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

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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.

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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.

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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

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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

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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

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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

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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

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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.

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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

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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

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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

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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).

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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

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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.

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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

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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

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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

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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

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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.

In my final series of experiments (Chapter 4), I wanted to assess whether there were gross

differences in energy consumption and expenditure between sham, T3 and T10 SCI

animals that could potentially explain the increased energy storage observed in the

27

previous experiments (Chapter 3). My second aim was to determine whether circadian

rhythms in energy expenditure and blood glucose were maintained after SCI. Lastly, I

wanted to assess whether carbohydrate metabolism was disordered as a result of SCI.

Given the importance of autonomic balance in the regulation of circadian blood glucose I

hypothesized that circadian glucose patterns would be abolished by SCI. The acute

response to blood glucose changes is partly effected by the neural control of the adrenals,

pancreas and liver; therefore, I hypothesized that the response to hypo- and

hyperglycemic challenge would be slower in high-thoracic SCI animals than either low-

thoracic or sham-injured animals.

28

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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.

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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.

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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.

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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

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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

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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

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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

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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.

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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).

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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.

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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

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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,

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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

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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

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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).

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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

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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

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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).

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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.

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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

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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).

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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

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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.

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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)

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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.

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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.

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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)

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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

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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

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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

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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

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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

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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.

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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). In fact, we

found that this method provides even more sensitive measurements than MRI

quantification, which was still able to detect differences after SCI, but underestimated

visceral adipose tissue. Therefore, as long as these methods are not used interchangeably,

either could be used to document changes in adiposity after SCI depending on the

equipment and expertise unique to each laboratory.

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4 ENERGY EXPENDITURE, CIRCADIAN RHYTHMS, AND

CARBOHYDRATE METABOLISM AFTER THORACIC SPINAL CORD

INJURY2

4.1 Introduction

Our intermittent eating patterns require our bodies to have means to cope with both

surges and slumps in nutritional levels associated with alternating periods of feeding and

fasting. We are exquisitely sensitive to changes in carbohydrate levels and are well

equipped to store and retrieve energy based on demand. 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.

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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

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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

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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.

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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

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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.

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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.

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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.

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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

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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

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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.

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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).

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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.

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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.

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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).

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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).

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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

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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).

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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.

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(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

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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.

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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

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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).

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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

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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

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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).

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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).

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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

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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

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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

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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

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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. They

provide a medium where both injury level and severity can be determined conclusively.

The combined efforts of clinical and experimental researchers should clarify the

metabolic sequelae of SCI, their etiologies, and the most effective means to maintain

good health despite these changes.

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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

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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

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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

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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-

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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

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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

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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

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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

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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.,

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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

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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

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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).

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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

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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

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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.

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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

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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. They provide further impetus to not be so

enthralled with our motor outcomes that we forget about the important roles of the

autonomic nervous system, and the differences that may arise from autonomic injury

despite similar motor dysfunction.

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APPENDIX: ANIMAL CARE CERTIFICATE