maximum decrease in systolic BP (d) was significantly different from zero only in animals with T3 SCI.  This decline in BP was more severe at one week than at one month (p<0.0001) or three months after injury (p<0.0001). Symbols: * denotes significantly different from one week post-injury, p<0.0001.   57    58 maximum decrease in SAP was not statistically different from zero in the sham-injured animals (p=0.15) or in the low-thoracic SCI groups, one week (p=0.83), one month (p=0.068) or three months post-injury (p=0.086). Average HR responses to tilting are shown in Figure 2.2.  One week post-injury, animals with T10 SCI showed an increase in HR beginning shortly after the start of tilting (Figure 2.2a).  Animals with T3 SCI, on the other hand, exhibited bradycardia in the early part of the tilt and only increased their HR in the second half of the tilt (Figure 2.2a).  At one month post-injury, sham-injured animals showed a rapid increase in HR in response to tilting (Figure 2.2b).  In animals with T3 SCI, tilting also triggered tachycardia, but this response was slower and less pronounced than sham-injured animals (p=0.0498, Figure 2.2b). The maximum HR change during tilting from pre-tilting baseline (Figure 2.2d) was significantly different from zero in sham-injured animals (p=0.023), animals with T3 SCI at one (p=0.026) and three months post-injury (p=0.047), and animals with T10 SCI one week (p=0.015) and three months post-injury (p=0.043).  The peak HR response was quite variable in animals with T3 SCI at one week post-injury and in animals with T10 SCI at one month post-injury; neither of these groups had a maximum HR different from zero (p=0.063, p=0.073). Colorectal distension (CRD) triggered hypertension in animals with high-thoracic SCI (Figure 2.3a,b).  Animals with low-thoracic SCI also exhibited some hypertension during CRD; however, this was significantly less than animals with high thoracic SCI (p=0.0015).  The hypertension in animals with high-thoracic SCI was accompanied by   59 Figure 2.2 Animals exhibited tachycardia in response to an acute orthostatic stress. One week post-injury (a) Animals with T10 SCI (n=6) exhibited modest tachycardia during tilting that resolved shortly after the return to a horizontal position.  Animals with T3 SCI (n=5) did not exhibit tachycardia until the second half of the tilt.  One month post-injury (b) sham-injured animals (n=5) showed a rapid increase in heart rate (HR). Animals with T10 SCI (n=4) exhibited a modest increase in HR, but the rate of increase appeared slower than sham-injured animals.  Animals with T3 SCI (n=5) showed a slight increase in HR during tilting.  Three months post-injury (c), both animals with T3 SCI (n=5) and those with T10 SCI (n=6) showed an increased HR during tilting.  The maximum increase in HR (d) was significantly greater than zero in all animals except for T3 SCI animals one week post-injury (p=0.063) and animals with T10 SCI one month post-injury (p=0.073).  For the most part, there were no differences in the degree of tachycardia; however, at one month post-injury, the increase in HR was more pronounced in sham-injured animals than in animals with T3 SCI (p=0.0498). Symbols: * denotes significantly different from sham-injured group (p<0.05).   60    61 Figure 2.3 Colorectal distension elicited hypertension and bradycardia in animals with high-thoracic SCI, while animals with low-thoracic SCI experienced milder hypertension accompanied by tachycardia At one month post-injury, animals with high-thoracic SCI exhibited pronounced increases in blood pressure (BP) in response to colorectal distension (CRD) (representative beat-to-beat BP trace (a),b).  Animals with low-thoracic SCI, on the other hand, displayed small and transient changes in blood pressure in response to CRD (representative beat-to-beat BP trace (c),b).  The maximum change in systolic BP was significantly higher in animals with high-thoracic SCI compared to those with low- thoracic SCI (b).  The heart rate (HR) response to CRD was bradycardia in animals with high-thoracic SCI, while animals with low-thoracic SCI exhibited tachycardia. Symbols:  * denotes significant difference from animals with T10 SCI (p<0.05); grey bar indicates duration of colorectal distension (CRD).     62   63 bradycardia, while animals with low-thoracic SCI exhibited tachycardia during CRD (Figure 2.3d).  These responses were significantly different from each other (p=0.015). CRD elicited pronounced hypertension and bradycardia at both one and three months following T3 SCI.  The peak hypertension and bradycardia were quantitatively indistinguishable at one and three months after T3 SCI (Table 2.2).  There was no difference in peak SAP, DAP, MAP, HR or mean change in these variables between the two groups (p>0.05 for all comparisons).  While peak changes were indistinguishable, animals with T3 SCI recovered pre-distension SAP more quickly at one month than at three months post-injury (Figure 2.4a, b).  At one month post-injury the SAP returned to baseline levels within 30 seconds following the deflation of the distension stimulus (black arrow, Figure 2.4a).  In contrast, at three months after injury, animals did not recover pre- distension SAP until 110 seconds after the alleviation of distension (grey arrow, Figure 2.4a).  Examination of representative beat-to-beat traces during CRD (Figure 2.4c,d) revealed one factor that likely contributed to the slow recovery three months post-injury, namely BP increases in this group in response to deflation of the balloon catheter. One month after T3 SCI, penile sheath retraction induced BP changes characteristic of AD.  A representative BP trace (Figure 2.5) shows the increase in both SAP and DAP in response to this stimulation.  The maximum SAP during stimulation ranged from 144.8 mmHg to 180.2 mmHg (Table 2.3).  Tachycardia was present at the time of peak SAP. Peak SAP during penile sheath retraction was significantly higher than pre-stimulation (p=0.0042); that is to say, the peak change in SAP was significantly greater than zero. Peak DAP (p<0.0024) and MAP (p=0.003) were both higher than pre-stimulation values.   64 Table 2.2 Blood pressure and heart rate responses to colorectal distension were equally pronounced at one month and three months following T3 SCI. The severity of the blood pressure (BP) increase triggered by colorectal distension (CRD) did not change between one and three months post-injury.  Beat-to-beat BP was averaged over one-second intervals throughout the 60-second period of CRD.  Maximum systolic (SAP), maximum diastolic (DAP), and maximum mean arterial BP (MAP) were calculated during this period.  BP changes were quantitatively similar in both groups (p>0.05 for all comparisons).  Change in systolic BP was reported compared to the 30- second average prior to distension.  The increase in SAP and concomitant decrease in heart rate (HR) were not different at one and three months post-injury. Abbreviations: bpm, beats per minute; DAP, diastolic arterial pressure; HR, heart rate; MAP, mean arterial pressure;. SAP, systolic arterial pressure.     65    T3 SCI HR (bpm) SAP (mmHg) DAP (mmHg) MAP (mmHg) Maximum 439.7 ±12.15 166.3 ± 4.15 133.6 ± 3.92 144.5 ± 3.79 1 month (n=5) Change -60.41 ± 18.86 45.34 ± 4.43 32.34 ± 2.60 36.48 ± 3.00 Maximum 429.0 ± 16.23 180.8 ± 4.52 144.5 ± 5.75 156.5 ± 4.70 3 months  (n=6) Change -59.68 ± 15.00 52.88 ± 8.12 40.16 ± 6.70 44.35 ± 7.06   66 Figure 2.4 Colorectal distension elicited pronounced hypertension and bradycardia in animals with T3 SCI at one and three months post-injury. At one and three months post-injury, systolic arterial pressure (SAP) (a) increased with CRD and began to return to baseline when distension was alleviated.  One month after injury, SAP returned to pre-distension levels more rapidly than at three months post- injury (arrow indicates SAP is no longer elevated compared to pre-distension levels). The HR (b) in both groups showed reflex bradycardia during the period of distension, which slowly returned to pre-distension levels after the balloon was deflated. Representative blood pressure (BP) recordings of animals with T3 SCI that were subjected to CRD at one or three months post-injury are shown in c and d.  At one month post-injury (c) BP typically began to return steadily to baseline levels after CRD was alleviated.  At three months post-injury we often observed a second rise in BP when the balloon catheter was deflated (d). Symbols:  arrow indicates SAP is no longer elevated compared to pre-distension levels.   67     68 Figure 2.5 Representative arterial blood pressure recording of an animal with T3 SCI undergoing penile sheath retraction one month post-injury. Both systolic and diastolic blood pressure were elevated during sexual stimulus (penile sheath retraction). Symbols: grey bar indicates duration of sexual stimulus.   69      70 Table 2.3 Sexual stimulation elicited hypertension and tachycardia in animals with T3 SCI. Penile sheath retraction was performed for a period of 60 seconds.  Beat-to-beat blood pressure (BP) was averaged over one-second intervals, and maximum systolic (SAP), diastolic (DAP), and mean arterial BP (MAP) were calculated during this period.  Change in SAP was reported compared to the 30-second average prior to stimulation.  Heart rate (HR) was reported at the time of peak SAP. Abbreviations: bpm, beats per minute; DAP, diastolic arterial pressure; HR, heart rate; MAP, mean arterial pressure; SAP, systolic arterial pressure.    71  T3 SCI HR (bpm) SAP (mmHg) DAP (mmHg) MAP (mmHg) Baseline 522.7 ± 13.2 132.5 ± 5.2 106.7 ± 4.8 115.3 ± 4.8 Maximum 554.5 ± 10.5 160.1 ± 5.6 131.6 ± 4.5 140.9 ± 4.6 1 month (n=7) Change 31.8 ± 5.1 27.5 ± 6.1 24.84 ± 4.96 25.62 ± 5.34      72 2.4 Discussion Here we present the first comprehensive assessment of baseline CV variables and CV responses in SCI animals over a range of time post-injury.  We employed a variety of clinically relevant stimuli that are known to elicit CV dysfunction during activities of daily living of individuals with SCI.  Resting BP and HR were similar across time and level post-injury, except for a decreased SAP one week post-injury in animals with T3 SCI and an increased SAP at one month following T10 SCI.  OH was present in high, but not low, thoracic SCI and was most severe in the acute period (one week) post-injury. The hypertension and bradycardia characteristic of AD was present in animals with high- thoracic SCI but not those with low-thoracic SCI.  AD was similar at one and three months post-injury, but the hypertensive episode resolved more quickly in the one month group. To date, OH has only been investigated in animals with intact nervous systems.  The majority of these studies have focused on the mechanisms associated with CV deconditioning and orthostatic intolerance after spaceflight (Hawkey, 2003; Williams et al., 2009).  Aerospace research has established the head-up tilt is as a robust model of orthostatic stress.  This model has been shown to reliably induce hypotension in rats exposed to simulated microgravity (Sun et al., 2004; Tarasova et al., 2007).  It has also been a useful tool to investigate the physiological changes involved in the typical response to orthostasis (Raffai et al., 2005; Ramsey et al., 2007).  These studies, involving conscious rats, have typically been performed using a plastic restraint cylinder combined with a protractor to determine the angle of elevation (Martel et al., 1996; Ramsey et al., 2007; Bedette et al., 2008).  Due to the significant amount of handling that   73 our animals received throughout their recovery, we chose to perform the tilt on our arms with a simple loose restraint.  We found this to be less stressful for the animals as they were less likely to slip and experience muscle spasms during tilting.  The profound and repeatable decreases in BP that we observed lead us to believe that this maneuver is robust and reproducible.  Orthostatic instability has never, to our knowledge, been described in animals with SCI. OH was most severe in high-thoracic SCI animals acutely after injury.  This agrees well with what has been documented clinically (Sidorov et al., 2008).  The diminished response to orthostatic stress at one and three months after injury suggests that there could be some form of compensation, so that the BP fall is reduced even in the continued absence of functional descending sympathetic pathways.  Plasma volume expansion, for example could improve orthostatic tolerance (Groomes and Huang, 1991). Conversely, the dramatic fall in BP one week after T3 SCI is unlikely to be due to hypovolemia or other surgery-associated complications, as we did not see an effect one week after T10 SCI. Another possibility is that animals sustained an increase in muscle spasms associated with tilting, which would increase venous return to the heart.  We did not notice a major increase in tilt-induced muscle spasms over time following SCI, however, we did not formally measure them.   Sham and T10 SCI groups both showed an increase in BP during tilting.  This is similar to that observed in individuals with low level injuries and those without neurological injury undergoing the “sit-up test” (Claydon and Krassioukov, 2006).   74 During the “sit-up test”, we observed an increased HR in individuals with cervical SCI, thoracic SCI, and in able-bodied controls (Claydon and Krassioukov, 2006).  Similarly, here we observed tachycardia in all groups undergoing an acute orthostatic stress.  Reflex tachycardia was equally pronounced in sham-injured animals and those with T3 SCI at one week post-injury, despite the more pronounced hypotensive stimulus in the latter. Furthermore, the baroreflex-mediated effects on HR appeared to be delayed in the latter. We have recently shown that individuals with cervical SCI show a larger reflex delay in response to a “sit-up maneuver” (Claydon and Krassioukov, 2008), possibly due to a delay in the cardiac baroreflex response (Claydon et al., 2005).  Although our animals have a T3 SCI, this injury can disrupt some cardiac sympathetic innervation, which could disrupt the sympathetic portion of the cardiac baroreflex response.  More research is clearly warranted in this area, especially with regards to the effects of SCI in the high- thoracic region, where cardiac sympathetic control is partially preserved. Since its inception, CRD has proven to be a robust stimulus for producing AD (Krassioukov and Weaver, 1995).  This clinically relevant maneuver is reproducible and fairly simple to perform, and remains the most widely-used procedure for studying AD worldwide.  Here we have shown that CRD does not induce AD in animals with low- thoracic SCI; the mild hypotension and tachycardia was significantly different from the hypertension and bradycardia exhibited by animals with high-thoracic SCI.  Other visceral stimuli, such as a distended bladder, have also been shown to be potent inducers of AD in animals with high- but not low- thoracic SCI (Rivas et al., 1995).  Together these results suggest that the investigation of AD after SCI or after therapeutic intervention may be a useful tool for determining the level of autonomic lesion   75 To date, the majority of studies using CRD to examine AD have observed the CV response in animals up to one month post-injury (Krassioukov and Weaver, 1995; Leman et al., 2000; Mayorov et al., 2001; Krassioukov et al., 2002; Landrum et al., 2002; Marsh and Weaver, 2004; Hou et al., 2008), while some have observed animals to 6 to 8 weeks post-injury (Mayorov et al., 2001; Collins and Dicarlo, 2002; Marsh and Weaver, 2004; Laird et al., 2006; Kalincik et al., in press; Laird et al., 2009).  Many SCI research groups are starting to follow SCI animals out to more chronic time periods after injury; therefore, we thought that it was important to determine whether AD is changed quantitatively or qualitatively with time post-injury.  In many ways, the response to CRD was similar at one and three months post-injury; peak BP and HR changes were indistinguishable. However, the average BP and HR responses over time show that the normalization of SAP and HR occurs much more quickly at one month than three months post-injury.  The alleviation of AD, typically achieved shortly after the removal of the offending stimulus, is clinically important because it means that the blood vessel endothelium is no longer exposed to acute mechanical stress. Sexual activity has recently emerged as a salient trigger for AD clinically.  In humans, self-stimulation and vibratory penile stimulation have been documented to increase SAP by as much as 107 mmHg, and similar increases are likely present during sexual activity at home (Sheel et al., 2005; Claydon et al., 2006; Courtois et al., 2008).  Sexual stimulation can also elicit malignant AD which is characterized by significant and prolonged elevations in arterial BP that are difficult to manage (Elliott and Krassioukov, 2006).  These examples demonstrate the need for documentation and research in this area.  There is currently only one study of animal evidence describing CV responses to   76 sexual stimulation in female rats following SCI (Sansone et al., 1997).  This study found that vaginocervical stimulation elicited hypertension and bradycardia in rats with mid- thoracic, but not lumbar, SCI (Sansone et al., 1997).  In our study, we utilized penile sheath retraction as a sexual stimulus in male rats.  Unlike CRD and vaginocervical stimulation, during sexual stimulation we observed modest tachycardia. Despite the technical challenges associated with autonomic assessment in animals, AD is beginning to be included as an outcome measure following interventions for SCI (Kalincik et al., in press).  Our current work elucidates two important aspects of CV autonomic testing after SCI: the addition of an orthostatic stress test and the importance of multimodal testing.  While CRD has already been well characterized as a model, an acute orthostatic stress has never been applied to spinal cord injured animals.  The inclusion of OH assessment provides a second physiological measure to strengthen conclusions made regarding the degree of autonomic dysfunction after SCI.  The head-up tilt provides a simple and complementary assessment of CV function.  As there are a multitude of possible triggers of AD present in activities of daily living, it is also important to have multiple modes of afferent stimulation available to test this condition experimentally.  If a treatment is put forward to prevent or alleviate AD, it must be robust, and therefore must reduce the severity of BP changes, regardless of the stimulus. A variety of clinically relevant stimuli must be ready to test these potential treatments in animal models of SCI. We acknowledge that there are limitations to physiological testing performed in animals. The behavioural responses of rodents are shaped by their nature as a prey species and we are conscious of the element of stress during testing.  In our baseline evaluation of BP   77 and HR, we observed an increased SAP in animals with T10 SCI at one month compared one week post-injury.  This elevation could be the result of increased stress in the one week group compared to the one month group, as the latter would have been handled a greater number of times before physiological testing; however, the fact that the DAP and MAP were not different between groups suggests that, overall, BP was not profoundly different.  Secondly, during sexual stimulation, the HR response was unlike the bradycardia most often seen clinically.  While this response during AD can be variable, with some individuals experiencing tachycardia, there are several reasons that our animals might be more likely to experience tachycardia.  The injury level in our experiment (T3) partially preserves sympathetic cardiac innervation; therefore tachycardia could have been manifested as part of the normal response to stress. Furthermore, after testing a number of different ways to expose the perineal area, we found that the most effective was to hold the animals vertically; however, this also presented an orthostatic stress which could be responsible for eliciting tachycardia induced by baroreflex withdrawal.   These examples highlight the inherent complexities in animal physiological research and speak to the importance of minimizing stress in experimental protocols. There have been exciting and constructive developments in autonomic spinal cord injury research over the past few years.  First, in collaboration with ASIA and ISCOS, comprehensive autonomic evaluation standards have been established to be used in clinical diagnosis and research studies after SCI (Alexander et al., 2009b).  Secondly, an international panel, The Spinal Cord Outcomes Partnership Endeavor (SCOPE), has recently evaluated the methodology and validity of some of the most common general   78 autonomic function outcome measures (Alexander et al., 2009a).  While no one tool has emerged as a clear forerunner, this evaluation provides a sense of the reliability and prevalence of the tools currently in use.  It also provides evidence of a concerted movement towards a more rigorous approach to autonomic testing. We believe that it is critical for our animal models keep pace with what is important clinically and medically after SCI.  Here we have described the CV response to orthostatic stress and sexual stimulation, both of which are novel and clinically relevant models for assessment of autonomic dysfunctions following SCI.  Sexual stimulation, similar to CRD, incited a persistent elevation of arterial BP, consistent with the presence of AD.  A head-up tilt demonstrated CV changes consistent with OH.  Both of these could be used as outcome measures to test therapeutic interventions after SCI.  The use of these analogous assessments in our clinical and experimental subjects should facilitate the translation of our understanding of CV changes after SCI between the bench and the bedside.   79 2.5 References  Alexander MS et al. (2009a) Outcome measures in spinal cord injury: recent assessments and recommendations for future directions. Spinal Cord 47:582-591. Alexander MS et al. (2009b) International standards to document remaining autonomic function after spinal cord injury. Spinal Cord 47:36-43. Anderson KD (2004) Targeting recovery: priorities of the spinal cord-injured population. J Neurotrauma 21:1371-1383. Bedette D, Santos RA, Fontes MA (2008) Cardiovascular reactivity after blockade of angiotensin AT1 receptors in the experimental model of tilting test in conscious rats. Br J Pharmacol 153:966-971. 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Ramsey MW, Behnke BJ, Prisby RD, Delp MD (2007) Effects of aging on adipose resistance artery vasoconstriction: possible implications for orthostatic blood pressure regulation. J Appl Physiol 103:1636-1643. Rivas D, Chancellor M, Huang B, Salzman S (1995) Autonomic dysreflexia in a rat model of spinal cord injury and the effect of pharmacological agents. Neurourology and Urodynamics 14:141-152. Sampson EE, Burnham RS, Andrews BJ (2000) Functional electrical stimulation effect on orthostatic hypotension after spinal cord injury. Arch Phys Med Rehabil 81:139-143. Sansone GR, Bianca R, Cueva-Rolon R, Gomez LE, Komisaruk BR (1997) Cardiovascular responses to vaginocervical stimulation in the spinal cord- transected rat. Am J Physiol 273:R1361-1366. Sheel AW, Krassioukov AV, Inglis JT, Elliott SL (2005) Autonomic dysreflexia during sperm retrieval in spinal cord injury: influence of lesion level and sildenafil citrate. 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Yarkony GM, Katz RT, Wu YC (1986) Seizures secondary to autonomic dysreflexia. Arch Phys Med Rehabil 67:834-835.      84 3 CARDIOMETABOLIC RISK FACTORS IN EXPERIMENTAL SPINAL CORD INJURY1 3.1 Introduction During the last three decades, significant improvements in critical care after spinal cord injury (SCI) have dramatically reduced mortality in the acute period (Strauss et al., 2006). However, mortality rates in the chronic period (>2 years following injury) have changed very little, and people with SCI have a significantly reduced life expectancy (Garshick et al., 2005; Strauss et al., 2006).  The cardiovascular (CV) sequelae of SCI play an important role, since cardiovascular disease (CVD) or dysfunction accounts for a significant proportion of morbidity and mortality in chronic SCI (Garshick et al., 2005; Strauss et al., 2006).  Importantly, the spinal cord-injured population is afflicted with CVD both at a higher prevalence and earlier than the able-bodied population (reviewed by Myers et al., 2007). Spinal cord injury alters body composition by precipitating changes in both lean and fat mass.  It is well-accepted that people with SCI have decreased lean mass, due to a number of factors including disuse atrophy of paralyzed skeletal muscles (Grimby et al., 1976; Castro et al., 1999), decreased bone density (Wilmet et al., 1995), and a reduction in total body water (Nuhlicek et al., 1988).  Recent evidence shows that people with SCI carry increased visceral adipose tissue in the abdomen compared to able-bodied controls, despite matching waist circumference (Edwards et al., 2008; Maruyama et al., 2008). This is particularly salient because visceral adipose tissue is associated with increased CV  1 A version of this chapter has been published.  Inskip JA, Plunet W, Ramer LM, Ramsey JB, Yung A, Kozlowski P, Ramer MS, Krassioukov AV (2010) Cardiometabolic risk factors in experimental spinal cord injury. J Neurotrauma 27:275-85.   85 risk and incidence of metabolic syndrome, and is an independent predictor of all-cause mortality in the able bodied population (Kuk et al., 2006; Gater, 2007). There is also evidence that visceral adipose tissue is pathological in rodents: surgical removal of visceral adipose tissue increases longevity (Muzumdar et al., 2008).  Therefore, the increased visceral fat observed in the SCI population could conceivably be a contributor to their reduction in lifespan. Despite the high proportion of CVD, metabolic syndrome, and increased adiposity after SCI, body composition is rarely reported in this population.  Part of this can be attributed to the fact that there are problems with body composition analysis after SCI; commonly- used tools such as body mass index (BMI) and waist circumference are based on assumptions about body composition that do not hold after SCI (Jones et al., 2003; Buchholz and Bugaresti, 2005; Edwards et al., 2008; Wilt et al., 2008).  These traditional measurements underestimate the level of body fat in this population and, as a result, CVD and cardiometabolic risk are likely under-recognized clinically (Gater, 2007). Health care providers and the SCI community are increasingly cognizant of the high incidence of CVD (Garshick et al., 2005) metabolic dysfunction (Lee et al., 2005) and dyslipidemia in this population (Nash and Mendez, 2007).  A number of comprehensive reviews have been published on these subjects (Gater, 2007; Myers et al., 2007; Bauman and Spungen, 2008).  However, cholesterol and blood lipid levels remain underreported more generally in the SCI field.  This shortage of systematic documentation combined with the lack of large cohort studies makes it difficult to conclude whether cholesterol and lipoprotein levels vary predictably with level or completeness of injury (Wilt et al., 2008).  The limited evidence that we do have precludes decisions about the most   86 appropriate predictors of cardiometabolic risk following SCI, and leaves us with no choice but to rely on the measures established for the able-bodied population.  Of the so- called atherogenic triad, including increased low-density lipoprotein cholesterol (LDL- C), decreased high-density lipoprotein cholesterol (HDL-C) and increased triglycerides, some clinical SCI research reports a small decrease in HDL-C (Brenes et al., 1986; Bauman et al., 1992; Krum et al., 1992; Bauman et al., 1999b).  However, in some of these studies LDL-C is also lowered (Bauman et al., 1992; Bauman et al., 1999b), making the total cholesterol/HDL-C ratio unchanged, or even improved.  This provides another indication that the traditional cardiometabolic markers might not be the most appropriate for determining risk in the SCI population. Weight management and nutrition are lifelong challenges for individuals with SCI. Acutely after injury, nutritional deficits are common (Rodriguez et al., 1997; Dvorak et al., 2004); in the chronic setting, on the other hand, it is the positive energy balance that appears to be most problematic for the majority of individuals with SCI (Gater, 2007; Weaver et al., 2007; Rajan et al., 2008).  While rodents are widely used in research to model motor and sensory function after SCI as well as test repair strategies, it is not known whether these experimental animals exhibit the same metabolic problems as humans in the chronic setting. If we are to begin to understand the etiology of obesity and metabolic dysfunctions after SCI, discover appropriate markers for cardiometabolic risk, and begin to address interventions, it is imperative to have a rat model in which to do so.  There has been very little animal research done in this regard (Primeaux et al., 2007; Rouleau and Guertin, 2007).  The aim of these experiments was to determine if rats with SCI develop features   87 of global cardiometabolic risk.  We chose to incorporate two of the most common diagnostic assessments used clinically: serum lipid profiling and body composition analysis.  Each of these outcomes was assessed using two methods, one that demands significant technological investment and one that can be done fairly inexpensively and readily adopted by other laboratories.  In order to determine whether these risk factors vary by injury level, we examined lipid profile and body composition in rats with T3 or T10 complete SCI.  Both lipid profiling and body composition were quantified in the acute (one week) and subacute (one month) periods post-injury to observe the effects of time on these variables. 3.2 Materials and methods All animal procedures were performed in accordance with the guidelines of the Canadian Council for Animal Care and approved by the University of British Columbia Animal Care Committee.  Sixty-two male Wistar rats (250-350g) were used in these experiments. 3.2.1 Surgery and animal care Prophylactic enrofloxacin (Baytril; 10 mg/kg, subcutaneous (s.c.), Associated Veterinary Purchasing (AVP), Langley, BC) was administered for three days prior to surgery.  Rats were anesthetized with ketamine hydrochloride (Vetalar; 70 mg/kg, intraperitoneal (i.p.), AVP) and medetomidine hydrochloride (Domitor; 0.5 mg/kg, i.p., AVP).  Both buprenorphine (Temgesic; 0.02 mg/kg, s.c., AVP) and ketoprofen (Anafen; 5 mg/kg, s.c., AVP) were also administered immediately prior to surgery.  A complete transection was performed at T3 (n=30) or T10 (n=8) or sham surgery was performed at T3 (n=24).   88 For complete spinal cord transection at the third thoracic segment (T3), a dorsal midline incision was made in the skin and superficial muscles overlying the C8-T2 vertebrae. Muscles over the T2 vertebra were blunt dissected to isolate the T2 dorsal process, which was removed with rongeurs.  At the T2-T3 intervertebral gap, the dura was opened with microscissors and the spinal cord was cut using extra fine scissors. Complete transection was confirmed under the dissecting microscope by clear division of the rostral and caudal stumps of the cord, and Gelfoam (Pharmacia & Upjohn Company, Pfizer, New York, NY) was placed in this space to minimize bleeding.  The muscle layer was sutured continuously (Vicryl; 4-0) and the skin was closed with interrupted sutures (Prolene; 4- 0).  Sham surgery included the both the partial laminectomy of the T2 vertebra and durotomy at T3, but the spinal cord was not damaged. For complete spinal cord transection at T10, a midline incision was made in skin and superficial muscles over the lower thoracic spine.  Muscles overlying the T9 vertebrae were separated by blunt dissection, and a partial laminectomy was performed to expose the spinal cord prior to transection. After surgery, anesthesia was reversed with atipamezole hydrochloride (Antisedan; 1 mg/kg, s.c., Novartis, Mississauga, ON).  Animals were given warmed Lactated Ringer’s solution (5 mL, s.c.) and were placed in a temperature-controlled chamber (Animal Intensive Care Unit, HotSpot for Birds, Los Angeles, CA).  Both buprenorphine (Temgesic; 0.02 mg/kg, s.c.) and ketoprofen (Anafen; 5 mg/kg, s.c.) were administered once daily for three days to manage post-operative pain.  Antibiotic treatment (enrofloxacin, Baytril; 10 mg/kg, s.c.) was also continued for three days to reduce the incidence of bladder infection.   89 During the first week post-injury, manual bladder expression was performed three times daily.  After one week, this was reduced to twice daily until spontaneous bladder function returned (about 10 days post-injury).  Skin sutures (Prolene; 4-0) were removed one week after surgery to minimize skin irritation. Rats were monitored daily for the first two weeks after surgery, and once every two days thereafter.  This included assessment of body weight, activity level, social behaviour, lesion state and clinical signs of morbidity. When bladder infections developed, animals received a three day course of enrofloxacin (10 mg/kg, s.c.).  A combination of topical antibiotics (Triple Antibiotic Ointment, including polymixin B sulfate, bacitracin zinc and gramicidin; twice daily) and hydrocortisone cream (Cortate; 0.5% hydrocortisone, twice daily) were used to treat signs of dermatitis; this was occasionally necessary in sham-injured animals scratching at their lesion site or in SCI-animals with small pressure sores on the ventral aspect of their hindlimbs. Rats were caged in stable groups of four animals to avoid the potential effects of isolation on food consumption and allow for the benefits of social housing (Patterson-Kane, 2002; Conour et al., 2006).  Cages were fitted with rubber matting to facilitate mobilization of animals with SCI (Multy Tile, Multy Industries, Atlanta, GA).  The fenestrated matting was layered between standard cedar chips to allow for both cushioning and moisture absorption. Modified low-reaching bottles ensured easy access to water. All animals received an enriched standardized diet that was initiated three days pre- operatively to allow for acclimation.  As gastrointestinal stasis is common acutely after SCI (Tong and Holmes, 2009); we like to provide soft, palatable, nutrient-rich food throughout this period to encourage the return of gastrointestinal motility and maintain   90 energy and protein levels. Three times daily, each cage received 60 mL complete meal replacement shake (strawberry), 40 g nutritive transport gel (Charles River Laboratories International Inc., Wilmington, MA), 60 g fruit (orange and apple), 12 g cereal (Cheerios) or 24 g commercially available rat treats (alternating), and 40 g kibble (LabDiet, Rodent Diet 5001), all three times daily.  Rat kibble was also available ad libitum on the cagetop hopper.  In the first post-operative week there was often some leftover transport gel and always some kibble in the cage after feedings: any remaining food was removed from the bottom of the cage at the next feeding.  At two weeks post-injury, animals could easily move about their cage and were able to eat the regular rat kibble.  At this time, we continued to provide a varied diet of fruits and cereals, but removed the transport gel, and meal replacement milkshake. 3.2.2 Magnetic resonance imaging Magnetic resonance imaging (MRI) was performed at the UBC MRI Research Centre using a 7 Tesla (T) preclinical MRI system (BioSpec 70/30 USR, Bruker BioSpin, Ettlingen, Germany) one month after T3 SCI (n=6) or sham surgery (n=6).  Animals were killed with an overdose of isoflurane (5% in 100% O2, 1L/min) immediately prior to imaging.  Coronal images were captured using a 2 mm slice thickness (no gap) with an in-plane resolution of 391x 391 µm. A total of 20-25 slices were acquired for each animal, spanning the depth of the body.  MR images were acquired using spin-echo technique with the repetition time TR of one second and the echo time of 14 miliseconds. Two sets of images were acquired; in the first set signals from water and fat were in phase. In the second set of images, the refocusing pulse was shifted by 368 microseconds,   91 which resulted in the fat signal being at 135 degrees in phase with relation to the water signal. Total imaging time for each animal was approximately 20 minutes. 3.2.3 MRI post-processing and image analysis A series of water-fat images were generated from the spin-echo images for each animal using a modified asymmetric Dixon method (Xiang, 2006).  Dorsal-most and ventral- most images were removed from analysis as the anatomy was often difficult to discern. Images were subjected to a threshold and the number of positive pixels per image was automatically counted (SigmaScan Pro, Version 5.0). The total amount of fat per image was subdivided into subcutaneous or visceral using anatomical landmarks; visceral fat was defined in each image as the fat bordered laterally by the abdominal muscle, rostrally by the rib cage, and caudally by the top of the testes; subcutaneous fat was calculated by subtracting the total number of visceral fat pixels from the total number of fat pixels per image.  Total fat mass was calculated by volume reconstruction: slice thickness was used to calculate the volume of fat per slice, and slices were combined to obtain a volume of fat per animal.  The volume of adipose tissue was converted into weight using a standard densitometric conversion for white fat (0.901 g/cm3) (Fidanza et al., 1953; de Souza et al., 2001). 3.2.4 Visceral white adipose tissue dissection Visceral white adipose tissue (vWAT) was dissected from animals at one (n=5 T3 SCI; n=4 sham) or four weeks (n=6 T3 SCI; n=7 sham; n=8 T10 SCI) post-injury.  Brown adipose tissue was not included in this analysis as it is sparse in adult humans and serves primarily a thermogenic and energy dissipation role rather than lipid storage in rodents (Krief et al., 1993; Lowell and Flier, 1997).  Visceral WAT included both retroperitoneal   92 and perigonadal fat depots.  The retroperitoneal fat depot included all of the fat surrounding the kidney and adrenal gland; extending rostrally to encircle the adrenal gland, caudally to the vas deferens, and dorsally to the posterior abdominal wall. The perigonadal fat depot was dissected from around the vas deferens and testes, and often included folds that extended rostrally into the peritoneum.  Fat depots were weighed immediately following dissection.  Subcutaneous adipose tissue was not quantified by dissection due to the technical difficulties associated with separating it from the overlying dermal layer. As a significant number of laboratories studying spinal cord injury are interested in neural morphology, and need to preserve tissue by fixation, it was necessary for us to replicate our results using perfusion-fixed animals. A second group of rats with T3 SCI (n=4) was euthanized at four weeks post-SCI with an overdose of chloral hydrate (trichloroacetaldehyde hydrate; 1 g/kg, i.p) and perfused prior to visceral fat dissection (with PBS, followed by 4% paraformaldehyde in 0.1 M phosphate buffer). 3.2.5 Blood lipid profiling Blood samples were examined via standard clinical laboratory blood lipid analysis or a home cholesterol test system – both tests (described below) use colorimetric methods to assess lipid concentrations.  Samples for laboratory lipid analysis were collected from the abdominal aorta after overdose of isoflurane.  Samples were refrigerated for 40 minutes at 4°C, centrifuged at 3000 rpm for 10 min, and serum was removed by pipette for storage at -80°C.  Samples for use with the home cholesterol test system were collected from the tail vein, and whole blood was used for testing, as per manufacturer’s   93 instructions.  All samples were collected when rats were in a non-fasting state, near the start of the light cycle. Laboratory blood lipid analysis was conducted on serum samples using standard enzymatic colorimetric tests for cholesterol, triglycerides and HDL-C with a Dade Behring RxL Max Analyzer (Dade Behring, Newark, DE) by the Department of Pathology and Laboratory Medicine at Vancouver General Hospital.  Blood was collected one week (n=6 T3 SCI; n=6 sham) or four weeks (n=6 T3 SCI; n=6 sham; n=5 T10 SCI) after injury.  Results were reported in mmol/L: due to the range limitations of the equipment used, which is designed for human samples, cholesterol results less than 1.30 mmol/L were reported as < 1.30, triglyceride results less than 0.17 mmol/L were reported as < 0.17, HDL-C results less than 0.26 mmol/L were reported as < 0.26.  LDL levels were calculated, where possible, using the Friedewald method (Friedewald et al., 1972). In a separate group of animals, one month post-injury (n=6 T3 SCI; n=7 sham), blood lipids were assessed using a home cholesterol test system (CardioChek Analyzer, Indianapolis, IN) with Lipid Panel Test Strips (PTS, Indianapolis). Total cholesterol, HDL cholesterol and triglyceride levels were reported in mg/dL, and converted to mmol/L using standard conversion factors: again, due to the range limitations of the equipment, total cholesterol less than 100 mg/dL was reported as < 100 mg/dL, triglyceride results less than 50 mg/dL were reported as < 50 mg/dL, HDL-C results less than 15 were reported as < 15 mg/dL.  LDL-C levels were calculated, where possible, using the Friedewald method (Friedewald et al., 1972).   94 3.2.6 Statistical analysis Statistical analysis was performed using SigmaStat (Version 3.0).  All data are expressed as means ± standard error of the mean (SEM).  For all statistical outcomes, results were considered significant if p<0.05. One-way repeated measures ANOVA (RM ANOVA) was used to calculate group mean body weight changes from baseline for each group, and the Holm-Sidak method was used to conduct pairwise comparisons against baseline.  The nonparametric Friedman RM ANOVA was used to analyse groups that were not normally distributed.  Rank-based ANOVA was used to compare mean group body weights at the end of the experiment (30 days post-injury). The amount of WAT in sham and T3 SCI rats, quantified using MRI, was compared using Student’s t test (two-tailed).  One-way analysis of variance (ANOVA) was used to compare the amount of fat obtained by dissection, and pairwise comparisons were made using the Holm-Sidak method.  A bivariate correlation was used to demonstrate the relationship between MRI and dissection results, using the Pearson product-moment correlation coefficient.  Regression analysis was performed on a Bland-Altman plot of the difference in % vWAT dissection (% vWAT dissection - % vWAT MRI) against the average % vWAT by the two methods (Bland and Altman, 1986).  The difference between the MRI and dissection results were analysed using a one-sample t-test (two- tailed). All blood lipid statistics were performed using data expressed in molar concentration (mmol/L).  Lipid data obtained from the Department of Pathology were normally distributed and were compared using one-way ANOVA combined with the Holm-Sidak   95 test to detect pairwise differences at relevant time points (one week and one month post- injury).  Data obtained using the home cholesterol test were compared using Student’s t test if they were normally distributed; otherwise, the nonparametric Mann-Whitney Rank Sum Test was used. 3.3 Results Animals with T3 SCI showed significant changes in weight over time (F(16,22)=157.03, p<0.001): at three days following T3 SCI, rats had a reduction weight compared to pre- injury baseline (p=0.007, Figure 3.1); however, their weights returned to pre-operative values by 7 days post-injury.  T10 injured rats also showed changes in weight over time (F(7, 22)=40.46, p<0.001) including early signs of weight loss (p=0.025 two days after SCI compared to baseline weight); however, they returned to their pre-operative weights 6 days after injury.  Sham surgery did not result in any significant loss of body weight. At the end of the experiment (30 days post-injury) there were significant differences between groups (H=8.308, df=2, p=0.016): the mean weight of the T3 SCI group was not significantly different from the sham-injured group (p>0.05).  In contrast, mean weight of the T10 SCI group was significantly less than sham-injured group (p<0.05). At one month after injury MRI was performed to quantify white adipose tissue (WAT) in sham and T3 SCI rats (representative fat images are shown in Figure 3.2). Rats with complete T3 SCI had significantly more visceral WAT as a percentage of body weight compared to sham-injured pair-fed animals (p=0.02, Figure 3.2c).  High-thoracic-injured rats also had more subcutaneous WAT than sham-injured controls (p=0.03, Figure 3.2c). Both groups in this experiment started at the same weight (sham 300.17 ± 4.00; T3 SCI 312.00 ± 7.39, p=0.19) and at one month post-injury there was still no difference in body   96 Figure 3.1 Rats with high-thoracic SCI and those with low-thoracic SCI recover to their pre-operative weights shortly after injury. Rats with high-thoracic SCI lost a significant amount of weight immediately after injury (n=30).  By 8 days post-injury, this group returned to their pre-operative weight.  Rats with low-thoracic SCI also showed a significant weight loss after injury; however, they resolved to pre-operative weight by only 6 days post-injury (n=8).  Sham-injured animals did not lose any weight post-operatively (n=24). Symbols: black bar denotes significant difference of T3 SCI rats from pre-operative weight (p<0.05); grey bar denotes significant difference of T10 SCI rats from pre- operative weight (p<0.05).   97    98 Figure 3.2 Rats with high-thoracic SCI carried increased visceral and subcutaneous fat one month post-injury. Representative abdominal MRI images of water (a,c) and fat (b,d) in a sham-injured rat (a,b) and a rat with T3 SCI (c,d).  Fat images were generated using an asymmetric Dixon method: in these images, visceral and subcutaneous fat depots are clearly divided by the abdominal wall. Volumetric fat measurements were made from a series of images spanning the depth of each animal (20-25 slices; slice thickness = 2mm).  While there was no difference in body weight between sham- and spinal cord-injured rats at one month post-injury (e), both visceral and subcutaneous fat accounted for a greater percentage of body weight in rats with T3 SCI. * denotes significant difference from time-matched sham-injured animals.   99   100 weight between sham-injured and T3 SCI animals (Figure 3.1c).  These data are reminiscent of clinical findings using computed tomography, which document increased visceral fat in individuals with chronic SCI (Edwards et al., 2008). Visceral adipose tissue, including both retroperitoneal and perigonadal fat depots, was also measured by dissection and wet weight.  Significant differences were found in the amount of visceral fat accumulated with time after injury (F(5,28)=7.61, p<0.001). While there was no difference between sham and T3 SCI rats in the amount of visceral fat as a percentage of body weight one week after injury (p=0.315), one month after injury, T3 SCI rats had significantly more visceral fat than sham-injured controls (p=0.005, Figure 3.3a). Rats with low-thoracic SCI (T10) did not show increased visceral fat compared to sham-injured controls (p=0.924). A separate group of animals was used to determine whether perfusion with fixative prior to dissection would alter adipose tissue weight. Visceral fat weighed after perfusion made up 4.06 ± 0.32 % of body weight in T3-injured animals.  This was indistinguishable from the results obtained by fresh dissection in T3-injured animals (p>0.05). Analysis of visceral fat using preclinical MRI may not be feasible for the majority of laboratories. To ascertain whether the dissection of visceral fat was a good approximation of the amount of visceral fat as quantified by MRI, we examined the relationship between the amount of visceral fat quantified by MRI and by dissection.  In this data set, both measurements of visceral fat were performed in the same group of animals; animals were euthanized, MRI images were acquired, and then the dissection was performed.  There was a good correlation between the amount of visceral adipose tissue measured by MRI and that measured by dissection (r=0.92, p<0.0001, Figure 3.3b).  However, the mean   101 Figure 3.3 Visceral white adipose tissue dissection and wet weight measurement was sensitive to changes after SCI. Visceral white adipose tissue (vWAT), obtained by fresh dissection and measured by wet weight, was increased at one month following T3 SCI compared to sham-injured rats (a). Similar results were obtained in a second group of rats one month after T3 SCI (b); however, there was no change in visceral fat of animals with low-thoracic (T10) SCI. The amount of vWAT obtained by MRI correlated well with the amount obtained by dissection in the same group of animals (c). Symbols: * denotes significant difference from time-matched sham-injured animals and animals with low-thoracic SCI (both p<0.05).   102    103 difference between the two measurements was significantly more than zero (2.63 ± 0.22 %, t=11, df=10, p<0.0001).   The Bland-Altman analysis indicated that MRI quantification tended to underestimate visceral adipose tissue compared to dissection and that there was some discrepancy between MRI and dissection measurements of vWAT: 9% of points fell beyond two standard deviations away from the mean difference in % vWAT dissection (% vWAT dissection - % vWAT MRI) (Figure 3.3c).  Therefore, while both dissection and MRI provide sensitive measures of visceral adipose tissue, they should not be used interchangeably as there is some discrepancy between them. Blood lipid profiling is a standard component of clinical assessment of CV health and a well-accepted indicator of risk of CVD.  To date, clinical data on blood lipid profiles following SCI are limited (Brenes et al., 1986; Bauman et al., 1992; Krum et al., 1992; Bauman et al., 1999b), precluding a consensus on appropriate lipid profile indicators of CV risk for individuals with SCI. Blood lipid profiles are rarely reported in experimental SCI (Rouleau and Guertin, 2007); although rats are the most commonly used animal model, there are no available data on the blood lipid profile of rats after SCI. Blood lipids were first analysed using standard colorimetric hospital laboratory techniques. Blood samples were collected from non-fasted rats, and serum was separated for analysis. At one week post-injury, T3 SCI and sham-injured rats had similar levels of non-fasting serum triglycerides, HDL-C and LDL-C (Table 3.1).  However, at one month after surgery, the T3 SCI group had significantly higher triglycerides than both sham- injured (p=0.004) and T10 SCI (p<0.001) (Table 3.1).  This is particularly interesting, given that non-fasting triglycerides have emerged as important independent predictors of   104 Table 3.1 Non-fasting serum lipid concentrations in high-thoracic, low-thoracic and sham-injured rats reveal that rats with high-thoracic SCI have elevated triglyceride levels one month post-injury. No differences were observed between sham-injured rats and those with T3 SCI at one week post-injury.  At one month post-injury, animals with T3 SCI had increased serum triglyceride levels compared to both sham-injured rats and those with T10 SCI.  Animals with T10 SCI had significantly less HDL-C compared to sham-injured animals. Symbols: * indicates significant difference from time-matched sham-injured group; † indicates significant difference from T3 SCI group.  Note that LDL-C could not be calculated for this group using the Friedewald equation. Abbreviations: CHOL, cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol.   105   1 week 1 month  Sham (n=6) T3 SCI (n=6) Sham (n=6) T3 SCI (n=6) T10 SCI (n=5) Total CHOL (mmol/L) (mg/dL) 1.63 ± 0.09 (62.67 ± 3.61) 1.69 ± 0.16 (65.30 ± 6.31) 1.54 ± 0.12 (59.26 ± 4.65) 1.49 ± 0.07 (57.46 ± 2.88) 1.41 ± 0.08 (54.53 ± 3.06) HDL-C (mmol/L) (mg/dL) 0.62 ± 0.04 (23.97 ± 1.69) 0.67 ± 0.04 (25.84 ± 1.45) 0.59 ± 0.03 (22.75 ± 1.15) 0.55 ± 0.03 (21.28 ± 1.23) 0.47 ± 0.04* (18.20 ± 1.57) Calc. LDL-C (mmol/L) (mg/dL) 0.48 ± 0.17 (18.59 ± 6.66) 0.57 ± 0.22 (22.14 ± 8.58) 0.47 ± 0.11 (18.22 ± 4.36) x 0.55 ± 0.10 (21.34 ± 3.89) Triglycerides (mmol/L) (mg/dL) 1.39 ± 0.30 (123.11 ± 26.57) 1.34 ± 0.29 (118.54 ± 25.79) 1.17 ± 0.14 (103.92 ± 12.13) 2.45 ± 0.42* (216.85 ± 37.48) 0.78 ± 0.13† (69.44 ± 11.79)   106 CV events in several recent prospective clinical trials (Bansal et al., 2007; Nordestgaard et al., 2007).  Animals with T10 SCI, exhibited similar triglyceride levels to sham- controls (p>0.05), but lower levels than animals with T3 SCI (p<0.05), possibly suggesting a healthier cardiovascular profile.  On the other hand, their HDL-C levels were less than sham-controls, which confers reduced atheroprotective effects (Brewer, 2004).  The LDL-C levels in T3-injured rats at one month following SCI could not be calculated using the Friedewald formula because total cholesterol levels were undetectable with the eqipment used.  This highlights some of the limitations of applying clinical measurements to rodents. We next wanted to determine whether these results were reproducible using a home cholesterol test system (CardioChek).  These systems are relatively inexpensive and commercially available in the United States.  Their main advantage is that they provide immediate data from very small amounts of whole blood (such as the drop obtained from a tail vein): this permits repeated testing in the same animals without risk of morbidity due to blood loss.  CardioChek lipid profile analysis was performed on separate groups of T3 SCI and sham-injured pair-fed rats.  There was no difference in body weight between these two groups (p>0.05).  Using the CardioChek meter, triglyceride levels were significantly higher in T3 SCI rats than in sham-injured pair-fed control rats (Table 3.2; p=0.045).  There was no difference in HDL-C levels between sham- and T3-injured rats (p>0.05).  Total cholesterol levels in both groups fell below the detection range of the CardioCheck system; as a result, we were unable to calculate LDL-C levels.   107 Table 3.2 Non-fasting blood lipids measured using a home cholesterol test system showed similar results to laboratory quantification. Using a freely available home cholesterol test system (CardioChek Analyzer, Indianapolis, IN), rats with T3 SCI had significantly more triglycerides than sham-injured pair-fed controls.  There was no difference in HDL cholesterol between the two groups. Total cholesterol in both groups fell below the detection threshold of this system (<2.57 mmol/L).  As a result, we were unable to obtain the LDL cholesterol using this method, which is normally calculated using the Friedewald equation. Symbols: * indicates significant difference from time-matched sham-injured group.  x indicates LDL-C could not be calculated for this group using the Friedewald equation. Abbreviations: calc., calculated; CHOL, cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol.   108   Sham 1 month (n=7) T3 SCI 1 month (n=6) Total CHOL mmol/L (mg/dL) < 2.57 (<100) < 2.57 (<100) HDL-C mmol/L (mg/dL) 0.66 ± 0.13 (25.57 ± 4.82) 0.77 ± 0.10 (29.83 ± 4.03) Calc. LDL-C mmol/L (mg/dL) x x Triglycerides mmol/L (mg/dL) 1.31 ± 0.26 (115.57 ± 22.91) 2.72 ± 0.61 (241.33 ± 54.13)    109 3.4 Discussion Obesity is a common and worrisome secondary complication of SCI (Gater, 2007).  The accumulation of visceral adipose tissue, a known determinant of CV risk, has the potential to contribute to the early onset of CVD and shortened lifespan in this population (Myers et al., 2007; Garshick et al., 2005; Strauss et al., 2006).  In order to understand the etiology of increased adipose tissue and metabolic dysfunctions after SCI, and begin to address interventions, it is essential to have animal models of these aspects.  While rodents’ motor function has been well characterized after SCI, their autonomic function, including CV function, has been comparatively understudied (Maiorov et al., 1997a; Maiorov et al., 1998; Cameron et al., 2006; Lujan and DiCarlo, 2007; Inskip et al., 2009). Research addressing CV risk and metabolic changes after rodent SCI has been even more scant (Rodenbaugh et al., 2003; Primeaux et al., 2007; Rouleau and Guertin, 2007).  Here we show that, like humans, rats have increased visceral fat deposition one month after complete high-thoracic SCI.  We also demonstrate that rats with T3 SCI exhibit non- fasting hypertriglyceridemia.  These hallmarks of cardiometabolic risk are absent acutely after injury, but develop in the subacute period one month after SCI.  Over the same time period, rats with a complete low-thoracic injury do not accumulate visceral fat.  On the whole, their lipid profiles are not distinctly improved or worsened by their injury: their triglyceride levels are less than animals with T3 SCI, suggesting a healthier cardiovascular profile, yet their HDL-C levels are less than sham-injured animals, which confers less protection against atherosclerosis. The question of sheer energy imbalance after SCI is an important one that warrants further investigation.  It is tempting to conclude that the reduced energy expenditure   110 without compensatory change in nutrient intake is at the root of the pathophysiology of obesity after SCI.  Indeed, energy expenditure is reduced after SCI due to the reduction in activity and basal metabolic rate – both related in part to a reduced muscle mass (Buchholz et al., 2003a).  However, clinical findings in individuals with SCI indicate that total body fat is high even in young elite athletes, and visceral adiposity is increased in highly active members of the community (Edwards et al., 2008; Mojtahedi et al., 2009). The fact that these individuals have increased adiposity even with increased energy expenditure suggests there may be additional metabolic alterations beyond the raw changes in input-output balance.  Interruption of descending autonomic pathways and resultant loss of sympathetic regulation of fat tissue could be among the major factors precipitating altered lipolysis and redistribution of fat tissue after SCI.  Recent evidence has shown that sympathetic nerve activity, recorded from nerve endings in WAT, is partly responsible for mediating central glucagon-like peptide’s effects on lipid storage (Nogueiras et al., 2009).  The disruption of central neural control as a result of SCI could also alter lipid storage.  It is clear that more research is needed to untangle the effects of altered energy expenditure and of injury itself on metabolism after SCI.  Rats with high- thoracic SCI provide an appropriate model in which to do this, as energy expenditure can be modulated by many different exercise regimes and diets that are routinely used in the laboratory.  These types of experiments could inform our nutrition recommendations and exercise prescriptions for people with SCI. Our results with low- and high-thoracic SCI rats suggest that there might be metabolic changes associated with SCI beyond the raw changes in the input-output energy balance. Both T3 and T10 SCI animals were fed the same enriched diet throughout the   111 experiment.  All animals exhibited flaccid hindlimb paralysis and mobilized with their forelimbs only.  Nonetheless, rats with low-thoracic SCI did not exhibit either feature of cardiometabolic risk that we tested for; they did not develop visceral obesity, nor did they show any evidence of hypertriglyceridemia.  It is possible that rats with T10 SCI have a slightly higher basal metabolic rate and increased energy expenditure than rats with high- thoracic SCI due to their increased trunk muscle innervation and activity. However, we would expect that the difference between sham and T10-injured animals would be greater than the difference between T3- and T10-injured animals, given that both hind limbs are denervated by thoracic SCI. One possibility is that the loss of descending autonomic neural control over visceral adipose tissue, with spinal outflow exiting from the thoraco-lumbar cord, perturbs fat storage and release in T3 SCI rats.  It follows that low-thoracic SCI preserves descending neural control to a portion of the visceral adipose tissue rostral to the lesion, and thus does not disrupt fat storage and release in this area.  Given that T10 complete transection still eliminates descending neural control over caudal vWAT (gonadal/epidydimal WAT) (Youngstrom and Bartness, 1995), it is possible that rats with T10 SCI do experience cardiometabolic changes, but that these develop more slowly than in high-thoracic SCI due to the difference in descending sympathetic control.  It would be interesting to follow T10 SCI rats over a more chronic time period to see if they simply develop cardiometabolic changes more slowly; here we have only observed a very short time frame relative to the decades that many people live with SCI.  Taken together, our results suggest that there is a level-dependence associated with the development of visceral obesity and non-fasting hypertriglyceridemia following SCI – at least in the subacute   112 period after injury.  The lower thoracic cord may represent a critical spinal locus for regulated lipid metabolism. Alternatively, there may be a threshold number of thoracic spinal segments receiving descending autonomic neural control that are critical to regulate lipid metabolism.  An intermediate thoracic injury model would be needed to differentiate between these possibilities. While descending control of adipose tissue is lost caudal to complete SCI, innervation (by both sensory and sympathetic axons (Fishman and Dark, 1987; Youngstrom and Bartness, 1995)) is likely preserved.  Normally, sympathetic nerve endings are found in both adipose tissue parenchyma and vasculature (Slavin and Ballard, 1978; Giordano et al., 2005; Giordano et al., 2006), and sympathetic nerve activity is an important trigger for lipid mobilization in both rats and humans (Gross and Migliorini, 1977; Arner et al., 1990).  After autonomically complete SCI, resting muscle (Wallin and Stjernberg, 1984) and visceral (Osborn et al., 1987; Maiorov et al., 1997a) sympathetic nerve activity are low below the lesion, as are plasma catecholamines (Mathias et al., 1976; Mizushima et al., 2003; Claydon and Krassioukov, 2006; Wecht et al., 2008).  It is likely that there is increased storage and reduced lipid mobilization as a result.  Indeed, after sympathetic denervation we know that white adipose depots increase in size (Youngstrom and Bartness, 1998).  Reflex bursts of sympathetic activity that often accompany high SCI likely subserve some lipid mobilization (Karlsson et al., 1997; Niijima, 1998, 1999); however, the overall level of sympathetic activity will still be quite low. Previous work documenting body composition and adiposity after rodent spinal cord injury reported that T3 SCI results in significant weight loss, with animals not returning to pre-operative weights until 5 weeks post-injury (Primeaux et al., 2007). These animals   113 maintained chronically low weights, and even 16 weeks post-injury were not heavier than their pre-operative weights (Primeaux et al., 2007).  In contrast with the current findings, SCI rats had significantly less visceral adipose tissue than sham-injured controls (Primeaux et al., 2007).  Although the injury was the same, the pattern of post-operative weight gain we observed was much different: rats with T3 SCI regained their pre- operative weight by one week post-injury, and their weights were statistically indistinguishable from sham controls one month after injury. While it is unlikely to explain all of the discrepancies between our results, there are important diet differences between the two studies.  Rats in the study by Primeaux and colleagues were fed a strictly chow-based diet; in our experiments, all rats were fed a diet more reminiscent of the unrestricted human “cafeteria-style” diet.  In the presence of this enriched diet, rats with complete T3 SCI showed significantly more visceral fat than pair fed sham control rats. An intriguing possibility is that, following SCI, if the system is challenged by a large caloric load, there are more triglycerides stored than can be mobilized by reflex sympathetic activity: the system can no longer cope, such that triglyceride storage vastly outweighs mobilization. In addition to increased visceral fat, complete T3 SCI rats also displayed increased non- fasting triglyceride levels – a finding which could suggest that lipid clearance is perturbed.  Indeed, postprandial hypertriglyceridemia has been reported previously in humans with SCI after a high caloric milkshake (Nash et al., 2005), also suggesting that lipid metabolism can be disrupted by SCI.  This is a clinical concern, as postprandial lipemia is associated with endothelial dysfunction and the development of atherosclerosis (Groot et al., 1991; Patsch et al., 1992).  As elevated triglycerides are rarely elevated in   114 the absence of other atherogenic features (Grundy, 1998; Despres, 2007), the increased non-fasting triglyceride levels that we have observed at one month after T3 SCI could represent the onset of cardiometabolic changes after SCI.  Interestingly, as in the case of visceral fat, rats with T10 SCI did not show increased non-fasting triglycerides compared to age-matched pair-fed controls.  Again, we cannot rule out the possibility that this group is simply slower to develop metabolic changes after SCI.  However, the established relationship between poor postprandial triglyceride clearance and visceral obesity (Couillard et al., 1998; Blackburn et al., 2003) suggests that visceral obesity itself could drive the poor triglyceride clearance after T3 SCI – a feature absent after low-thoracic SCI. The results from our animal experiments strongly suggest that in clinical studies we should stratify our subjects based on their neurological injury level, and not simply their limb function.  The division of clinical subjects as paraplegic or quadriplegic results in heterogeneous groups, with a range of injury levels.  This division does not give us any information regarding the true neurological injury level.  In our results, while both high- and low-thoracic SCI affect lower limb function and result in paraplegia, these injuries have very different effects on fat accumulation and triglyceride levels.  Therefore, at the very least, neurological injury level must be reported in studies of lipid metabolism – ideally in combination with level of sympathetic dysfunction.  With the use of a complete SCI used in this experimental study, we knew the level of injury and that the injury was sympathetically complete; we cannot make these same assumptions in clinical research, necessitating sympathetic testing.   115 Despite the prevalence of CVD in people with SCI and the overwhelming impact on lifespan, cardiometabolic outcomes are rarely documented in experimental animal models of SCI.  Here we have shown that specialised equipment is not always necessary for the assessment of cardiometabolic health in animals; fairly simple and affordable techniques exist that are sensitive to the changes that occur after SCI. Serum HDL-C and triglycerides can be quantified in a minimally invasive way using standard human home testing kits.  Increased visceral adiposity can be detected by gross fat pad dissection and wet weight either before or after fixation (depending on existing protocols).  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These physiological compensations are modulated by a combination of endocrine and neural signals.  Spinal cord injury (SCI) can mechanically interrupt the neural pathways between the brain and the peripheral organs but it spares the humoral lines of communication.  We have a limited understanding of the way that the body regulates energy metabolism in the absence, or with the partial disruption, of descending neural control. It is important to appreciate the unique anatomy of the autonomic nervous system in order to understand how it can be affected by SCI at different levels.  Parasympathetic outflow to the visceral organs exits the central nervous system at the level of the brainstem; as a result, it is generally spared by SCI.  Sympathetic outflow exits the cord between T1 and L2 levels (Strack et al., 1988); therefore, high- and low-thoracic spinal cord injuries can have profoundly different effects on the sympathetic nervous system (SNS).  High-thoracic injuries can disrupt most descending control from the brain to the  2 A version of this chapter will be submitted for publication.  Inskip, J.A., Plunet, W., Ramer, L.M., Krassioukov, A.V. and Ramer, M.S. (2010) Energy expenditure, circadian rhythms, and carbohydrate metabolism after thoracic spinal cord injury.   124 preganglionic sympathetic neurons residing in the spinal cord, while low-thoracic injuries leave the bulk of the SNS under descending neural control from the brain.  Most organs are innervated by both parasympathetic and sympathetic nerves, and require the coordinated activity of these two branches in order to function normally.  Following SCI, there is typically a loss of balance between these two branches; immune function in the spleen for example, is impaired by high-, but not mid-, thoracic SCI – likely an effect of sympathetic dysregulation (Lucin et al., 2007).  We do not know whether the level of SCI, and resultant SNS dysfunction, affects whole body carbohydrate metabolism. The intact autonomic nervous system also plays an essential effector role in a number of daily rhythms governed by the biological clock in the suprachiasmatic nucleus (SCN) (Chan et al., 1991; Warren et al., 1994; Makino et al., 1997; Cailotto et al., 2005).  The circadian rise in blood glucose, for example, which occurs just before the start of the active period in both rats and humans, is the result of increased hepatic glucose production mediated by an increase in sympathetic activity to the liver (Kalsbeek et al., 2004; Cailotto et al., 2005).  The liver requires a balance of autonomic activity; if either the sympathetic or parasympathetic innervation is disrupted the circadian glucose rhythm is abolished (Cailotto et al., 2008).  It is possible that after high-level SCI, which effectively separates the distal SNS from the brain, the endogenous rise in blood glucose could be abolished. The maintenance of circadian rhythms is important for many aspects of health. Chronic disruption, phase-shift, or total loss of these biological patterns has been correlated with a number of health complications, including obesity, diabetes, metabolic syndrome and cardiovascular disease (CVD) (Aronson, 2001; Ruger and Scheer, 2009).  Given the high   125 incidence of these pathologies among the SCI population (Bauman and Spungen, 1994; Bauman et al., 1999a; Lee et al., 2005; Gater, 2007), and evidence that several hormonal and hematogenous circadian rhythms are disrupted or absent after SCI (Krum et al., 1991; Munakata et al., 1997; Iversen et al., 2002; Hjeltnes et al., 2005), we hypothesized that other circadian rhythms, including blood glucose, activity and core temperature might also be disrupted by SCI. We have recently shown that T3 SCI in rats results in increased adipose tissue and hypertriglyceridemia as early as one month post-injury (Inskip et al., 2010).  The purpose of our current study was three-fold: 1) to quantify some of the major components of energy expenditure and energy consumption by animals with high-thoracic (T3) SCI, low-thoracic (T10) SCI and sham-injured controls; 2) determine whether major circadian rhythms are maintained following T3 and T10 SCI; 3) examine carbohydrate metabolism after T3 and T10 SCI. Given that the liver and pancreas receive the bulk of their sympathetic innervation from mid-thoracic cord (T6-T12), the essential role of these organs in glucose metabolism, and the high prevalence of carbohydrate and metabolic complications in clinical SCI, we hypothesized that carbohydrate metabolism would be compromised by high-thoracic SCI, resulting in greater latency to clear glucose from the blood, and poorer responsiveness to insulin. 4.2 Materials and methods All animal procedures were performed in accordance with the guidelines of the Canadian Council for Animal Care and approved by the University of British Columbia Animal   126 Care Committee.  Male Wistar rats (250-350g, n=36) were obtained from Charles River Laboratories, Montreal, QC. 4.2.1 Surgery and animal care Prophylactic enrofloxacin (Baytril; 10 mg/kg, subcutaneous (s.c.), Associated Veterinary Purchasing (AVP), Langley, BC) was administered for three days prior to surgery.  Rats were anesthetized with ketamine hydrochloride (Vetalar; 70 mg/kg, intraperitoneal (i.p.), AVP) and medetomidine hydrochloride (Domitor; 0.5 mg/kg, i.p., AVP).  Both buprenorphine (Temgesic; 0.02 mg/kg, s.c., AVP) and ketoprofen (Anafen; 5 mg/kg, s.c., AVP) were also administered immediately prior to surgery.  A complete transection was performed at T3 (n=12) or T10 (n=12) or sham surgery was performed at T3 (n=12). A complete spinal cord transection was performed at the third (T3) or tenth (T10) thoracic segment.  In both surgeries, a dorsal midline incision was made in the skin and superficial muscles overlying vertebrae were separated by blunt dissection. For T3 transection, the T2 dorsal spinous process was removed, and the durotomy and transection was performed in the T2-T3 intervertebral gap.  For T10 transection, a partial laminectomy of the T9 vertebrae was performed, followed by durotomy and transection. Complete transection was confirmed under the dissecting microscope by the clear division of the rostral and caudal stumps of the cord, and gelfoam (Pharmacia & Upjohn Company, Pfizer, New York, NY) was inserted in this space.  The muscle and skin layers were closed with sutures.  Sham surgery included both the partial laminectomy of the T2 vertebra and durotomy at T3.   127 After surgery, anesthesia was reversed with atipamezole hydrochloride (Antisedan; 1 mg/kg, s.c., Novartis, Mississauga, ON).  Animals were given warmed Lactated Ringer’s solution (5 mL, s.c.) and were placed in a temperature-controlled chamber (Animal Intensive Care Unit, HotSpot for Birds, Los Angeles, CA).  Both buprenorphine (0.02 mg/kg, s.c.) and ketoprofen (5 mg/kg, s.c.) were administered once daily for three days to manage post-operative pain.  Antibiotic treatment (enrofloxacin, 10 mg/kg, s.c.) was also continued for three days to reduce the incidence of bladder infection. During the first week post-injury, manual bladder expression was performed three times daily.  After one week, this was reduced to twice daily until spontaneous bladder function returned (about 10 days post-injury).  Skin sutures were removed one week after surgery to minimize skin irritation.  Rats were monitored daily for the first two weeks after surgery, and every second day thereafter.  This included assessment of body weight, activity level, social behaviour, lesion site and clinical signs of morbidity.  A three-day course of systemic antibiotics (enrofloxacin, 10 mg/kg, s.c.) was administered to animals showing signs of bladder infection.  If necessary, topical antibiotics (Triple Antibiotic Ointment; polymixin B sulfate, bacitracin zinc and gramicidin) and hydrocortisone cream (Cortate; 0.5% hydrocortisone) were used daily to treat dermatitis. 4.2.2 Telemetry A subset of animals (n=5 per group, one animal per cage) had a telemetric capsule (ER- 4000 E-Mitter, Mini Mitter, OR) implanted into the abdominal cavity, for collection of core temperature and activity data.  Capsules were implanted one week prior to SCI to obtain preoperative recording and minimize infection from the incision site being   128 dragged through bedding. Animals were given prophylactic antibiotics (enrofloxacin, 10 mg/kg s.c.) for three days prior to surgery. Animals were anesthetized using isoflurane (Aerrane; 5% induction, 1-2% maintenance in 100% O2, AVP) and administered buprenorphine (0.02 mg/kg, s.c.) prior to surgery.  A small midline incision was made and the abdominal muscle was opened with an incision along the linea abla fascia.  The sterilized telemetric probe, a sealed bio-inert capsule made of laboratory grade glass with biocompatible silastic coating (total volume=1.2mL, total weight <2g), was inserted into the abdominal cavity and placed dorsal to the small intestine just off the midline.  The small intestine was deflected back into place and the abdominal muscles and skin were sutured closed. Cages were kept on the telemetry base (ER-4000 receiver), which acquires data from the implanted capsule and emits radiowaves to power the E-Mitter.  Activity was recorded in counts, which provide a qualitative measure of movement: each count is the result of a change in signal strength between the transponder and receiver (Harkin et al., 2002). Therefore, there is no unit of magnitude or direction associated with these units. Temperature and activity data were collected every minute using the VitalView™ data acquisition system.  Minute data was averaged over each hour to get a sense of daily patterns.  In order to look at gross changes over time, but maintain daily rhythm information, hourly data were averaged weekly.  Total daily activity was also calculated by combining the average hourly activity throughout each day and averaged weekly.   129 4.2.3 Housing All rats were pair-housed, as single housing is stressful for rodents, and they neither behave normally nor display normal eating habits when stressed.  Cages (38x20x59 cm) were kept on top of telemetric recorders in a room maintained at 22°, with a 12h:12h light-dark cycle (lights on at 07:00h).  Cages were fitted with a fenestrated rubber matting to facilitate mobilization of animals with SCI (Multy Tile, Multy Industries, Atlanta, GA).  Modified low-reaching water bottles ensured easy access for all animals. All animals received a standardized diet that was initiated three days pre-operatively to let them acclimate to the novel food.  Three times daily, each cage received 30 mL complete meal replacement shake (strawberry), 20 g nutritive transport gel (Charles River Laboratories International Inc., Wilmington, MA), 30 g fruit (orange and apple), 6 g cereal (Cheerios) or 12 g commercially available rat treats (alternating), and rodent kibble ad libitum (LabDiet, Rodent Diet 5001).  Two weeks post-injury, the meal replacement shake and nutritive transport gel were removed from the diet.  Rodent kibble and water were provided ad libitum for the duration of the experiment. 4.2.4 Calorie consumption Twenty-four hour calorie consumption was measured once a week.  To maintain as many features as possible consistent with normal behaviour, consumption was measured in pairs.  On the day of testing, animals were transferred into clean cages, with a standard amount of food present.  Food was administered throughout the day as above.  At the end of the 24-hour period, animals were transferred to clean cages, all remaining food was weighed, and calories were calculated as per nutrition information provided by the manufacturers.   130 4.2.5 Blood sampling All blood samples were taken from the distal third of the lateral tail.  The surface of the tail was prepared using a cotton swab with 70% ethanol, and venipuncture was performed using a 25G needle, inserted at a shallow angle.  Non-fasting blood biomarkers were tested near the beginning of the light cycle pre-injury, at one month and three months after injury.  Animals were briefly anesthetized (4% Isoflurane in 95% O2 at 1L/min) and loosely restrained using a clean tea towel.  Glucose testing was performed using human handheld monitors.  Blood was collected using the disposable strips that accompany the glucose monitor (which requires <0.01mL).  Fasting blood biomarkers (12-hour overnight fast) were tested using the same protocol and equipment. 4.2.6 Glucose tolerance test A glucose tolerance test (GTT) was performed preoperatively, at one month and three months post-injury. A glucose solution (40% D-(+)-Glucose (G8270, Sigma-Aldrich, Oakville, ON) in sterile dH20) was prepared the day before each test and left at room temperature overnight.  This ensured that the glucose was predominantly in its β-form, which is more readily transported than its α-isomer (Nelson and Cox, 2005).  Following a 12-hour overnight fast, rats were administered an i.p. glucose load (2g/kg body weight in distilled water) (Nakhooda et al., 1983; Zisman et al., 2000; Diehl et al., 2001).  Blood glucose was measured immediately before, and 30, 60, 90 and 120 minutes after the i.p. glucose injection using a human glucose meter (Accu-chek Aviva, Roche, Mannheim, Germany).  A rapid return to baseline glucose levels indicates higher glucose sensitivity; this is measured by the raw glucose levels at each time point or by the overall area under the glucose curve (AUC) during the two-hour test.   131 4.2.7 Insulin tolerance test An insulin tolerance test (ITT) was performed preoperatively, at one month and three months post-injury.  Following a 12-hour overnight fast, rats were administered a bolus i.p. insulin injection (0.75 IU/kg in 0.1% NaCl at room temperature) (Zisman et al., 2000; Ai et al., 2005; Zhang et al., 2008).  Blood glucose was measured immediately before, and 30, 60 and 120 minutes after the i.p. insulin injection using a human glucose meter (Accu-chek Aviva).  The ITT provides an index of the effects of insulin on both the liver and the peripheral tissues.  The rate constant for the disappearance of glucose (KITT) was calculated as the slope of the reduction in log transformed blood glucose values over the linear portion of the decline in glucose, multiplied by 100.  Therefore, a larger rate constant indicates greater insulin mediated glucose disposal. 4.2.8 Circadian glucose At three months post-injury blood samples were taken via tail venipuncture every two hours for 24 hours and measured using a glucometer (Accu-chek Aviva). 4.2.9 Data analysis All data are presented as means ± standard error of the mean (SEM).  Data analysis was performed using Prism 5 for Mac OS X (GraphPad Software Inc., San Diego, CA).  Two- way repeated measures analysis of variance (RM-ANOVA) was used to compare calorie intake, temperature, activity, and blood glucose data between groups.  When a main difference between groups was found, the Bonferroni post-test was used for pairwise comparisons between groups.  Where time effects were noted, one-way RM-ANOVA   132 was used to identify circadian rhythms within groups, with the Bonferroni post-test to compare baseline light-period values (07:00h) and dark-period (19:00h-06:00h) values. 4.3 Results 4.3.1 Calorie consumption and body weight All groups of animals consumed a similar amount of calories throughout the experiment; there was no effect of SCI on total food consumption (Figure 4.1a, p>0.05).  More calories were consumed in the early weeks compared to the later weeks post-injury (Figure 4.1a, all p<0.0001). Following surgery, animals with T3 SCI and those with T10 SCI both lost a moderate amount of weight (Figure 4.1b); it took 11 and 12 days, respectively, for these groups of animals to return to their pre-operative weights.  Sham animals did not lose any weight subsequent to surgery (Figure 4.1b).  This weight divergence lasted throughout the experiment to three months post-injury, when sham-injured animals weighed more than both animals with T3 SCI (p<0.01) and those with T10 SCI (p<0.001). 4.3.2 Core temperature and circadian temperature rhythms Core temperature was depressed during a part of the light phase in the first week following high-thoracic SCI compared to pre-injury temperature at the same time (p=0.0284, Figure 4.2a,b).  However, there was no significant effect of SCI on core temperature (p>0.05).  During the first week post-injury, circadian temperature changes were maintained in T3 SCI (p<0.05 between 07:00h and 19:00h) and T10 SCI animals (p<0.001), but these patterns were abolished in sham animals (p>0.05, Figure 4.2b). By two weeks post-injury, all groups displayed nocturnal increases in core temperature and   133 this pattern was maintained for the duration of the experiment. Throughout the 12-week experiment, there was a slight rise in temperature just around 09:00h (Figure 4.2b-e). Figure 4.1 Calorie consumption and body weight throughout the 12-week experiment. Calorie consumption (a) was measured once a week over a 24-hour period.  Calories were obtained from an enriched diet including complete meal replacement shake, nutritive transport gel, fruit, cereal, commercially available rat treats and rodent kibble. There was no overall effect of SCI on calorie consumption. Body weight (b) was recorded daily for the first two weeks post-injury, every two days for the next two weeks, and every four days for the duration of the experiment.  Both T3- and T10-injured animals showed some weight loss following injury; however, preoperative body weights were recovered by 11 and 12 days post-operatively, respectively.  For the majority of the experiment, sham-injured animals remained heavier than animals with T3 SCI but not T10 SCI.  At 12 weeks post-injury, sham-injured animals were heavier than both animals with T3 SCI and those with T10 SCI.   134   135 Figure 4.2 Core temperature was decreased by T3 SCI but was normalized by two weeks post-injury. Core body temperature was recorded using a telemetric transponder implanted into the abdominal cavity.  Preoperative temperatures (a) were indistinguishable between groups. The nocturnal increase in body temperature was evident in preoperative temperatures.  At one-week post-injury (b) the core temperature of animals with T3 SCI was significantly lower than preoperative values.  The nocturnal increase in temperature was present in animals with T3 SCI and those with T10 SCI, but was absent in sham-injured animals. At two-weeks post-injury (c) preoperative temperatures were restored and the nocturnal rise in temperature was present in all groups.  These patterns were maintained to six- weeks post-injury (d).  At this time, the thermic effect of food can also be seen, with a peak in body temperature occurring just after the morning feeding (between 09:00 and 10:00h).  Similar temperature patterns were maintained through to the end of the experiment (12 weeks post-injury, e).    136    137 This peak in temperature, coincident with a peak in activity (see Figure 4.3), was likely an effect of the disruption associated with the first daily dose of food and cage changing. 4.3.3 Circadian activity patterns The circadian rhythm in locomotive activity was evident in data collected preoperatively (Figure 4.3a).   There was a significant main effect of time on activity levels (p<0.0001) and one way RM-ANOVA post-tests revealed an increase in activity in the dark period compared to light period in all groups (sham, p<0.0001; T3, p<0.0001; T10, p<0.0001). RM-ANOVAs were conducted for each group individually to determine whether activity levels demonstrated nocturnal rhythmicity.  The nocturnal increase in activity level was abolished in the first week following T3 SCI (p>0.05; Figure 4.3b).  However, this rhythm was restored by two weeks post-injury (p<0.001) and was maintained for the rest of the experiment (Figure 4.3c-e).  Nocturnal activity rhythms were preserved in animals with T10 SCI in the first week after injury (p=0.0012) and throughout the experiment, except for at three weeks post-injury, when there was no difference in activity levels between dark and light periods.  Nocturnal activity was increased in sham-injured animals at one week post-injury (p<0.0001) and this pattern was maintained throughout the experiment (all p<0.05; Figure 4.3). The effects of enriched food (first given each day at 09:00h) were seen reflected in the activity levels, which showed a coincident rise at this time (Figure 4.3).  The resultant overall pattern was biphasic, with peak activity levels at approximately 09:00h and 23:00h daily.    138 Figure 4.3 Nocturnal activity patterns were not disrupted by high- or low-thoracic SCI. Activity levels were recorded as a surrogate measure of energy expenditure using telemetry.  Activity counts were averaged every minute, and the average counts per minute were graphed for each hour during the day.  Preoperative activity levels were not different between groups (a).  All groups displayed greater activity levels during the dark cycle (a).  One week post-injury (b) the nocturnal increase in activity level was abolished in animals with T3 SCI, but was present in animals with T10 SCI and sham controls. All groups demonstrated a rise in activity levels during the dark cycle at two weeks post- injury (c).  Similar trends were maintained through six (d) and 12 weeks post-injury (e). In all weeks post-injury (b-e), a rise in activity levels was observed just after the morning feeding, which occurred between 09:00 and 10:00h.  This is likely an effect of excitement and physical activity in response to the enriched food that was given at this time.     139    140 4.3.4 Total daily activity Preoperative total daily activity was equal between sham, T3 and T10 SCI groups (all p>0.05, Figure 4.4).  As expected, average total daily activity levels were significantly decreased in the first week after both T3 and T10 SCI compared to sham-control activity during this time (T3, p<0.001; T10, p<0.001, Figure 4.4).  Total activity remained low in T3 SCI animals compared to sham, at two (p<0.01), three (p<0.05) and six weeks post- injury (p<0.05; Figure 4.4).  At all other weeks (4,5,7-12), there was no difference in average daily activity levels between animals with T3 SCI and those with sham injury (all p>0.05).  The total activity of animals with T10 SCI was quite variable in the early weeks post-injury (Figure 4.4).  After the first week post-injury, the average daily activity was indistinguishable from sham activity levels at weeks 2-6, 8, and 10-12 post-injury (all p>0.05); activity levels were lower in animals with T10 SCI at 7 and 9 weeks post-injury (both p<0.05).  Total activity levels were indistinguishable between animals with T3 SCI and those with and T10 SCI throughout the 12-week experiment (p>0.05 every week post-injury). The comparison of post-operative activity levels for each group relative to their pre- injury levels was also informative, and perhaps more appropriate given the variability in activity levels between animals (Refinetti, 1989).  One-way RM-ANOVA revealed that T3 activity was reduced at all weeks compared to pre-injury (p<0.01 at all time points), while T10 activity was reduced for the majority of time post-injury (at one week post- injury, and weeks 5-12 (all p<0.05).  Sham activity levels were not different between pre- and post-operative periods (all p>0.05).   141 Figure 4.4 Average daily activity was reduced by both high- and low-thoracic SCI acutely after injury. Preoperative average daily activity was not different between groups.  Total activity levels of animals with T3 SCI and animals with T10 SCI were significantly reduced in the first week post-injury.  Total activity in both of these groups gradually returned to levels that were not statistically different from sham-injured animals.  There was no difference between the activity levels of animals with T3 SCI and those with T10 SCI throughout the experiment. Symbols: grey bar indicates significant difference between animals with T3 SCI and sham controls (p<0.05); black bar indicates significant difference between animals with T10 SCI and sham controls (p<0.05).   142      143 4.3.5 Circadian blood glucose Blood glucose in all three animal groups displayed the classic diurnal pattern, peaking just before the start of the dark cycle and the onset of the activity period (Sham n=11; T3 n=10; T10 n=9; Figure 4.5).  There was a significant effect of time on blood glucose (p<0.001); one-way RM-ANOVAs and pair-wise comparisons between 07:00 and 19:00h confirmed significant circadian blood glucose rhythms in sham (p<0.0001), T3 SCI (p<0.0001), and T10 SCI (p<0.001).  Qualitatively, animals with T10 SCI did not show a gradual increase in glucose throughout the afternoon, however, quantitative analysis revealed no significant effect of SCI on blood glucose patterns (p>0.05). 4.3.6 Glucose sensitivity Preoperative intraperitoneal (i.p.) glucose injection resulted in a rapid rise in blood glucose, followed by a gradual return towards baseline glucose levels over 90 minutes. There was no effect of SCI on blood glucose levels during the test (p>0.05, Figure 4.6a). Similarly, the total area under the curve (AUC), which provides a single measurement of the overall glucose response, was not different between groups (p>0.05, Figure 4.6d).  A smaller AUC suggests more rapid clearing from blood, and therefore a greater sensitivity to glucose administration, which is likely, but not definitively, due to greater insulin sensitivity. At one month post-injury, there was a difference in blood glucose levels between groups (p<0.0001).  Animals with T3 SCI had reduced glucose levels compared to sham-injured animals at 30, 60 and 90 minutes post-injection (all p<0.001).  Blood glucose was normalized by 120 minutes post-injection (p>0.05).  Animals with T10 SCI showed a similar trend: they had reduced glucose levels compared to sham-injured animals at 30   144 Figure 4.5 Circadian blood glucose variations were not disrupted by high- or low- thoracic SCI. At three months post-injury, circadian blood glucose was recorded every two hours using a human glucometer.  All animals displayed the classic peak in blood glucose just prior to the beginning of the active period.  There was no effect of SCI on blood glucose concentrations. Symbols: white bar denotes significant difference in blood glucose levels of sham controls from baseline morning levels (07:00h) (p<0.05); black bar denotes significant difference in blood glucose levels of animals with T3 SCI from baseline levels (07:00h) (p<0.05); grey bar denotes significant difference in blood glucose levels of animals with T10 SCI from baseline levels (07:00h) (p<0.05).   145        146 Figure 4.6 Animals with high- and low-thoracic SCI displayed improved glucose tolerance compared to sham-injured animals. An intraperitoneal (i.p.) glucose injection was used to assess glucose tolerance (glucose tolerance test, GTT.  When conducted preoperatively, the GTT resulted in a rise in blood glucose followed by a gradual return to baseline levels (a).  There was no difference in baseline blood glucose levels between groups.  One month post-injury (b), animals with T3 SCI exhibited smaller rise in glucose levels compared to both sham-injured animals and animals with T10 SCI, suggesting improved glucose tolerance.  Animals with T10 SCI exhibited a smaller increase in glucose levels compared to sham controls. At three months post-injury (c), animals with T3 SCI had lower glucose levels than sham controls throughout the majority of the GTT, while animals with T10 SCI had lower glucose levels compared to sham controls in the later half of the test. The total area under the curve (AUC) of the glucose response was also calculated (d). This measure provides an overall assessment of glucose sensitivity, with a lower AUC indicating greater glucose sensitivity.  Animals with T3 SCI and those with T10 SCI had lower AUCs than sham-injured animals at one and three months post-injury.  Animals with T3 SCI were more sensitive than those with T10 SCI at one month post-injury. Symbols: ** denotes difference between both sham-injured animals and animals with T3 SCI and sham-injured animals and those with T10 SCI (p<0.05); x denotes difference between sham-injured animals and those with T3 SCI (p<0.01); # denotes difference between sham-injured animals and those with T10 SCI (p<0.01); * denotes difference between the two indicated groups (p<0.05). Abbrevations: Pre-op, preoperative; AUC, area under the curve.   147    148 (p<0.001), 60 (p<0.01) and 90 minutes post-injection (p<0.05), but there was no difference by the end of the test (120 minutes; p>0.05).  There was an early difference between animals with T3 SCI and those with T10 SCI; the former had lower fasting glucose levels at the beginning of the test (p<0.05), and lower glucose levels at 30 minutes post-injection (p<0.001, Figure 4.6b).  The total AUC reflected these differences; one month post-injury, animals with T3 SCI were more sensitive to glucose than animals with T10 SCI (p<0.05) and sham controls (p<0.001, Figure 4.6d).  Animals with T10 SCI were more glucose sensitive than sham-injured animals (p<0.01). Similar trends persisted at three months post-injury (Figure 4.6c).  Animals with T3 SCI had reduced blood glucose compared to sham-injured animals at 30 (p<0.01), 60 (p<0.001), and 90 minutes post-injection (p<0.001, Figure 4.6c). Animals with T10 SCI also had reduced glucose levels compared to sham controls; however, this occurred later in the test, at 60 (p<0.01), 90 (p<0.05) and 120 minutes post-injection (p<0.01, Figure 4.6c).  Total AUCs also revealed that animals with T3 SCI and those with T10 SCI were both more sensitive to glucose administration than sham controls (p<0.01, p<0.05). There was no difference between the two groups with SCI (p>0.05). 4.3.7 Insulin-induced hypoglycemia Preoperative insulin injection did not reveal differences between groups in raw glucose response (p>0.05, Figure 4.7a) or rates of glucose disposal (slope, KITT) (p>0.05, Figure 4.7d).  At one month post-injury, there was a significant effect of SCI on the glucose response to insulin administration (p<0.0001, Figure 4.7b).  Blood glucose levels of T3 SCI animals were significantly lower than sham-injured animals at all times following insulin injection (all p<0.001), and those with T10 SCI (p<0.05 at 30 min, p<0.01 at 60   149 Figure 4.7 Animals with T3 SCI and animals with T10 SCI displayed rapid insulin- induced hypoglycemia at one and three months post-injury, respectively. An intraperitoneal (i.p.) insulin tolerance test (ITT) was used to assess the insulin sensitivity following SCI.  The glucose response to insulin was measured using a human glucometer.  The preoperative response to i.p.insulin was indistinguishable between groups (a).  At one month post-injury (b), animals with T3 SCI demonstrated a greater sensitivity to insulin, as seen by their larger drop in blood glucose levels throughout the test.  By three months post-injury (c), there were no differences in blood glucose levels between groups.  The rate of glucose disposal (slope, KITT, d) provides a single measure of insulin sensitivity, with a higher KITT indicating a more rapid glucose disappearance rate.  One month post-injury, animals with T3 SCI had a larger KITT than animals with T10 SCI and sham controls.  At three months following SCI this trend was altered and animals with T10 SCI showed the greatest sensitivity to insulin. Symbols: x denotes significant difference between animals with T3 SCI and sham controls (p<0.05); * denotes significant difference between the two indicated groups (p<0.05). Abbrevations: Pre-op, preoperatively; KITT, rate of glucose disposal.   150    151 min and 120 min).  There was no difference between glucose levels in sham-injured animals and those with T10 SCI (all p>0.05).  These relationships were also reflected by the overall rate of glucose disposal (KITT, Figure 4.7d). The pattern of insulin sensitivity that emerged at one month post-injury was abolished by three months post-injury, at which time there was no overall effect of SCI on blood glucose levels during the insulin tolerance test (p>0.05, Figure 4.7c).  However, animals with T10 SCI experienced a more rapid decline in blood glucose in response to insulin compared to sham controls (p<0.05). 4.3.8 Fasting and non-fasting blood glucose To determine whether the insulin-induced hypoglycemia results were applicable to endogenously-triggered hypoglycemia, we measured blood glucose levels in a fed state and following a 12-hour fast.  Preoperative fasting glucose was not different between groups (sham 6.04 ± 0.27, T3 6.02 ± 0.25, T10 5.87 ± 0.15, p=0.872). At one month post-injury, there was no difference between groups in fasting or non- fasting glucose levels  (p>0.05, Table 4.1).  All groups experienced a decrease in blood glucose in response to fasting (sham p<0.0001, T3 SCI p<0.0001, T10 SCI p=0.0011). Animals with T3 SCI did not show an accentuated hypoglycemic response, as was seen in the acute insulin tolerance test, which was also performed at one month post-injury. At three months post-injury, non-fasting glucose levels were significantly lower in animals with T10 SCI than in sham-injured animals (p<0.001, Table 4.1).  Again, sham- injured animals suffered mild hypoglycemia when fasting (p=0.03).  Animals with T3   152 Table 4.1 Animals with SCI experienced mild hypoglycemia in response to a 12- hour fast at one month post-injury, but not at three months post-injury. Blood glucose levels were assessed in a non-fasting situation and following a 12-hour fast.  All animals exhibited mild hypoglycemia in response to a 12-hour fast at one month post-injury.  At three months post-injury, sham-injured animals again exhibited mild hypoglycemia in response to fasting.  At this time, animals with T3 SCI and those with T10 SCI did not experience any change in blood glucose. Symbols: * denotes significantly different from non-fasting values at the same time post- injury (p<0.05); † denotes significantly different from sham control group (p<0.05).   153  Time post-injury Blood glucose (mmol/L) Sham (n=11) T3 SCI (n=10) T10 SCI (n=9) Non-fasting 7.82 ± 0.29 7.57 ± 0.25 7.09 ± 0.19 1 month 12-hour fast 5.68 ± 0.17* 5.33 ± 0.11* 6.24 ± 0.143* Non-fasting 6.64 ± 0.14 6.21 ± 0.11 5.91 ± 0.06† 3 months 12-hour fast 6.34 ± 0.13* 5.97 ± 0.20 6.14 ± 0.14    154 SCI and T10 SCI, on the other hand, did not show decreases in blood glucose (T3 SCI p=0.29 and T10 SCI p=0.16). 4.3.9 Fasting and non-fasting blood ketones Circulating ketones are typically low in fed humans and rodents (Robinson and Williamson, 1980).  During prolonged fasting, ketone body production by the liver is increased and ketones are used by some tissues as an alternative energy source to glucose (Olpin, 2004).  Consistent with the general role of ketones, all groups showed low ketone levels under non-fasting conditions at one month post-injury (Figure 4.8a).  There was no difference in ketone levels between groups (p>0.05).  Compared to non-fasting values, all groups showed an increase in ketones following a 12-hour fast (all p<0.001, Figure 4.8b). At this time, animals with T3 SCI had significantly higher blood ketones compared to both sham-injured animals and those with T10 SCI (both p<0.001).  Animals with T10 SCI, on the other hand, had lower blood ketones than sham-injured animals (p<0.05).  At three months post-injury, animals with T3 SCI also showed significantly higher blood ketones compared to sham-injured animals (p<0.05, Figure 4.8c). 4.4 Discussion Here we present novel data about metabolic function after SCI, an understudied topic, especially in experimental SCI.  We demonstrate that calorie consumption is similar in animals with complete T3 SCI, animals with complete T10 SCI and those with sham injury.  We also show that activity levels are equally depressed in animals with T3 SCI and those with T10 SCI.  Interestingly, the normal circadian patterns in core body temperature, activity levels and blood glucose were generally unaffected by high- and   155 Figure 4.8 Animals with high-thoracic SCI exhibit a greater ketone response to fasting.  At one month post-injury, non-fasting ketone levels were equally low in all groups (a). A 12-hour fast triggered a ketone response in all groups (b).  Animals with T3 SCI showed a significantly greater rise in blood ketones compared to animals with T10 SCI (p<0.05) and sham-injured animals (p<0.0001).  At three months following post-injury (c), fasting ketones were again elevated compared to sham-injured animals (p<0.05), but there was no difference between T3 and T10 injured animals. Symbols: * denotes significant difference between the two indicated groups (p<0.05).    156    157  low-thoracic SCI.  Finally, unlike the pathological carbohydrate metabolism associated with diabetes that has been documented clinically, glucose tolerance and insulin sensitivity were both increased in animals with SCI.  This work contributes to the development of animal models and translational research in the area of metabolism after SCI. Energy intake is an important component of any discussion regarding energy balance. We recently showed that animals with T3 SCI accumulate more adipose tissue than animals with T10 SCI and sham-injured animals.  One goal of our current study was to determine whether this excess energy might have been partly due to an increase in energy intake.  One previous report found that, when provided with rodent kibble ad libitum, calorie consumption was consistently higher in rats with complete T3 SCI compared to sham controls (Primeaux et al., 2007).  However, with our enriched food diet in addition to kibble ad libitum, we did not find any difference in calorie consumption between groups.  Body weight was decreased by SCI, but preoperative weights were recovered, in both groups with SCI, within two weeks post-injury (Figure 4.1) consistent with our previous work (Inskip et al., 2010).  Sham-injured animals gained more weight in the experiment than animals with T3 SCI (Figure 4.1b). In the current experiment, activity levels provided a surrogate measurement of energy expenditure from physical activity.  Acutely after SCI, both animals with T3 SCI and those with T10 SCI displayed reduced daily activity.  Activity levels were equally suppressed by T3 and T10 SCI; therefore, an extreme imbalance in energy expenditure due to reduced activity is unlikely to explain the increased adiposity that we have recently observed in the former, but not the latter, at one month post-injury (Inskip et al., 2010).   158 Of course, activity units are not the ideal measurement of activity-related energy expenditure.  Even with the same level of activity, it is likely that animals with SCI have altered costs of physical activities.  Recent research in energy expenditure after SCI has revealed that the standard non-invasive equipment used to estimate energy expenditure, which is calibrated to able-bodied controls, underestimates the energy demands of some tasks and overestimates the energy expenditure associated with other tasks (Collins et al., in press; Hiremath and Ding, 2009).  We expect that there are also differences in energy expenditure associated with physical activity in animals after SCI.  On the one hand, they may have a higher cost of movement in some respects due to the fact that they mobilize with their forelimbs only; however, it is conceivable that their energy expenditure is reduced in other respects, due to the fact that only small muscles are recruited to perform the task compared to sham-injured animals, who recruit the large hindlimb muscles as well.  The use of indirect calorimetry would allow us to determine the overall calorie consumption associated with movement.  This analysis might also allow us to determine whether there are differences in calorie consumption between animals with high- and low-thoracic SCI.  From our current results, we can only conclude that there are no differences between animals with T3 SCI and those with T10 SCI at the whole body activity level. The activity data also revealed that there were no major changes in the timing of activity periods and resting periods throughout the experiment: rats with SCI displayed the typical increase in nocturnal activity.  The nocturnal increase in core body temperature also remained present after high- and low-thoracic SCI, even during the first week post-injury, when acute hypothermia is present (Laird et al., 2006).  Taken together, the patterns in   159 activity levels and body temperatures indicate that the circadian rhythm is not grossly abolished or phase-shifted by SCI. We were surprised to observe the classic circadian rhythm in blood glucose in all groups of animals, given the imbalance between the parasympathetic and sympathetic neural control of the liver that is present after high-thoracic SCI.  A recent study investigating the effects of liver denervation found that when either sympathetic or parasympathetic innervation to the liver was disrupted, so too was the typical circadian glucose pattern (Cailotto et al., 2008).  There are several possible reasons for this discrepancy.  In the former study, the sympathetic nerves were cut at the level of the hepatic artery.  This injury severs the axons of the sympathetic ganglionic neurons leading to the degeneration of the distal tip of the neurons within the liver, accompanied by increased fibroblast proliferation (Albino-Teixeira et al., 1990), changes in microcirculation (Colle et al., 2004) and major decreases in norepinephrine content (Berg et al., 1990). Therefore, the hepatic environment is quite different from that after SCI, where the peripheral sympathetic circuitry, and likely the local hepatic environment, is preserved.  This difference could contribute to the circadian glucose rhythm after SCI and not after hepatic sympathetic denervation.  With the hepatic microenvironment intact, it is possible that the rhythm could be maintained by hormonal rather than neural cues. Systemic glucose levels are actively maintained, with basal levels of glucose mobilization and uptake occurring even at rest (Woerle et al., 2003).  The balance between mobilization and uptake is modulated by hormonal and neural cues and can be rapidly altered to respond to changes in internal energy states (Nonogaki, 2000).  The uptake role becomes particularly important after meals, when there is an surge of glucose absorbed   160 into the blood (Jenkins et al., 1982).  Glucose uptake occurs predominantly in the muscle, liver and adipose tissue.  Given the muscle atrophy that exists below the level of the SCI (Dupont-Versteegden et al., 1998; Gregory et al., 2003; Biering-Sorensen et al., 2009), and resultant decrease in metabolically active tissue, there should be reduced demand for glucose in the periphery, resulting in slower clearance of glucose from the blood.  On this basis, we expected that glucose clearance would be slower after SCI, especially given the high prevalence of insulin resistance and obesity seen clinically.  However, we, like one other study conducted in a similar model (Primeaux et al., 2007), found opposite results in an animal model of SCI.  One possible explanation is that we did not observe our animals for a sufficient length of time post-injury; the human equivalent of three rat months is approximately 7.5 years (Ruth, 1935).  The studies that have documented insulin resistance in SCI (and reported time post-injury) have been conducted on individuals that were, on average, 12-18 years post-injury (Duckworth et al., 1980; Bauman and Spungen, 1994; Bauman et al., 1999a).  Furthermore, when individuals are grouped on the basis of glucose tolerance and insulin resistance, there is a significant difference in years post-injury (but not average age) between the insulin-resistant diabetic subjects and those with normal glucose tolerance (Duckworth et al., 1980).  These examples highlight the possibility that the typical timelines of experimental SCI are simply not long enough to mimic the chronic pathological changes in glucose homeostasis seen clinically.  More research will be needed to determine if this is the case. During periods of energy deficit, such as insulin-induced hypoglycemia, glucose is mobilized primarily from hepatic glycogen stores. The pancreas also plays an important role in the response to hypoglycemia, by increasing glucagon secretion and reducing   161 insulin secretion.  As the liver and the pancreas receive the bulk of their sympathetic innervation from the celiac ganglia and superior mesenteric ganglion, we hypothesized that this response might have been perturbed in animals with T3 SCI but not those with T10 SCI or sham controls.  However, this was not the case.  Insulin-induced hypoglycemia was pronounced in high-thoracic SCI animals one month after injury.  This was observed quantitatively in the rapid rate of blood glucose decline and qualitatively in the abnormal behaviour during the latter half of the test.  While all animals were lethargic during testing, animals with T3 SCI were almost completely inactive and, at the end of the test, few responded normally when fruit and treats were provided.  These animals clearly showed a deficit in their ability to respond to acute hypoglycemia.  However, the same animals did not display any abnormalities in their glucose response to overnight fasting (Table 4.1) suggesting that physiological, endogenous, longer-term responses are not perturbed by SCI. The ketone response (Figure 4.8) suggests that there could be a change in substrate metabolism following SCI.  Ketones, which provide an alternate fuel to glucose for peripheral tissues, are produced by the liver when carbohydrate levels are low.  The increased ketone response observed in animals with T3 SCI, suggest increased hepatic production of ketones (or decreased peripheral uptake of ketones).  Whether this production represents a normal compensation for low carbohydrate levels or a fundamental alteration in substrate metabolism post-injury, such as an increased reliance on ketones for metabolism, remains to be elucidated. Taken together, our results suggest that the loss of descending sympathetic control of the liver and pancreas does not directly contribute to the typical pathological changes that are   162 associated with SCI, such as impaired glucose tolerance and reduced insulin sensitivity after spinal cord injury.  This points to the role of other energetic changes, such as reduced physical activity levels (van den Berg-Emons et al., 2008), high fat intake (Groah et al., 2009) and increase in adipose tissue (Edwards et al., 2008; Maruyama et al., 2008), and supports the role of activity and lifestyle interventions in maintaining healthy metabolic profiles following SCI. The variation in injury level, completeness, and degree of autonomic nervous system injury can add some level of uncertainty into clinical metabolic research.  Animal models provide another valuable way to investigate the effects of SCI on metabolism.  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Robinson AM, Williamson DH (1980) Physiological roles of ketone bodies as substrates and signals in mammalian tissues. Physiol Rev 60:143-187.  Rüger M, Scheer FA (2009) Effects of circadian disruption on the cardiometabolic system. Rev Endocr Metab Disord 10: 245-60. Ruth E (1935) Metamorphosis of the pubic symphysis. I. The white rat (Mus norvegicus albinus). Anat Rec 64:7. Strack AM, Sawyer WB, Marubio LM, Loewy AD (1988) Spinal origin of sympathetic preganglionic neurons in the rat. Brain Res 455:187-191. van den Berg-Emons RJ, Bussmann JB, Haisma JA, Sluis TA, van der Woude LH, Bergen MP, Stam HJ (2008) A prospective study on physical activity levels after spinal cord injury during inpatient rehabilitation and the year after discharge. Arch Phys Med Rehabil 89:2094-2101. Warren WS, Champney TH, Cassone VM (1994) The suprachiasmatic nucleus controls the circadian rhythm of heart rate via the sympathetic nervous system. Physiol Behav 55:1091-1099. Woerle HJ, Meyer C, Dostou JM, Gosmanov NR, Islam N, Popa E, Wittlin SD, Welle SL, Gerich JE (2003) Pathways for glucose disposal after meal ingestion in humans. Am J Physiol Endocrinol Metab 284:E716-725. Zhang M, Lv XY, Li J, Xu ZG, Chen L (2008) The characterization of high-fat diet and multiple low-dose streptozotocin induced type 2 diabetes rat model. Exp Diabetes Res 2008:704045.  Zisman A, Peroni OD, Abel ED, Michael MD, Mauvais-Jarvis F, Lowell BB, Wojtaszewski JF, Hirshman MF, Virkamaki A, Goodyear LJ, Kahn CR, Kahn BB (2000) Targeted disruption of the glucose transporter 4 selectively in muscle causes insulin resistance and glucose intolerance. Nat Med 6:924-928.    167 5 DISCUSSION The results presented in this thesis demonstrate the wide range of cardiovascular (CV) and metabolic changes that can be elicited by spinal cord injury (SCI).  The effects of SCI on CV physiology are not always readily apparent.  Both resting blood pressure (BP) and heart rate (HR) can be remarkably similar between animals with SCI and neurologically-intact controls.  It is in the minute-to-minute CV control, normally effected by a combination of central control and reflex pathways, that the SCI-associated dysfunctions emerge more clearly.  Indeed, the capacity of the CV system to respond to physiological perturbations can be severely compromised by SCI.  Here I have shown that the BP response to either an orthostatic stress or a visceral stimulus reveals a pronounced divergence between CV control in animals with high- and low-thoracic SCI. Furthermore, time was found to play an important role in the degree of these dysfunctions. The metabolic system also has an important role in the short-term maintenance of homeostasis.  The body must respond to changes in endogenous energy levels by altering the balance of energy storage and retrieval.  These actions are carried out by a combination of neuronal and hormonal pathways, mediating a wide variety of roles in carbohydrate and lipid metabolism.  Many of these anatomical pathways travel via the spinal cord and are therefore vulnerable to injury.  Here I have shown that lipid metabolism is altered by SCI, and that visceral white adipose tissue (vWAT) accumulation and blood triglyceride levels are related to the level of thoracic SCI.  In the time frame that I observed, this relationship was not as clearly defined in carbohydrate metabolism, despite the differential disruption of neural pathways to visceral organs   168 involved in glucose metabolism.  Differences in the efficiency of carbohydrate metabolism over time post-injury suggest that the body continues to respond to the changing body composition beyond the acute post-injury period. The following discussion outlines the CV and metabolic outcomes of experimental SCI and how they relate to their clinical counterparts.  Both sections discuss the implications of the current research, their relevance to future studies and to experimental SCI research more broadly, including recommendations for refining specific methodologies.  Finally, some general insights and limitations of experimental SCI research are presented. 5.1 Cardiovascular function after experimental SCI Cardiovascular function is abnormal after SCI.  This is manifested clinically in acute episodes of hypertension or hypotension in response to specific stimuli.  The fact that rats with SCI exhibit some CV dysfunctions similar to those seen clinically has been appreciated for some time; however, orthostatic hypotension (OH) has never been modeled experimentally, despite its restrictive effects on the lives of individuals with SCI.  The results of Chapter 2 demonstrate the feasibility of incorporating novel physiological stimuli, such as an orthostatic stress, in our autonomic testing in animals. A reliable and reproducible orthostatic stressor is an important addition to our autonomic testing toolkit in animals, as many individuals who suffer from AD must also suffer from OH (Mathias and Frankel, 1999).  We now have a way to reproduce these conditions and model human SCI more closely.  With this model, we can also address questions regarding whether the pronounced range of BPs experienced by individuals with SCI affects the anatomical structure and function of the CV system.  Furthermore, this model   169 could potentially be used to evaluate the recovery of CV function following therapeutic interventions for SCI. Alternative sensory stimuli for eliciting AD are also important additions for experimental SCI research because there are myriad triggers present in the daily lives of individuals with this condition.  Therefore, a range of stimuli will be necessary if we are to test preclinical therapeutic interventions in the future.  It is important to establish the typical response to these novel stimuli before we test these therapies because not all noxious or non-noxious stimuli reliably trigger AD (Burton et al., 2008).  As mentioned in Chapter 2, the sexual stimulus utilized was complicated by the necessity of restraint and upright position.  However, the results provide proof of principle for using novel stimuli in experimental research.  The use of analogous assessments in the clinical setting and with our experimental animals will facilitate the translation of our understanding of CV dysfunction after SCI between the bench and the bedside. Further experiments will be necessary to determine whether the severity of OH in experimental SCI correlates with the degree of damage of sympathetic pathways, and therefore whether it could be an appropriate test to assess autonomic function, for example after a contusion injury or following therapeutic intervention.  Severity of injury is important because a large proportion of SCI researchers use incomplete injuries, including contusion or clip-compression injury.  Therefore, it would be important to validate the interaction between the severity of injury and the degree of OH.  Previous work from our lab has shown that the severity of AD correlated with the severity of clip compression injury acutely after T5 SCI (Maiorov et al., 1998).  Therefore, I would predict similar effects on OH, as both of these disorders occur, most commonly, when the   170 injury disrupts the descending sympathetic pathways to the splanchnic vascular bed. However, it is important to determine the natural course of CV dysfunction for each specific type and severity of injury in question, as animals with mild (20 g) or moderate (35 g) clip-compression injuries did not exhibit any signs of AD at one week post-injury, despite the same animals clearly showing signs one day after injury (Maiorov et al., 1998). In the current work, I evaluated only two discrete injury levels.  Further experiments will also be necessary if there is interest in fine-tuning the relationship between BP control and level of SCI.  Based on the results in Chapter 2, and the general understanding of the primacy of the splanchnic vasculature in BP control, the relationship between BP control and level of injury is unlikely to be a linear one. 5.2 Recommendations for future cardiovascular research in experimental SCI One of the disadvantages of using fluid-filled cannulae connected to an external transducer to record CV variables is that the results obtained provide only a brief snapshot of CV function.  This is especially relevant after SCI, when we know the CV system undergoes changes over time (Mathias and Frankel, 1999; Mayorov et al., 2001). Practically speaking, the recording protocol also creates several unavoidable stressors, including the fact that it must be performed shortly after surgery, demands animal handling, and restricts natural behaviour and movement.  In the future, I would prefer to use a telemetric system with an implantable fluid-filled cannula and radiotelemetric transponder.  This transponder could be implanted prior to SCI, which would reduce the confounding effects of surgery and stress, and allow me to obtain preoperative CV data. Furthermore, the use of telemetry would give me the capacity to ask a range of clinically-   171 relevant questions otherwise limited by the short-term nature of the external transducer set-up.  In my mind, the questions with highest priority are: does the severity of AD and OH change over time for individual animals; what is the incidence of AD during rehabilitation; in individual animals, is the severity of AD comparable when elicited by different kinds of sensory stimuli. The research herein also has implications for experimental SCI research more broadly. With CV autonomic function recently highlighted as a priority for individuals with SCI (Anderson, 2004), several research groups are beginning to include CV function as an outcome measure following a therapeutic intervention, either in its own right or as part of a battery of motor, sensory, and autonomic assessments (Kalincik et al., in press).  The current results, together with the clinical incidence and level dependence of CV dysfunction, support the use of a high-thoracic or cervical injury model in order to establish reliable, quantifiable abnormalities in BP.  This is necessary so that we can then focus on ameliorating these abnormalities. 5.3 Metabolic function after experimental SCI With new tools, greater awareness, and more widespread and frequent testing, the clinical community is beginning to appreciate the wide variety of metabolic changes in individuals with SCI.  On the other hand, the experimental research is lacking in this area. The overall objective of the experiments in Chapters 3 and 4 was to determine whether there were detectable changes in whole body metabolism after experimental SCI and, if so, whether these changes varied with thoracic injury level. The results of Chapter 3 reveal that the overall adiposity and blood triglyceride levels of animals with high- and low-thoracic SCI were distinctly different.  The results of   172 carbohydrate metabolism were not so discrete; overall, animals with SCI demonstrated more efficient glucose metabolism than sham-injured controls, but this effect was not clearly affected by the level of SCI. At first look, the discrepancy in carbohydrate metabolism between the results of Chapter 4, showing efficient carbohydrate metabolism after SCI, and the high clinical prevalence of diabetes reported elsewhere (Bauman and Spungen, 1994) appears pronounced and, more importantly, divergent.  However, a review of the literature suggests that there may be two distinct phenotypes of carbohydrate metabolism after SCI. Many of the widely cited studies that demonstrate an abnormally high prevalence of diabetes in individuals with SCI compared to neurologically intact individuals have been criticized for their sample selection and lack of appropriate controls.  One of the major problems with existing studies is that many were conducted in veterans with SCI (Bauman et al., 1996; Bauman and Spungen, 2000; Wilt et al., 2008).  On average, veterans have a higher prevalence of diabetes compared to the civilian population (Prakash et al., 2002; Lavela et al., 2006), which makes it difficult to dissociate the effect of SCI from general lifestyle effects.  However, other more rigorous studies have shown similar trends (Imai et al., 1996; Bauman et al., 1999a) On the other hand, several recent carbohydrate metabolism studies show efficient carbohydrate metabolism following SCI (Raymond et al., in press).  Typically, following a standard oral glucose load, blood glucose increases rapidly followed by a gradual return to baseline (Chernecky and Berger, 2004).  In individuals without any carbohydrate metabolic abnormalities baseline glucose levels are typically restored by two hours after loading (Chernecky and Berger, 2004).  In a recent SCI study, a significant proportion of   173 individuals with paraplegia exhibited very low blood sugar levels (2-5 mmol/L) by the two-hour mark (Raymond et al., in press).  While these specific individuals were more active than other subjects with SCI, a review of glucose tolerance tests performed in endurance and sprint athletes reveal that even these elite athletes do not drop to such low glucose levels, and rarely overshoot their resting glucose levels at the end of this test (Dela et al., 1991; Niakaris et al., 2005; Fontana et al., in press).  Therefore, some aspect of homeostatic regulation appears to be abnormal in a subset of individuals with SCI and in the animals tested in Chapter 4.  We can also see this demonstrated during exercise, where healthy controls can maintain their blood glucose, while individuals with SCI display moderate hypoglycemia (Kjaer et al., 2001).  Finally, anecdotes of frequent episodes of hypoglycemia suggest that the lack of glucose control can also be a common reality for some individuals with SCI (K. Anderson-Ennis, unpublished observations). Ultimately, there appear to be two divergent phenotypes of glucose metabolism after SCI. Whether these outcomes are a product of time (with one invariably leading to the other), or represent discrete populations with fundamental differences in neurological injury level, or are secondary to differences in activity levels, remains unclear.  These last two possibilities are especially difficult to dissociate, as the former tends to negatively influence the latter (Carlson et al., 2009; Raymond et al., in press).  Curiously, the increased insulin sensitivity of animals with T3 SCI at one month post-injury wanes by three months post-injury.  This could support the idea that pathological alterations in carbohydrate metabolism occur with time after injury.  Of course, longitudinal studies will be essential to support or refute this conclusion.  Longer-term studies are also recommended based on the disparity between the relative duration of injury between our   174 rodent and human research in this area (see 5.6 Insights and limitations of experimental SCI research, below). The glucose, insulin and, ketone results raise interesting questions about general substrate metabolism after SCI.  Ketone body production is inversely correlated to the availability of carbohydrates (Robinson and Williamson, 1980).  During prolonged fasting, the liver increases the production of ketone bodies, which can be used as oxidative fuels in peripheral tissues (Robinson and Williamson, 1980).  Ketones are especially important in the nervous system, which is restricted in the range of fuels that it can utilize (Robinson and Williamson, 1980).  The increased ketone levels observed in animals with high- thoracic SCI suggest increased hepatic production or decreased peripheral use.  These changes may be the result of animals with SCI having a heightened sensitivity to glucose changes, so that a similar decrease in glucose elicits a greater ketogenic response. Irrespective of the mechanism, the pronounced ketone response together with recent clinical evidence of other alterations in substrate metabolism, such as the reduced ability to mobilize fats (Kjaer et al., 2001; Knechtle et al., 2004) and the failure of carbohydrate ingestion to improve performance (Temesi et al., 2009), suggests that there may be fundamental alterations in substrate metabolism post-injury. There was no evidence of global disturbances in circadian metabolic function in animals with SCI.  With respect to the sleep-wake cycle, measured by physical activity levels, this is perhaps not surprising, given that the neural circuit between the principal circadian oscillator in the suprachiasmatic nucleus and the pineal gland was not disrupted (Larsen et al., 1998).  However, I expected that the core body temperature would be disrupted by SCI, given the role of the ANS in mediating body temperature through its control of the   175 superficial vasculature and the high prevalence of hypothermia in patients with SCI (Khan et al., 2007).  The results of Chapter 4 suggest that the core body temperature rhythms were not disrupted by SCI.  However, I cannot say conclusively that the endogenous temperature rhythm was not affected because the sleep-wake cycle can indirectly regulate body temperature (Hastings et al., 2003), which could effectively mask any alterations in temperature patterns. The blood glucose rhythm also persisted after SCI.  I was surprised by this given the necessary role of balanced autonomic activity in glucose rhythmicity that has been reported elsewhere (Cailotto et al., 2008).  However, it is possible that the presence of this rhythm could be driven by the feeding protocol (see 5.4 Recommendations for future metabolic research using this model, below).  It would be interesting to use similar outcome measures in a higher-level injury that disrupts central control of the pineal gland.  I suspect we would observe altered sleep-wake cycles in these animals and corresponding dysregulation of core body temperature patterns.  Based on the results of Chapter 4, I would not predict any differences in glucose patterns. 5.4 Recommendations for future metabolic research using this model For future studies of metabolism after SCI, the existing experimental protocols could be modified in a number of ways.  These simple modifications would minimize potential confounds and make the results more robust.  Feeding and fasting have consequences on metabolism, alertness and activity (Murphy et al., 2003).  Rodents typically consume the majority of their daily calories during the dark period when they are most active (Sidlo et al., 1995).  Feeding rodents during their light period when they are typically sleeping can disrupt typical activity patterns and induce internal desynchrony (Salgado-Delgado et al.,   176 2010).  Therefore, presenting palatable foods during the light cycle, as was done in the experiments in this thesis, was a poor choice in terms of maintaining the natural rhythms of the laboratory rat, and introduced some abnormalities in physiological rhythms.  The effect of feeding was observed in the rise in core temperature and activity levels coincident with the first feeding of the day (Figure 4.1, 4.2).  Ideally, we should keep the feeding schedule consistent with the natural setting and, wherever possible, analogous to clinical situation.  This would mean providing the enriched food during the dark cycle, when rodents are typically consuming the bulk of their daily calories.  One way to facilitate this would be to reverse the light/dark cycle in the animal unit so that rodents and researchers are awake and active at the same time. Reversing the light/dark cycle would also make it possible to fast the animals during the light cycle ahead of blood tests.  This would make the physiological state of the rats during fasting similar to humans, who are asked to fast overnight prior to testing. Furthermore, this would ensure that animals were fasted for a true period of 12 hours.  In my existing fasting regimen, animals were fasted during the dark period when they normally consume their food.  Therefore, it is possible that animals were fasted for longer than 12-hours, if they had not yet eaten much of their daily calories when their food was removed at the end of the light cycle.  The reversal of the light/dark cycle would have a number of positive effects on maintaining more normal chronology of nutrient intake and would make the experimental protocol more robust. Ideally, circadian rhythms would have been tested in the absence of other entrainment features (Cailotto et al., 2005; Cailotto et al., 2008).  As a result of not controlling the light so that it was constant throughout the 24-hour period, or providing food at regular   177 intervals throughout the 24-hour period, I cannot say definitively that the circadian glucose rhythm was truly endogenous, i.e. that there were not other external entrainment factors that were responsible for generating it.  One of the most obvious possibilities for driving the glucose rhythm is the feeding schedule.  To conclusively determine whether the endogenous glucose rhythm is still present after SCI, it would be wise to conduct two further experiments: one where the animals are still given their enriched food, but in their typical eating (dark) period; in the second experiment, animals should be housed in dim lighting (24-h per day) and given their diet at equal intervals throughout the 24-h period (so-called “unmasking” conditions).  The first experiment would inform us about whether the rhythm is present in the typical eating schedule (similar to the clinical setting), while the second would reveal whether the rhythm is truly driven endogenously. 5.5 Metabolic implications for future experimental SCI research The altered fat and carbohydrate metabolism demonstrated by the results herein raise several issues germane to treatments and protocols currently being used in experimental SCI research.  In the past few years there has been an emergence of metabolic interventions as therapeutic targets for neuroprotection and neurological recovery following neurological damage, including SCI.  These interventions include dietary restriction (Plunet et al., 2008; Maalouf et al., 2009), nutritional composition changes (Streijger et al., 2009), administration of omega-3 polyunsaturated fatty acids (King et al., 2006; Huang et al., 2007; Michael-Titus, 2007) and cholesterol-lowering statin therapies (Holmberg et al., 2008; Mann et al., 2009).  The results of Chapters 3 and 4 demonstrate that rats with high-thoracic SCI can exhibit an altered metabolic state, most evident in their increased adipose tissue, hypertriglyceridemia and altered responses to exogenous   178 insulin and glucose administration.  As a result of their altered physiology, standard therapies might not have the same effects on rodents with high-thoracic SCI as they do in neurologically intact animals.  Similarly, normally routine protocols, such as insulin administration (Yang et al., 2009) or every-other-day-fasting (Plunet et al., 2008) likely have altered effects in this system.  It is important to keep these issues in mind when selecting treatments for animals with SCI and interpreting the results of experimental trials. 5.6 Insights and limitations of experimental SCI research Large-scale clinical metabolic and CV SCI research studies are complicated and expensive to execute.  An additional complication specific to clinical SCI research is the difficulty in recruiting a sufficient number of subjects with similar injury levels.  In attempt to enroll a sufficient number of subjects and obtain adequate statistical power, researchers often widen the inclusion criteria, introducing significant variability in the level, completeness, and time after injury.  Pooling these variables can make it practically impossible to conclude whether effects are injury dependent, especially if the specific injury details are not reported for the reader.  It is particularly difficult to get a clear picture of the effect of autonomic injury on CV and metabolic function from current clinical research.  Even fewer studies report the level and completeness of ANS injury – even when testing autonomic outcomes (Handrakis et al., 2009).  Perhaps this can partly be attributed to the fact that there is still some debate as to which ANS assessments provide accurate, sensitive, and valid measures in the SCI population (Alexander et al., 2009a).   179 Experimental research provides an important, controlled way to characterize CV and metabolic function after SCI.  Complete transection injuries, in particular, provide certain and consistent damage to autonomic pathways.  These studies can be used to inform clinical research about salient biomarkers and level-dependent features of injury.  For example, the results of this thesis suggest that level of injury is not trivial – aspects of CV control, lipid and glucose metabolism can be quite different between high- and low- thoracic SCI.  However, to date, a significant proportion of clinical research has been muddied by the combination of widely different injury levels (Wilt et al., 2008).  In the future, the careful documentation and systematic reporting of our clinical subjects, ideally with the inclusion of an assessment of the integrity of autonomic pathways, may allow us to make more meaningful and robust conclusions about the associations between injury level, CV dysfunction and metabolic changes. Of course, the use of animal models has its own limitations.  In the context of this thesis, some of these limitations are inherent in the use of an animal model, while others could be improved by modifying experimental protocols to reduce the presence of confounding variables, for example, or to more closely follow the timeline of clinical SCI. The anatomical differences between rodents and humans are unavoidable, but important to acknowledge.  In terms of BP control, the tail is a unique appendage with a considerable degree of capacitance (Smith et al., 1998) and therefore potential to affect systemic BP.  However, the spinal outflow to the tail vasculature is located in segments (T11-L2), making it equally affected by both the high- and low-thoracic transection used in this series of experiments.  Therefore, the tail is unlikely to have affected the inter- group differences in orthostatic tolerance.  A second important consideration is that   180 rodent physiological testing can easily be confounded by the introduction of stimuli apart from the direct variable being tested.  Stimuli common to the research setting, such as noise (Baldwin et al., 2007), handling (Balcombe et al., 2004), and restraint (Irvine et al., 1997; Sharp et al., 2002), can all affect BP and HR quite significantly.  Therefore, in the current experiments every attempt was made to minimize these stimuli.  One of the key features of colorectal distension (CRD) is that the initiation of stimulation (once the catheter is inserted) does not require any manipulation of the animal.  This minimizes the animals’ stress and its confounding effects on BP.  Sexual stimulation, on the other hand, was complicated by the necessity of restraint and orthostasis.  If alternative stimuli to CRD are needed in the future, I would propose the use of more simple stimuli such as superficial somatic stimulation; in my opinion, the benefits of minimizing artifacts outweighs the clinical significance of sexual stimulation as a trigger for AD.  Finally, the use of telemetry (as discussed above) would provide a way to determine whether the CV variables during testing are different from baseline values.  This could inform us about how stressful our procedures are, and provide feedback so that we can modify them to minimize stress. Experimental SCI researchers are not typically asked to justify the age of animals used in their experiments or the duration of injury.  However, these specifications can be important to the clinical relevance and ultimately to the translatability of the research.  In this thesis, like most experimental SCI research, the animals that were studied were still growing at the time of injury (“Wistar rat datasheet”, 2010).  This is problematic because it is possible that SCI could disrupt normal growth in addition to precipitating its direct effects on the body; therefore, some of the differences I have observed could be due to   181 altered growth patterns rather than SCI per se.  Secondly, at the time of injury, my animals were approximately 12-13 weeks; the typical lifespan of a male Wistar rat is approximately 2 years (Goodrick et al., 1983).  Given that the population that we are trying to model is primarily between 20 and 40 years old (Pickett et al., 2006), there is a significant disparity between our animal model and the clinical reality in the age at injury relative to total lifespan.  Ideally, we should begin experimental studies when rodents are fully developed and have started their adult life (around 6 months) (Maeda et al., 1985; Yu et al., 1985). Finally, reproducing the duration of injury is another important consideration for experimental SCI research.  SCI is a lifelong condition, yet a brief look at articles that employ a rat model of “chronic spinal cord injury” suggest that majority of studies consider three to eight weeks to be chronic.  Indeed, this may be an appropriate timeline for the motor, sensory and urological changes that occur following experimental SCI, however some of the metabolic changes likely only develop in the months post-injury and could be altered beyond that.  Therefore, asking questions about metabolism after experimental SCI demands a re-evaluation of current experimental timelines and endpoints.  Possibly related to metabolism after injury, there is also impetus to examine longevity after experimental SCI because individuals with SCI live an average of 13-25 years less than able-bodied people, depending on their injury level and severity (Strauss et al., 2006).  Ultimately, it would be ideal to include longevity as an outcome measure in these experiments, using survival as a final end-point.  Arguably, the costs and personnel associated with this type of longitudinal research quickly become prohibitive, especially in our short-term “publish-or-perish” academic environment.   182 5.7 Concluding remarks Translational biomedical research demands well-defined animal models that are faithful to the human condition.  We can use these models to characterize dysfunction, test preclinical therapies, guide clinical research, and ultimately to inform patient management.  Animal models of motor function after SCI have been well-studied and characterized; research in this area vastly outnumbers the research on autonomic outcomes of SCI (Inskip et al., 2009).  CV and metabolic dysfunctions both have significant effects on quality of life and functional independence after SCI.  Experimental models of these systems have the potential to provide a wealth of information regarding the nature of these dysfunctions and possible strategies to mitigate their effects. The results herein reveal that rodents with high-thoracic SCI experience deficits in BP control as a result of their injury.  Animals with low-thoracic SCI, on the other hand, do not display significant alterations in CV control.  Experimental SCI can also perturb metabolic homeostasis in a level-dependent fashion.  In animals with complete high- thoracic SCI this manifests as a gross increase in adipose tissue and higher blood triglycerides.  These changes are not present after complete low-thoracic SCI, suggesting that changes in lipid metabolism may be level-dependent.  Carbohydrate metabolism does not appear to be pathologically altered by SCI in the three-month period employed here. Given the results of this thesis, I encourage researchers, both clinical and experimental, to clearly report injury levels when assessing CV or metabolic function.  I also advocate that more metabolic and CV outcomes be incorporated into preclinical SCI research, given the critical relationships between metabolic function, CV disease, and longevity.  The insights gained from clinical and experimental SCI research should help to improve the   183 management of CV and metabolic complications after SCI.  Part of this management may include the development of SCI-specific guidelines for CV and metabolic health.  There are more and more examples of dietary modifications, exercise regimes, and drugs that elicit altered responses in individuals with SCI.  These examples suggest that we may not be able to generalize the evidence obtained using able-bodied subjects to individuals with SCI. The results of this thesis serve as a reminder to be prudent when comparing studies conducted in animals or humans with different injury levels, or combining groups of subjects with distinctly different injuries.  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J Gerontol 40:657-670.       189 APPENDIX: ANIMAL CARE CERTIFICATE  

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