Assessing Heart Rate Variability as a Surrogate Measure of Cardiac Autonomic Function in Chronic Traumatic Spinal Cord Injury

Transcription

1 Assessing Heart Rate Variability as a Surrogate Measure of Cardiac Autonomic Function in Chronic Traumatic Spinal Cord Injury by Rasha El-Kotob A thesis submitted in conformity with the requirements for the degree of Master of Science in Rehabilitation Science Graduate Department of Rehabilitation Science University of Toronto Copyright by Rasha El-Kotob 2015

2 Assessing Heart Rate Variability as a Surrogate Measure of Cardiac Autonomic Function in Chronic Traumatic Spinal Cord Injury Abstract Rasha El-Kotob Master of Science in Rehabilitation Science Graduate Department of Rehabilitation Science 2015 Individuals with a spinal cord injury (SCI) are at greater risk of cardiovascular disease (CVD) than able-bodied individuals. A major CVD contributing factor is the presence of autonomic disturbances, but the SCI-related changes in cardiac autonomic function are poorly understood. Heart rate variability (HRV) has been reported to non-invasively assess the cardiac autonomic nervous system (ANS). The following thesis involves investigating resting HRV in 56 subjects with a traumatic chronic SCI with the aim to 1) describe the overall distribution of HRV in SCI; 2) determine whether there are HRV differences based on level and/or severity of injury; and, 3) determine whether there is a relationship between parasympathetic and sympathetic frequency measures. The results revealed that HRV is variable between-subjects, there were no significant HRV differences based on level and/or severity of impairment, and the low frequency-to-high frequency ratio (LF:HF), may not be an applicable measure in traumatic chronic SCI. ii

3 Acknowledgments I would like to thank my supervisor, Professor Molly Verrier, for accepting me as a Master s student, introducing me to the world of research, providing me with true mentorship, and stimulating my interests. I would also like to thank my co-supervisor, Dr. Sunita Mathur, for her constant genuine encouragement and for offering me valuable on-going guidance even beyond the scope of my research. I took pleasure in conducting my Master s study and I owe it to both of my supervisors. I would like to acknowledge the members of my advisory committee, Dr. Catherine Craven, Dr. Dave Ditor, and Dr. Paul Oh for their insightful input and even assistance regarding the planning and execution of my research work. Thank you to the research staff, graduate students and co-op students at Toronto Rehabilitation Institute-UHN, Lyndhurst Centre. I was fortunate to work in such a fruitful research environment with such knowledgeable colleagues. I would also like to especially thank Dr. Masae Miyatani for allowing me to run a secondary data analysis on her collected data. I am forever grateful for my parents who have always believed in me and never stopped cheering me on. Thank you to my siblings, for being the reasons why I smile even during the stressful times. Finally, I would like to express my gratitude to my university sweetheart and devoted husband- not only did you support me relentlessly, but also you sincerely showed an immense interest in my research which undeniably contributed to my eagerness. I would also like to acknowledge the funder of this research: The Canadian Institute of Health Research (Grant #: TCA ). iii

4 Table of Contents Acknowledgments... iii Table of Contents... iv List of Tables... vii List of Figures... viii List of Appendices... ix Glossary... x Chapter 1: Introduction Spinal cord injury Cardiovascular disease in spinal cord injury The function of the autonomic nervous system in spinal cord injury Heart rate variability Literature review The findings on heart rate variability in spinal cord injury Factors affecting heart rate variability Study rationale Chapter 2: Objectives and Hypothesis Objectives Primary Objective Secondary Objective Tertiary Objective Hypothesis Primary Hypothesis Secondary Hypothesis iv

5 2.2.3 Tertiary Hypothesis Chapter 3: Methodology Overview Study variables Heart rate variability indices and related factors Subject selection Electrocardiogram recordings Medications Heart rate variability analysis Statistical Analysis Objective 1: Heart rate variability frequency distributions Objective 2: Comparison of heart rate variability based on level and/or severity of injury Objective 3: Assessing the LF and HF indices Chapter 4: Results Subject selection Frequency distributions of the heart rate variability indices Heart rate variability comparisons across level and/or severity of injury Comparison of heart rate variability related factors across cohorts Assessing the heart rate variability frequency domain indices: LF, HF and LF:HF Relationship between LF and HF Relationship between LF, HF, LF:HF and influencing factors Predicting LF and HF from heart rate variability-related factors Chapter 5: v

6 5 Discussion Implications and future directions Study limitations Chapter 6: Conclusions References Appendices vi

7 List of Tables Table 1. HRV time domain and frequency domain measures*... 8 Table 2. Factors reported to influence HRV Table 3. Potential HRV-related variables selected from the primary data Table 4. Demographics and vital signs of the participants in total sample and per cohort Table 5. Descriptive statistics for each HRV index in the entire sample (N=56) Table 6. Comparison of HRV indices based on level of injury Table 7. Comparison of HRV indices based on severity of injury Table 8. Comparison of HRV indices based on level and severity of injury Table 9. Relationship of the LF and HFindices based on level or severity of injury Table 10. Relationship of the LF and HF indices based on level and severity of injury Table 11. The relationship between LF, HF indices and the scalar HRV-related factors Table 12. Multiple linear regression analysis to predict LF for the entire sample (R 2 =0.039) Table 13. Multiple linear regression analysis to predict HF for the entire sample (R 2 =0.009) Table 14. Comparison of inter-individual variations in HRV between healthy subjects and chronic traumatic SCI vii

8 List of Figures Figure 1. Possible contributors to greater CVD risk in individuals with SCI Figure 2. Parasympathetic and sympathetic innervations of the heart and peripheral muscles Figure 3. Representative example of HRV analysis using LabChart (v.7.0) Figure 4. Representative example of Poincaré Plot before and after the application of 45Hz low pass filter Figure 5. CONSORT flowchart reflecting the inclusion and exclusion of the final data sample. 27 Figure 6. Frequency distribution of LF:HF Figure 7. Boxplot representing LF:HF distribution Figure 8. Boxplot representation, with and without the outliers, of LF:HF based on level and severity of SCI Figure 9. Boxplot representation of LTPAQ-SCI based on level and severity of injury Figure 10. Boxplot representation of LEMS based on level and severity of injury Figure 11. Boxplot representation of SCIM-III based on level and severity of injury Figure 12. The relationship between LF and HF for total sample Figure 13. Possible contributors to greater CVD risk in individuals with chronic traumatic SCI.48 viii

9 List of Appendices Appendix A Appendix B Appendix C Appendix D Appendix E Appendix F Appendix G Appendix H Appendix I Appendix J Appendix K ix

10 Glossary Absolute VO 2 peak: Highest value of oxygen uptake attained during an incremental exercise test. Expressed in litres of oxygen per minutes (L/min) Activities of daily living (ADL): refers to daily self-care activities with an individual s place of residence, in outdoor environments, or both. American Spinal Cord Injury Association (ASIA) impairment scale (AIS): a five point scale (A-E), where A corresponds to a complete injury, B-D is an incomplete injury, E is normal motor and sensory function Autonomic nervous system (ANS): the system of nerves and ganglia that innervates the blood vessels, heart, smooth muscles, viscera, and glands and controls their involuntary functions, consisting of sympathetic and parasympathetic branches Body mass index (BMI): An index for assessing overweight and underweight, obtained by dividing body weight in kilograms (kg) by height in meters squared (m 2 ). A measure of 25 kg/m 2 or more is considered overweight Bootstrapping: The sampling distribution of a statistic is estimated by taking repeated samples from the data set in order to ensure that analytical models are reliable and will produce accurate results Bradycardia: a slow heart rate, usually less than 60 beats per minute (bpm) Cardiac disease: congenital or acquired disease of only the heart Cardiorespiratory fitness: the ability of the circulatory and respiratory systems to supply oxygen to skeletal muscles during sustained physical activity Cardiovascular disease (CVD): congenital or acquired disease of the heart and blood vessels Chronic: having for a long duration Complete Injury: No motor or sensory function in the lowest sacral segments (S4-S5) x

11 Co-morbidities: Two or more diseases present simultaneously in a patient Distribution: A graph plotting values of observations on the horizontal axis Differences in HRV-related factors: Includes differences in age, sex, body mass index (BMI), waist circumference (WC), time post injury, current smoking status, smoking history, cardiorespiratory fitness level (absolute VO 2 peak, relative VO 2 peak, peak heart rate), leisure time physical activity questionnaire-spinal cord injury (LTPAQ-SCI), lower extremity motor score (LEMS), spinal cord independence measure (SCIM-III), number of co-morbidities, family history of heart disease and sleep apnea. Fast Fourier transform (FFT): Mathematical transformation of a function of time into a function of frequency. Heart rate variability: commonly used term to describe the oscillation of the heart rate and is determined by measuring the R peak to R peak intervals, also referred to as NN intervals, in an electrocardiograph (EGG) High frequency (HF): A heart rate variability frequency domain measure representing vagal modulation of the heart Hypotension: Low resting blood pressure; in men systolic blood pressure less than 110mmHg and in women systolic blood pressure less than 100mmHg Incomplete injury: motor and/or sensory function preservation below neurological level of injury and includes sacral segments (S4-S5) Leisure time physical activity questionnaire (LTPAQ): Total number of minutes of physical activity, not including activities of daily living, performed over the past week Low frequency (LF): A heart rate variability frequency domain measure representing parasympathetic and sympathetic, although more indicative of the latter, modulation of the heart xi

12 Lower extremity motor score (LEMS): Measures voluntary motor strength in five myotomes each scored out of five for a bilateral total of 50. A score of 30 or more suggests that the individual is likely to walk Mean Ranks: The data is ranked from lowest to highest to eliminate the effect of outliers Neurological level of injury (NLI): This is the lowest segment where motor and sensory function is normal in both the left and right side of the body Paraplegia: complete or partial loss of sensation and movement in legs and in part or all of the trunk due to an injury below the cervical vertebrae Peak heart rate: Highest value of heart rate attained during an exercise test. Expressed in beats per minute (bpm). Physical activity: Any bodily movement produced by the skeletal muscles that increases heart rate and breathing and requires energy expenditure Physical capacity: a measure of ability to perform Physical fitness: A state of physiological well being Poincaré Plot: A diagram in which each R-R interval is plotted as a function of the previous R- R interval. The values of each pair of successive R-R interval define a point in the plot. Proportion of the number of interval differences of the consecutive NN intervals greater than 50ms (pnn50): The proportion of the number of interval differences of the consecutive R peak to R peak intervals greater than 50ms derived from an electrocardiogram. A heart rate variability time domain measure that represents cardiac parasympathetic modulation Relative VO 2 peak: Highest value of oxygen uptake attained during an incremental exercise test. Expressed in milliliters of oxygen per kilogram of subject s bodyweight per minute (ml/kg/min) xii

13 Sleep apnea: Sleeping disorder in which breathing repeatedly stops and starts. Obstructive sleep apnea occurs when the throat muscles relax and Central sleep apnea occurs when the brain doesn t send proper signals to the muscles that control breathing. Spinal cord independence measure (SCIM-III): Measures independence, out of a total of 100, in performing activities of daily living. Spinal cord injury: An injury that damages the spinal cord, due to trauma or disease, and results in complete or partial paralysis. Statistical Power: The power of a test is the probability that a given test will find an effect assuming that one exists in the population. Square root of the mean squared differences (RMSSD): square root of the mean squared differences of the consecutive R peak to R peak intervals derived from an electrocardiogram. A heart rate variability time domain measure that represents cardiac parasympathetic modulation Surrogate measure: a measurement taken with the intent to gain insight into a variable that is either impractical to measure directly, or in principle impossible to measure. Sympatho-vagal balance: The interaction between the sympathetic and parasympathetic modulation of the heart. Tachycardia: Rapid heart rate, usually greater than 100 beats per minute (bpm) Tetraplegia: complete or partial paralysis of all four limbs due to an injury at the cervical vertebrae Traumatic: injury occurred due to physical damage to the spinal cord Waist circumference (WC): a measure of the distance around the abdomen with the aim to assess abdominal fat for chronic disease risk such as type 2 diabetes, high cholesterol, high blood pressure, and heart disease xiii

14 1 Chapter 1: 1 Introduction The likelihood of developing cardiac disease is 4.01 times greater in individuals with a spinal cord injury (SCI) than individuals without a SCI. 1 It has been speculated that a possible reason could be the disruption of the cardiovascular autonomic nervous system (ANS) as evidenced by the prevalence of autonomic dysreflexia and orthostatic hypotension within the SCI population. 1-5 The underlying physiological mechanisms responsible for the ANS disruptions in SCI have not yet been fully determined. 6 Heart rate variability (HRV) has been reported to non-invasively measures the modulation of the cardiac ANS and has the potential to assess risk of cardiac disease. 7 Consequently in this study, HRV was assessed to examine the cardiac autonomic changes in chronic traumatic SCI. 1.1 Spinal cord injury In 2010, it was reported that around 86, 000 people were living with a SCI in Canada with the prevalence to increase to 121,000 by Also it was reported that there are around 4,300 new cases of SCI per year, 42% due to traumatic injuries and 58% due to non-traumatic causes. 8 Traumatic SCIs arise due to a physical cause, for instance motor vehicle accidents, falls, or acts of violence. 8 Non-traumatic injuries, on the other hand, occur as a result of diseases, infections or tumors that disrupt the normal functioning of the spinal cord. 8 The cardiac risk factors for nontraumatic SCI are more challenging to identify than for traumatic SCI since non-traumatic injuries include tumor-related, congenital/developmental, infectious inflammatory and ischemic causes. 1 In addition, given that most of the SCI studies mainly focus on traumatic injuries, 9 there is more information available regarding cardiac health within the traumatic group. There are also demographic differences between the two groups: mean age is higher in the non-traumatic than in the traumatic group, and although the proportion of females and males is the same in the nontraumatic group there are three times more males than females in the traumatic group. 9 Additionally, there are more incomplete injuries than complete injuries reported within the nontraumatic group than in the traumatic group. 9 There are less secondary complications, such as spasticity and pressure ulcers, in the non-traumatic group than in the traumatic group. 10 Finally,

15 2 the traumatic group improves with rehabilitation to a greater extent than the non-traumatic group. 9 The spinal cord, which is located within the spinal canal, provides motor and sensory information between the brain and the body In humans, the spinal cord is comprised of 31 segments: 8 cervical i, 12 thoracic, 5 lumbar, 5 sacral and 1 coccygeal. 11 Each segment receives sensory information from skin areas referred to as dermatomes, and each segment innervates a group of muscles referred to as a myotome. 12 After a SCI, the International Standards for Neurological and Functional Classification of Spinal Cord Injury (ISNCSCI) are used to evaluate the neurological level of impairment (NLI) and severity of the injury [American Spinal Cord Injury Association (ASIA) impairment scale (AIS)] in terms of motor and sensory function (Appendix A) AIS is measured by a five point scale (A-E), where A corresponds to a complete injury (no motor or sensory function in the lowest sacral segments), B-D is an incomplete injury (motor and/or sensory function preserved below neurological level of injury and includes sacral segments) and E is normal motor and sensory function , Cardiovascular disease in spinal cord injury Cardiovascular disease (CVD) has been identified as the leading cause of morbidity and mortality accounting for approximately 30-50% of deaths within the SCI population; in contrast to 5-10% in an age and sex matched able-bodied population. 2-5,15-16 There is supporting evidence in the literature indicating that individuals with a SCI are at an increased risk of cardio-metabolic syndrome (CMS) CMS is characterized by having three or more of the following five clinical features: central obesity (waist circumference men>120cm, women>88cm), hypertriglyceridemia ( 1.7mmol/l), low plasma high density lipoprotein cholesterol (men<1.03mmol/l, women<1.29mmol/l), hypertension ( 130/85mmHg or on relevant medications), and fasting hyperglycemia ( 100mg/dl or on relevant medications) All of the aforementioned risk factors lead to atherosclerotic plaque formation and earlier onset of CVD In addition, after a SCI, the often sedentary lifestyle, physical deconditioning and inflated post-injury inflammatory cytokines contribute to the pro-atherogenic outcome and CVD i The 8 th cervical nerve emerges between the 7 th cervical (C7) and the first thoracic (T1) vertebrae 9

16 3 development As the number of CMS risk factors increases, CVD vulnerability greatly increases. 19 Some of the CMS risk factors overlap with the traditional CVD risk factors such as sex, age, diabetes, blood lipid profile, elevated systolic blood pressure, smoking status, sedentary lifestyle, unhealthy diet for example diet high in saturated fats, and obesity. 5,21 Also, investigators have reported that the risk of developing CVD increases with level and severity of injury i.e. higher and complete injury. 16,22 Nevertheless, a recent study by Miyatani et al. 23 found that there was greater arterial stiffness, an emerging indicator of coronary artery disease, in subjects with paraplegia than tetraplegia. Surprisingly, only 48% of the subjects with arterial stiffness met the diagnostic criteria for CMS. 23 Therefore, the CMS and traditional CVD risk factors do not completely explain why individuals with a SCI are at such great risk. 5 Consequently, there must be additional unexplained factors that contribute to the high prevalence of CVD within the SCI population. Cardiovascular autonomic disruption is common after a SCI and the impairment has been reported to increase the risk of developing CVD. 2-5 In able-bodied subjects, a poorly balanced cardiovascular ANS measured via the assessment of vital signs, has been associated with myocardial infarction, congestive heart failure, life threatening arrhythmias, and atherosclerotic plaque progression The function of the autonomic nervous system in spinal cord injury In comparison to studies examining motor and sensory dysfunction post SCI, there are fewer studies examining disturbances in the ANS. 16,25 After a SCI, there is a disruption in the ANS resulting in abnormal regulation of heart rate, blood pressure, bladder, bowel and temperature regulation, as well as respiratory and/or sexual dysfunction. 6,26 The parasympathetic preganglionic neurons are situated in the brain stem, specifically in the nuclei of four cranial nerves: oculomotorius (III), facialis (VII), glossopharyngeus (IX), and vagus (X). 5-6,17,27 The vagus nerve supplies most of the internal organs with the exceptions of the genital organs, bladder, distal intestine and anus, which are innervated by the parasympathetic sacral (S2-S4) nerves. 5-6,17,28 There is no parasympathetic innervation of the peripheral blood vessels except for the vessels that supply the pelvic organs. 5-6,28 As for the sympathetic preganglionic neurons they are situated in the grey matter of the spinal column at T1-L2. 4-6,26,28

17 4 With respect to cardiovascular autonomic function, the sympathetic preganglionic neurons at T1- T5 innervate the heart and the blood vessels of the upper body, while T6-L2 innervate the blood vessels of the lower body. 4-5,11,25-26,29 The parasympathetic innervation of the heart, arises from the vagal nuclei of the brainstem. 4-5,11,25-26,29 Therefore, depending on the level of injury, sympathetic function may be disrupted resulting in impaired control of heart rate and/or blood pressure and hypotension and bradycardia are both prevalent after a cervical injury ,25,27 Parasympathetic activity, via beat-to-beat control, decreases heart rate and conversely sympathetic activity gradually increases heart rate. 17,29-30 The level and degree of SCI has been reported to be directly linked to the extent of cardiovascular autonomic dysfunction. 4,16-17,26 For instance, individuals with a complete cervical injury suffer from an absolute disconnection between the upper autonomic centres in the brain and the intermediolateral cell column at T1- L2. 17 Early after a SCI, sympathetic activity is quickly disrupted resulting in bradycardia and the vagus nerve is hypersensitive for at least 2-3 weeks. 17 Some treatment approaches that may be required to maintain an adequate heart rate involve either implanting a temporary pacemaker or administering atropine which is a competitive muscarinic acetylcholine receptor antagonist. 17 Unfortunately, the acute period of cardiac autonomic disruption does not necessarily normalize and may become a chronic issue, especially among individuals with complete cervical or high thoracic injuries. 5,6,17,25 The disrupted cardiovascular ANS is characterized by a low resting sympathetic tone and an unaffected resting parasympathetic tone leading to a reduced resting blood pressure and heart rate and an abnormal cardiovascular response to exercise. 26 Furthermore, 91% of individuals with high and complete injuries are more prone to autonomic dysreflexia (AD) than those with low (below T6) and incomplete injuries (27%). 16,26-27 AD arises from a sensory stimulus below the level of injury and results in episodes of hypertension (20-40 mmhg above baseline) accompanied by a baroreflex mediated bradycardia. 5,17,25-26 Similarly, orthostatic hypotension (OH) is also related to the level and severity of the SCI. The incidence of OH is as high as 74% in individuals with high (T5 and above) and complete SCI. 4,16,25,27 OH is characterized by a decrease in systolic blood pressure 20 mmhg and/or a decrease in diastolic blood pressure 10 mmhg from baseline, immediately after transferring from a supine to a seated position. 4-5 The exact mechanisms resulting in both AD and OH are not clearly

18 5 understood and are probably multifactorial, however the loss of sympathetic control has been reported to be a predominant factor. 4-6 To evaluate autonomic function, a guideline was recently published (2012) outlining the International Standards to Document Remaining Autonomic Function after Spinal Cord Injury (ISAFSCI). 6 It is recommended to use the guideline in addition to the ISNSCI, and it can be administered at any time following the injury. 6 In the autonomic standards assessment form (Appendix B), a general description of the remaining autonomic function is recorded for each system/organ. 6 For the urinary tract, bowel and sexual function there is a grading system similar to the ISNSCI scoring system. 6 Also, the assessment form incorporates self-reported history, if any, regarding the function of the ANS. 6 In terms of assessing the cardiovascular ANS, general autonomic control of the heart is reported as normal, abnormal (bradycardia, tachycardia and/or any other dysrhythmias), unknown, or unable to assess. Similarly, autonomic control of the blood pressure, is described as normal, abnormal (resting systolic blood pressure is below 90 mmhg, OH and/or AD), unknown or unable to assess. 6 Nonetheless, it is important to consider that the use of ISAFSCI has not yet been validated. 6 In addition, the autonomic assessment, particularly for the heart, lacks sensitivity and specificity and does not definitively determine the degree of cardiac autonomic function/dysfunction. 1.2 Heart rate variability HRV is the most commonly used term to describe the oscillation of the heart rate and is determined by measuring the peak R to R intervals, also referred to as NN intervals, on an electrocardiogram (ECG). 31 The sinoatrial (SA) node, located in the right atrium of the heart, is responsible for generating each heartbeat and its firing rate is modulated by the ANS ,32-34 It has been reported that HRV analysis can non-invasively reflect cardiac regulation via the ANS which controls heart rate through parasympathetic and sympathetic innervation of the heart. 24,29,33,35-36 Studies have shown that low HRV is an independent predictor of cardiovascular dysfunction and cardiovascular risk. 31,33-34,36-40 Diminished levels of HRV in able-bodied subjects, have been associated with heart failure, 38 diabetes, 38,40 hypertension, 38,40 abnormal cholesterol, 40 asymptomatic left ventricular dysfunction, 38 fatal arrhythmias, 41 and death due to cardiac causes. 31

19 6 HRV standardize guidelines were developed in 1996 by a Task Force composed of members from the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. 7 Based on the recommended guidelines, there are a number of methods that can be utilized to measure HRV: 1. Time domain measures- subdivided into statistical measures and geometric measures; 2. Frequency domain measures; and, 3. Non-linear measures. 7 In the published literature, the two most commonly applied measurement methods for HRV are the time domain statistical measures and the frequency domain measures (Table 1). Investigators may have a preference towards these methods since some of the parameters are thought to be physiological markers and therefore can be used to directly assess sympathetic and parasympathetic modulation of the heart. The statistical measures, the square root of the mean squared differences of the consecutive NN intervals (RMSSD) and the proportion of the number of interval differences of the consecutive NN intervals greater than 50ms (pnn50), are a reflection of cardiac parasympathetic modulation. 31,35,37 As for the frequency domain measures, the high frequency (HF) component has been reported to reflect cardiac parasympathetic modulation 24,31-32,40-41 while the low frequency (LF) component is controversial. Some claim that LF is both a marker of parasympathetic and sympathetic modulation 24,31-32,40-41 while others claim that it is more indicative of sympathetic modulation. 2,26,30,33,42-45 Further understanding of the simultaneous actions of the neurotransmitters on the heart rate may assist in elucidating the physiological interpretations. The LF:HF has been described as a measure of the sympatho-vagal balance of the cardiac autonomic nervous system; higher ratio indicating greater sympathetic activity and a lower ratio indicating lower sympatheticactivity. 2,29-30,33,42-44 It is important to note that HRV measures the modulation of the cardiac ANS rather than the mean level of autonomic activity. 2,30 Therefore, comparing HRV, for instance between able-bodied individuals and individuals with a SCI or pre and post exercise intervention, may be more informative than solely reporting the value on its own. The HRV time domain measures are calculated directly from the NN intervals on an ECG, 7 while the frequency domain measures are derived using either parametric (e.g. autoregressive model) or non-parametric [e.g. fast Fourier transform (FFT)] mathematical algorithms. 7 The FFT is the most commonly used and recommended measure as it is simple and quick to apply. 7,31,33

20 7 Using FFT, the NN intervals in the ECG are transformed to provide the amount of variation as a function of frequency. 7,31,33 In the calculated power spectrum, the total power represents the total variance and each frequency component corresponds to a specific bandwidth 7,41 (Table 1). The frequency components are measured in absolute values of power (ms 2 ). 7 There are currently no widely accepted HRV normative values, 37 and this is probably due to the dynamic nature of the sympatho-vagal system and the intrinsic and/or extrinsic factors that may influence it. 44 The Task Force provided normal HRV values of the frequency measures in healthy adults extracted from a short term recording. However, a systematic review paper by Nunan and colleagues 37 questions the Task Force s normal values since they were approximated from small sample size studies. In comparison with the literature, the Task Force LF and HF power values were higher; Task Force figures being 1,170ms 2 for LF power and 975 ms 2 for HF power while the literature reported 519 ms 2 for LF power and 657 ms 2 for HF power. 37 Also, the LF:HF ratio reported by the Task Force ( ) was lower than the ratio that was extracted from the literature (2.8). 37

21 8 Table 1. HRV time domain and frequency domain measures* Time Domain- Statistical Measures Frequency Domain Measures Statistical variables include: Frequency variables include: Standard deviation of the NN interval (SDNN) Ultra low frequency (ULF) Bandwidth: below Hz Square root of the mean squared differences of the consecutive NN intervals (RMSSD) Number of the interval differences of the consecutive NN intervals greater than 50ms (NN50) Very low frequency (VLF) Bandwidth: Hz Low frequency (LF) Bandwidth: Hz High frequency (HF) Bandwidth: Hz Proportion of the NN50 (pnn50) which is calculated by dividing the NN50 by the total number of NN intervals * Table was developed using the Task Force guidelines Literature review The findings on heart rate variability in spinal cord injury Assessing HRV in SCI is valuable as it can quantify the extent of cardiac autonomic dysfunction that is distinctively experienced by each individual, and can be regularly used to evaluate and monitor changes in a clinical setting over time. 2,29,42 Unlike HRV analysis, most ANS measurement tools are invasive and/or require specialized expertise and equipment making it difficult to assess routinely in a clinical setting; for instance administering a sympathetic skin response test ii or measuring resting plasma catecholamine concentrations. 2,4,16 HRV analysis is currently the only assessment tool that solely examines cardiac autonomic modulation in SCI. ii Sympathetic skin response involves the momentary change of the electrical potential of the skin with the aim to assess sympathetic function. The response may be either spontaneously or reflexively induced by applying an internal or external arousal stimulus 46

22 9 The psychometric properties of measuring HRV have been minimally studied, but HRV analysis in SCI has been shown previously to be a reproducible measure (LF:HF and LF, intraclass correlation coefficient (R)= ; HF, R=0.53). 47 Additionally, Claydon and colleagues 2 revealed that the HRV frequency indices (HF, LF, LF:HF), measured in the supine position, correlated with clinical measures of the cardiovascular autonomic function including sympathetic skin response, orthostatic cardiovascular response, and plasma catecholamine levels. A number of studies have used time domain measures to assess HRV in chronic SCI. Bunten and colleagues 42 did not observe any differences in the time domain measures between the complete and incomplete cervical SCI, thoracic (T1-L5) SCI and able-bodied subjects. As well, Wang and colleagues 29 reported no differences in RMSSD and pnn50 when comparing complete cervical injuries against complete low thoracic injuries (T10-L2). Provided that the cardiac parasympathetic innervations remain intact after a SCI, the investigators expected that the parasympathetic time domain markers would not be disrupted. 42 Bunten and colleagues 42 explain that there is parasympathetic predominance, but without an increase in parasympathetic activity. However, Rosado-Rivera and colleagues 15 reported that the low paraplegia group (T7-T12) displayed lower RMSSD values compared to able-bodied, high paraplegia (T2-T5) and tetraplegia (C4-C8) groups. Therefore, as expected, the low paraplegia group displayed the highest mean heart rate (83±12bpm) and mean heart rate was significantly higher than the ablebodied (70±9bpm) group and the tetraplegiac group (69±10bpm). 15 Frequency domain measures have also been used to evaluate HRV in chronic SCI. A study by Claydon and colleagues 2 showed that individuals with complete and incomplete cervical SCIs displayed lower LF values, in comparison to thoracic (T2-T11) SCI and able-bodied subjects. The reduced LF in individuals with SCI is most likely due to the loss of sympathetic control. 2,42 Similarly, Wang and colleagues 29 reported low LF values in individuals with complete cervical injuries versus those with complete thoracic (T10-L1) injuries. Individuals with incomplete cervical injuries had greater LF values than those with complete cervical injuries given that there was less damage to the descending sympathetic pathways. 30,42 They also showed that the LF power in the thoracic group was similar to the controls indicating undisrupted sympathetic cardiac autonomic control. However, when examined per case, the two subjects with high thoracic SCI (above T5) displayed lower LF values than the controls. 2 On the contrary; the study

23 10 by Bunten and colleagues 42 found that both the thoracic group (T1-L5) and cervical group had lower LF values than the able-bodied subjects. Similarly, Castiglioni et al. 48 reported reduced LF values in the thoracic group (T5-L4) with respect to the able-bodied group. As for the HF component, the results by Claydon et al. 2 revealed that it was higher in the complete and incomplete cervical group than in the thoracic SCI and control group. The increase in vagal tone explained the presence of bradycardia within the cervical group. 2,49-50 In addition, the lower LF:HF outcome in the cervical group compared to the thoracic group and the control group further suggests that there is parasympathetic predominance after a cervical SCI. 2,42 However, Grimm and colleagues 30 found that individuals with a complete cervical injury had lower HF values than those with an incomplete cervical injury, thoracic (below T7) injury and able-bodied individuals. Wang et al. 29 also reported lower HF in a cervical group when compared to a thoracic group (T10-L1). Both Wang et al. 29 and Grimm et al. 30 found no differences in the LF:HF between the cervical and thoracic injuries. They suggested that the lack of difference in LF:HF indicated that the cardiac ANS was trying to maintain sympatho-vagal homeostasis The study by Bunten and colleagues, 42 on the other hand, found no differences in the HF component between the three groups indicating normal resting vagal tone. In the thoracic group (T2-T11), Claydon et al. 2 found that the HF was lower, and LF:HF was higher than that observed in the able-bodied group. In addition, Rosado-Rivera et al. 15 suggested that a reduced HF and higher LF:HF is a possible explanation for the prevalence of elevated heart rates among individuals with high and low paraplegia. Unfortunately, it is unclear why vagal tone is reduced but some have hypothesized that it could be a compensatory reduction with the aim to maintain sympatho-vagal balance 2,15,29-30 or be due to cardiovascular deconditioning. 15 Castiglioni and colleagues, 48 on the contrary, reported no differences in the HF values and LF:HF between the able-bodied and thoracic group (T5-L4). The reasons for the HRV discrepancies reported in the literature are still uncertain, however, there are a number of factors, including the experimental paradigm used for ECG collection that could have a major influence Factors affecting heart rate variability The relationship between HRV and potential influencing factors has been examined in previous literature. Factors including age, sex, obesity, fitness level, sleep apnea, emotional state, and

24 11 smoking status have all been confirmed (Table 2). There is a well-established relationship between age and HRV, with younger individuals showing higher HRV. 32,37,40 The decrease of HRV with increasing age could be due to the reduction of both parasympathetic and sympathetic activity. 33 The relationship between sex and HRV remains unclear. A review by Nunan and colleagues 37 found that the chosen unit of measurement influenced the relationship between sex and HRV. There is a confirmed link between ANS dysfunction and obesity 40,51 as Alrefaie et al. 52 and Mehta et al. 53 found a relationship between body mass index (BMI) and HRV while Farah et al. 54 reported a negative correlation between waist circumference and HRV, but no relationship between BMI and HRV. Farah et al. 54 argue that central obesity measured via waist circumference is a better indicator of cardiac autonomic dysfunction than general obesity measured by BMI. 54 Melanson and colleagues 55 examined the effect of endurance training on HRV in previously sedentary subjects and found that engaging in regular physical activity increased HRV. As for sleep apnea, Flevari et al. 56 hypothesized that sleep disordered breathing increased autonomic tone. Also, Chalmers and colleagues 57 conducted a meta-analysis and found reduced HRV in individuals with anxiety disorders. Finally, Lee et al. 58 reported that smoking decreases cardiac parasympathetic activity and increases sympathetic function. Table 2. Factors reported to influence HRV Factors Age Sex Influence on HRV Negative relationship between age and HRV Females displayed lower time domain measures than males (8-11% lower) LF and HF lower in males when expressed in absolute units (ms 2 ) (14% and 8% lower than females, respectively) When the units were normalized, LF was higher in males (17% higher than females) and HF was comparable When expressed in log units, females had 20% lower LF, and 18% lower HF than males LF:HF lower in females than males regardless of the measurement unit

25 12 Obesity Fitness level Sleep apnea Emotional state Smoking status Individuals with body mass index greater than or equal to 30 kg/m 2 had lower RMSSD, HF and LF but similar LF:HF in comparison to non-obese control group Negative correlation between waist circumference and RMSSD (r 2 =0.15) and PNN50 (r 2 =0.16) RMSSD and HF increased above baseline after 12 weeks of moderate-vigorous intensity exercise Constant HF but higher RMSSD, pnn50, LF, and LF:HF in patients with positional obstructive sleep apnea People with panic disorder, post-traumatic stress disorder, generalized anxiety disorder, social anxiety disorder all showed lower HF values relative to the control group Smokers had lower HF and higher LF and LF:HF than non-smokers RMSSD the same in smokers and nonsmokers 1.4 Study rationale The combination of an increased risk of cardio-metabolic syndrome, physical deconditioning, increase in inflammatory cytokines, cardiac autonomic dysfunction, and barriers to a physically active lifestyle all lead to an increased risk of developing CVD after a SCI (Figure 1). The currently available autonomic evaluation guidelines, unlike HRV, provide general information regarding cardiac ANS activity but do not measure the extent of cardiac autonomic dysfunction. Since the degree of cardiac autonomic dysfunction in SCI depends on the neurological level of impairment and severity of injury, resting HRV measures may vary accordingly. Parasympathetic innervation of the heart is still intact after a SCI, as it arises from the brainstem, and therefore cardiac autonomic function is thought to be disrupted due to sympathetic damage (Figure 2). Given the location of sympathetic innervation [T1-T4(T5)], HRV is expected to be disrupted in individuals with a SCI above the level of T5 and the degree of disruption is expected to be greater in complete injuries. Unfortunately, due to the few studies in SCI and the

26 13 inconsistent HRV findings, the relationship between HRV and SCI remains unclear. The limitations of current literature are: small sample size, combining different etiology of SCI, and/or cohort selection (discrepancies in neurological level of impairment). Consequently, the cardiac autonomic changes contributing to ANS dysfunction in SCI, as measured via HRV, are yet to be fully determined. In this thesis, resting supine HRV was examined in a large and representative sample of chronic traumatic SCI while still considering autonomic innervations based on the anatomy of the cardiac ANS. Chronic SCI, as opposed to acute or sub-acute, is considered to be a stable state and as a result is the ideal phase to study the adaptive state of ANS in individuals with a SCI. 3 Also, given that there are important etiological, comorbidities and demographic differences between traumatic and non-traumatic SCI it was decided to examine HRV and influencing factors in traumatic SCI only. Figure 1. Possible contributors to greater CVD risk in individuals with SCI. The theoretical framework summarizes the relationship between SCI and CVD. After a SCI, increased risk of cardio-metabolic syndrome, elevated levels of inflammatory cytokines, lifestyle changes, disrupted ANS, and some non-modifiable factors all contribute to overall CVD development (modified from Figure 1.0 on page 128 in the Rehabilitation Environmental Scan Atlas: Capturing Capacity in Canadian SCI Rehabilitation. 59 )

27 Figure 2. Parasympathetic and sympathetic innervations of the heart and peripheral muscles. Sympathetic innervations arise from T1-T4/T5 cord segments. Consequently, level of injury may affect cardiac autonomic function as measured by HRV. 14

28 15 Chapter 2: 2 Objectives and Hypothesis 2.1 Objectives Primary Objective To describe the distribution of HRV indices in a population of individuals with a chronic traumatic SCI. Primary HRV index: Low frequency to high frequency ratio (LF:HF). Secondary HRV indices: Low frequency (LF), high frequency (HF), square root of the mean squared differences of the consecutive NN intervals (RMSSD), and proportion of the NN50 (pnn50) which is the percentage of pairs of adjacent NN intervals differing by more than 50ms Secondary Objective a) To determine whether there is a difference in HRV indices (primary and secondary) based on level of injury (above T5 and below T5). b) To determine whether there is a difference in HRV indices (primary and secondary) based on severity of injury (complete injury and incomplete injury). c) To determine whether there is a difference in HRV indices (primary and secondary) based on level and severity of injury (complete and equal to/above T5, complete and below T5, incomplete and equal to/above T5, and incomplete and below T5). d) To determine whether there are any differences in the selected HRV-related factors based on level and/or severity of injury. The selected HRV-related factors are: age, sex, body mass index (BMI), waist circumference (WC), time post injury, current smoking status, smoking history, cardiorespiratory fitness level (absolute VO 2 peak, relative VO 2 peak, peak heart rate), leisure time physical activity questionnaire-spinal cord injury (LTPAQ-

29 16 SCI), lower extremity motor score (LEMS), spinal cord independence measure (SCIM- III), number of co-morbidities, family history of heart disease and sleep apnea Tertiary Objective a) To determine whether there is a relationship between low frequency (LF) and high frequency (HF) indices in chronic traumatic SCI. b) To determine whether there is a relationship between LF, HF, LF:HF and HRV related factors in chronic traumatic SCI: age, BMI, WC, time post injury, cardiorespiratory fitness level (absolute VO 2 peak, relative VO 2 peak, peak heart rate), LTPAQ-SCI, LEMS, SCIM-III, and number of co-morbidities. c) To determine whether there is a relationship between age, waist circumference and peak heart rate and the LF or HF indices in the entire study sample and in individuals with a complete injury that is equal to/above T5. d) To determine whether there is a relationship between LF, HF, age at injury and resting systolic blood pressure in the entire study sample and in individuals with a complete injury that is equal to/above T Hypothesis Primary Hypothesis There will be a multimodal distribution of the HRV indices based on level and severity of injury Secondary Hypothesis a) Individuals with an injury equal to or above T5 will display lower HRV values than those with an injury below T5. b) Individuals with a complete injury will display lower HRV values than those with an incomplete injury.

30 17 c) Individuals with a complete injury equal to or above T5 will display the lowest HRV values. Alternately, individuals with an incomplete injury below T5 will display the highest HRV values indicating an undisrupted cardiac ANS. d) Age, sex, time post injury, current smoking status, smoking history, and family history of heart disease do not depend on the level or severity of injury and thus will not show any differences across the cohorts. However, BMI, WC, number of co-morbidities and presence of sleep apnea will be greater in individuals with a higher level of injury and/or a complete injury. On the contrary, cardiorespiratory fitness level, LTPAQ-SCI, LEMS, and SCIM-III will be lower in individuals with a higher level of injury and/or a complete injury Tertiary Hypothesis a) The LF and HF indices will display a high positive linear relationship since the role of the ANS is to maintain homeostasis b) Both LF and the LF:HF will display a positive linear relationship with age, BMI, WC, time post injury and number of co-morbidities and a negative linear relationship with cardiorespiratory fitness level (absolute VO 2 peak, relative VO 2 peak and peak heart rate), LTPAQ-SCI, LEMS, and SCIM-III. HF will display a negative linear relationship with age, BMI, WC, time post injury and number of co-morbidities and a positive linear relationship with cardiorespiratory fitness level, LTPAQ-SCI, LEMS, and SCIM-III. c) Age, WC and peak heart will predict LF and HF indices in the entire sample and in individuals with an injury equal to/above T5. d) In the entire sample and in individuals with an injury equal to/above T5: There will be a positive linear relationship between LF and age at injury and resting systolic blood pressure. Whereas there will be negative linear relationship between HF and age at injury and resting systolic blood pressure.

31 18 Chapter 3: 3 Methodology 3.1 Overview This study was a secondary data analysis of a primary data set from a recently published study that explored the associations between arterial stiffness and spinal cord impairment. 23 The inclusion criteria of the primary study were English speaking subjects between years of age living in the Greater Toronto Area with a chronic SCI (C1-T12, AIS A-D, 2 years post impairment) of traumatic and non-traumatic etiology. 23 The exclusion criteria, of the primary study, consisted of any subjects with a previous or current history of: angina, myocardial infarction, atypical chest pain, coronary artery bypass or revascularization, aortic stenosis, uncontrolled arrhythmia or left bundle branch block, hypertrophic cardiomyopathy, severe chronic obstructive pulmonary disease requiring oral steroids or home oxygen, diaphragmatic pacer, and stroke. 23 The subjects underwent medical screening, electrocardiogram, and chart review to ensure that they met the inclusion and exclusion criteria. 23 Overall, out of the 125 subjects who were screened, 100 consented to participate, 10 withdrew their consent, and three did not meet the inclusion criteria and thus a total final sample of 87 subjects met the inclusion criteria 23 ; 75 subjects had ECG data collected. Both primary and secondary studies were approved by the University Health Network Research Ethics Board (REB#: DE) and the secondary study was also approved by the University of Toronto Office of Research Ethics (REB#:30133). HRV, as measured via ECG, was collected in accordance with the Task Force iii guidelines. Subjects were asked to abstain from caffeine and nicotine, and fast for at least 8 hours prior to the ECG collection session. The subjects were also instructed to refrain from exercise 24 hours prior to the session. The ECG data were collected between 9:00am-1:00pm. The subject was transferred to a supine position onto a bed, in a quiet and temperature controlled (24 C) room and iii European Society of Cardiology and the North American Society of Pacing and Electrophysiology Task Force HRV guidelines developed in 1996

32 19 allowed to rest for 20 minutes before collecting continuous 3-lead ECG (lead II system) for ten minutes, at a sampling rate of 1000Hz (PowerLab/16SP; AD instruments, Inc., Bella Vista, Australia) Study variables Heart rate variability indices and related factors Five HRV indices were selected based on the literature findings: LF:HF (primary index), LF, HF, RMSSD and pnn50. In addition to collecting ECG, the demographics and health status of each subject were also recorded in the primary study. Variables that were hypothesized to have an influence on HRV were included in this study (see Table 3). Table 3. Potential HRV-related variables selected from the primary data Construct of interest SCI impairments Age Sex Medications Obesity Smoking status Measurement Method Time post injury (years), neurological level of injury, severity of injury (complete or incomplete), and etiology of injury (traumatic or non-traumatic) Age (years) Sex (male/female) Beta blockers, calcium channel blockers and any other cardiac rhythm drugs BMI (kg/m 2 ) and WC (cm) Current smoking status and smoking history (yes/no)

33 20 Family history of heart disease Sleep deprivation Family history of heart disease (yes/no) Sleep apnea (yes/no) Cardiorespiratory fitness Absolute VO 2 peak (L/min), Relative VO 2 peak (ml/kg/min) and peak heart rate (bpm) Physical status Self-reported physical activity: LTPAQ-SCI (min/week) Self-reported independence in ADL s: SCIM- III (/100) Measured motor impairment: LEMS (/50) Chronic disease Number of co-morbidities (/7) Abbreviations: BMI, body mass index; WC, waist circumference; LTPAQ-SCI, leisure time physical activity questionnaire; ADLs, activities of daily living; SCIM-III, spinal cord independence measure; LEMS, lower extremity motor score 3.3 Subject selection Electrocardiogram recordings All of the ECG recordings were reviewed visually with the assistance of an internist with expertise in cardiovascular stress testing and ECG monitoring (Dr. P. Oh). For each subject, the rate and rhythm (normal sinus rhythm, bradycardia, or tachycardia), presence of premature atrial and/or ventricular contractions, electrical artifact and visual variability observed in the RR intervals were reviewed. If the subject displayed frequent premature contractions (greater than ten per minute), arrhythmias, or excessive artifact that prevented the proper analysis of the RR intervals, they were excluded from the dataset for detailed analysis Medications Medications were reviewed in consultation with a physiatrist (Dr. C. Craven) and internist (Dr. Oh). Subjects taking medications which could have an influence on HRV (beta blockers, calcium

34 21 channel blockers that influence cardiac conduction iv such as diltiazem and verapamil, and any other cardiac anti-arrhythmic drugs such as amiodarone, procainanmide, encainide and flecainide) were excluded from the study. 3.4 Heart rate variability analysis HRV analysis was conducted using LabChart (version 7.0). According to the Task Force, the gold standard for HRV short term recording analysis is a five minute interval. Therefore, the ten minutes of ECG were divided into three segments of five continuous minutes; first five minutes (t=0 - t=300 seconds), middle five minutes (t=150 - t=450 seconds) and last five minutes (t=300 - t=600 seconds) with the aim to select the segment with the least noise interference. Each five minute ECG recording was then reviewed to confirm that all and only the R peaks were marked (Figure 3a). The Poincaré Plot v was checked to examine the normal and ectopic vi RR interval ranges (Figure 3b) and to detect any ectopic islands vii. Physiologically, ectopic indicates any cardiac activity not originating from the SA node. 60 Ectopic islands were detected in 26.79% of the subjects and occurred mainly due to technical error or unknown causes. The details of the ectopic islands per subject are summarized in Appendix C. To omit ectopic islands, according to the noise-omitting method of Young and colleagues, 61 the data was filtered with a 45Hz low pass filter (Figure 4). The following post-filtered variables were recorded for the three segments: average heart rate, SDNN, SDANN, RMSSD, NN50, pnn50, total power, VLF, LF, HF, LF:HF, and noise/ectopic/artifact percentages. After analyzing all three ECG segments, the segments for each subject with the highest percentage of normal i.e. lowest percentage of ectopic beats, was included in the analysis. If the percentage of normal and ectopic beats were equal in all three segments for a particular subject, then a segment was randomly chosen using a computer-based randomizer ( If only the ectopic beats were all equal (0%) then the highest percentage of normal was chosen (the one closest to 100%). iv Any calcium channel blocker ending with ine only influences blood pressure for example amlodipine and nifedipine; they decrease blood pressure, but do not affect heart rate v The Poincaré Plot is a LabChart software feature used to assess the lengths of the RR intervals by plotting the length of each RR interval against the length of the following RR interval vi Defined as ectopic by the LabChart program vii The term ectopic islands refers to the clustering of certain data points

35 22 a) b) Figure 3. Representative example of HRV analysis using LabChart (v.7.0). a. The threshold was set and the R peaks were determined. The RR intervals are also referred to as NN intervals. b. The Poincaré Plot was used to examine the lengths of the RR intervals. The interval ranges indicates whether each RR interval is within the normal or ectopic range.

36 Figure 4. Representative example of Poincaré Plot before and after the application of 45Hz low pass filter. The arrows in the first diagram indicate two clusters of data, referred to as ectopic islands. After the filter was applied, the ectopic islands were removed and the noise in the data was reduced from 9.2 % to 0%. 23

37 Statistical Analysis Statistical analysis was conducted using IBM SPSS Statistics v.22 and is described per study objective. If the data were not normally distributed a median was reported and if the data were normally distributed a mean was reported Objective 1: Heart rate variability frequency distributions To describe the distribution of HRV in chronic and traumatic SCI, descriptive statistics were reported [mean and standard deviation or median and interquartile range (IQR)]. The frequency distributions, for each HRV index, were also plotted and the distribution was described using skewness and kurtosis. To assess whether the data were normally distributed, a Kolmogorov- Smirnov (K-S) test was administered and boxplots were checked for major outliers (a minor outlier was defined as 1.5xInterquartile range (IQR) outside the central box and a major outlier as 3.0xIQR outside the central box). If K-S p>0.05 and there were no major outliers in the boxplots, then the data were considered normally distributed. Furthermore, if the primary HRV index (LF:HF) displayed any outliers, the characteristics of the subjects who were outliers were examined with the aim to postulate possible reasons Objective 2: Comparison of heart rate variability based on level and/or severity of injury HRV parameters and the HRV-related factors were compared between: a. Level of injury (below versus above T5), b. Severity of injury (complete versus incomplete) and c. Level and severity of injury. For normally distributed data, an independent t-test or ANOVA was administered. For non-normally distributed data, a Mann-Whitney or Kruskal-Wallis test was used to compare across cohorts. Following ANOVA or Kruskal-Wallis, if there was a significant difference a post-hoc test was administered and was adjusted for multiple comparisons. Furthermore, if the primary HRV index (LF:HF) displayed any outliers, the data was examined to ensure that the outliers were not responsible for the results observed. For the categorical HRV-related factors, if in the chi square output the expected frequencies in each cell was greater than five then a Pearson Chi-Square test was used, if less than five then a Fisher s exact test was chosen. An alpha of 0.05 was set as the level of significance.

38 Objective 3: Assessing the LF and HF indices The relationship between the LF and HF indices was examined using Spearman s rho correlation co-efficient for the entire sample and per cohorts (level and/or severity of injury). For the entire sample, Spearman s was also used in order to determine whether there is a relationship between LF, HF, LF:HF and the scalar HRV-related factors. The relationship between LF, HF, age at injury and resting systolic blood pressure was assessed using Spearman s for the entire sample and in the cohort considered to be the most vulnerable to CVD (complete and equal to/above T5). The strength of each relationship was assessed using the following descriptors: r= little or no relationship, r= fair relationship, r= moderate to good relationship, and r>0.75 good to excellent relationship. 62 A multiple linear regression analysis was used to examine the relationship of CVD risk factors (age, waist circumference and peak heart) and the LF and HF indices. The relationship was assessed for the entire sample and in the cohort most vulnerable to CVD (complete and equal to/above T5). If the assumption of linearity and normality were not met, bootstrapping was conducted. An alpha of 0.05 was set as the level of significance.

39 26 Chapter 4: 4 Results 4.1 Subject selection The primary data set consisted of 75 subjects with non-traumatic and traumatic injuries. The non-traumatic subjects (n=13) were excluded from the data set based on etiology of injury. After the resting ECG was reviewed for each subject, three subjects were excluded: Two subjects displayed frequent premature ventricular contractions (PVCs 10/min) and one due to technical difficulties with ECG data collection. In addition, three subjects were excluded based on the medications reported: Two subjects were on beta blockers, and one subject was taking both a beta-blocker and a calcium channel blocker diltiazem (Tiazac XL) which decreases heart rate. The final sample size included a total of 56 subjects which were then further subdivided based on level and severity of injury (Figure 5). The characteristics of the participants are summarized in Table 4.

40 27 Assessed for eligibility (N=75) Excluded (N=19) - Non-traumatic SCI (N=13) - ECG: PVCs 10/min (N=2); Technical error (N=1) - Medications: β-blockers (N=2); β-blocker and Ca 2+ channel blocker (N=1) Analyzed (N=56) Complete and equal to or above T5 (N=27) Complete and below T5 (N=11) Incomplete and equal to or above T5 (N=10) Incomplete and below T5 (N=8) Figure 5. CONSORT flowchart reflecting the inclusion and exclusion of the final data sample. A total of 56 subjects were included for analysis.

41 28 Table 4. Demographics and vital signs of the participants in total sample and per cohort Total Complete and Complete and Incomplete Incomplete Sample equal to/above below T5 and equal and below T5 T5 to/above T5 N Age (years) 46.75± ± ± ± ±14.12 Time post injury (years) Sex (males/females) 14.23± ± ± ± ± /12 22/5 9/2 8/2 5/3 BMI (kg/m 2 ) 26.13± ± ± ± ±3.11 WC (cm) 95.65± ± ± ± ±10.05 HR (bpm) 61.67± ± ± ± ±7.07 SBP (mmhg) ± ± ± ± ±14.51 DBP (mmhg) 71.38± ± ± ± ±13.47 All values are mean ± standard deviation or as otherwise indicated Abbreviations: BMI, body mass index; WC, waist circumference; HR, heart rate; SBP, systolic blood pressure; DBP, diastolic blood pressure 4.2 Frequency distributions of the heart rate variability indices Table 5 summarizes the LF:HF descriptive statistics for the entire sample. The frequency distribution was positively skewed and leptokurtic indicating that the values cluster at the lower end and it is a pointy and heavy-tailed distribution (Figure 6). The results showed that the data were significantly different than a normal distribution as K-S p<0.001 and there were major outliers present in the boxplot (two major and two minor) (Figure 7). The characteristics of the

42 29 subjects who were outliers are summarized in Appendix D, Table 1. To further examine the influence of outliers, they were removed and the data were reassessed for normality [N=52, Mdn=1.08(0.59, 2.45), IQR=1.86]. The frequency distribution of the LF:HF without the outliers is in Appendix D, Figure 1. The data were still not normally distributed since K-S test p=0.002, however, the distribution became less positively skewed (+1.13) and leptokurtic (+0.57). The descriptive statistics of the secondary HRV indices (LF, HF, RMSSD, pnn50) are summarized in Table 5, and none of the indices were normally distributed. The frequency distributions and the boxplots for each HRV index are in Appendix E. The LF, HF, and RMSSD distributions were positively skewed and leptokurtic, whereas the distribution of pnn50 was positively skewed and platykurtic. The boxplots displayed: Five minor outliers in LF, three major and two minor outliers in HF, three minor outliers in RMSSD, and none in pnn50. The descriptive statistics for total power can be found in Appendix F, Table 1. Table 5. Descriptive statistics for each HRV index in the entire sample (N=56) LF:HF LF HF RMSSD pnn50 Median (Lower, 1.21 (0.63,2.85) ms 2 (207.73, ) ms 2 (143.69, ) ms (20.03,59.03) 6.62% (1.23,23.35) Upper quartile) IQR ms ms ms 22.12% Skewness Kurtosis p-value p<0.001* p<0.001* p<0.001* p=0.007* p<0.001* Kolmogorov-Smirnov (K-S) test; p 0.05* Abbreviations: LF:HF, low frequency to high frequency ratio; LF, low frequency; HF, high frequency, RMSSD, square root of the mean squared differences of the consecutive NN intervals, pnn50, proportion of the number of interval differences of the consecutive NN intervals greater than 50ms; IQR, interquartile range

43 30 Figure 6. Frequency distribution of LF:HF. The data was positively skewed (+2.70) and leptokurtic (+8.45). Figure 7. Boxplot representing LF:HF distribution. The asterisks represent major outliers (3.0xIQR outside the central box) and the circles represent minor outliers (1.5x IQR outside the central box).

44 Heart rate variability comparisons across level and/or severity of injury The HRV indices were compared between two cohorts: Level of injury (above versus below T5) and severity of injury (complete versus incomplete injury). None of the HRV indices differed based on level of injury (Table 6) or severity of injury (Table 7). The comparisons of total power can be found in Appendix F, Tables 2 and 3. Table 6. Comparison of HRV indices based on level of injury Median (Lower, Upper quartile) HRV Index Below T5 Above/Equal to p Value (N=19) T5 (N=37) LF:HF 1.54 (0.76,3.32) 1.10 (0.60,2.71) LF (ms 2 ) (209.71, ) (187.32,330.22) HF (ms 2 ) (177.26, ) (133.73,883.00) RMSSD (ms) (21.45,61.54) (19.48,58.94) pnn50 (%) 8.08 (1.49,26.64) 5.24 (1.13,23.07) Mann-Whitney test; p 0.05* Abbreviations: LF:HF, low frequency to high frequency ratio; LF, low frequency; HF, high frequency, RMSSD, square root of the mean squared differences of the consecutive NN intervals, pnn50, proportion of the number of interval differences of the consecutive NN intervals greater than 50ms

45 32 Table 7. Comparison of HRV indices based on severity of injury Median (Lower, Upper quartile) or Mean ± Standard deviation HRV Index Complete Incomplete p Value (N=38) (N=18) LF:HF 1.60 (0.69,3.06) 0.96 (0.47,1.76) LF (ms 2 ) (223.75, ) HF (ms 2 ) (139.41,876.10) (104.85, ) (127.68, ) RMSSD (ms) ± ± pnn50 (%) 5.00 (1.14,22.10) 8.31 (1.78,26.95) Mann-Whitney test; Independent t-test; p 0.05* Abbreviations: LF:HF, low frequency to high frequency ratio; LF, low frequency; HF, high frequency, RMSSD, square root of the mean squared differences of the consecutive NN intervals, pnn50, proportion of the number of interval differences of the consecutive NN intervals greater than 50ms Similarly, when examined across the four cohorts, based on level and severity of injury, none of the HRV indices were significantly different (Table 8). The comparisons in total power can be found in Appendix F, Table 4. To examine the influence of the outliers in LF:HF (the primary HRV index) a boxplot with and without the outliers was examined (Figure 8) and the new values were re-tested for comparisons. There were still no differences across the cohorts (p=0.133) when the outliers were omitted. The characteristics of the subjects who were the initial outliers can be found in Appendix D, Table 1. Refer to Appendix G to see the boxplots across level and severity of injury for each HRV index.

46 33 Table 8. Comparison of HRV indices based on level and severity of injury Median (Lower, Upper quartile) or Mean ± Standard deviation HRV Complete and Complete and Incomplete and Incomplete and p Value Index equal to/above below T5 equal to/above below T5 (N=8) T5 (N=27) (N=11) T5 (N=10) LF:HF (0.55,2.91) (1.51,4.84) (0.72,1.93) (0.35,2.00) LF (ms 2 ) (207.07,741.10) (455.41, ) (102.30, ) (122.14, ) HF (ms 2 ) (124.05,879.65) (177.26,660.09) (125.28, ) (144.60, ) RMSSD (ms) ± ± ± ± (Welch) (Brown- Forsythe) pnn (%) (1.11,22.50) (1.49,15.43) (1.78,24.70) (0.86,28.12) Kruskal-Wallis test; One way ANOVA test; p 0.05* Abbreviations: LF:HF, low frequency to high frequency ratio; LF, low frequency; HF, high frequency, RMSSD, square root of the mean squared differences of the consecutive NN intervals, pnn50, proportion of the number of interval differences of the consecutive NN intervals greater than 50ms

47 Figure 8. Boxplot representation, with and without the outliers, of LF:HF based on level and severity of SCI. For each color-coded cohort there are two boxplots, the darker shaded boxes represent data with the outliers while the lighter shaded boxes represent the data after the initial outliers were omitted. The outlier numbers correspond with the subject numbers. 34

48 Comparison of heart rate variability related factors across cohorts SCIM-III (p=0.036), relative VO 2 peak (p=0.038) and peak heart rate (p=0.033) were all greater in individuals with an injury below T5. However, time post injury (p=0.501), BMI (p=0.883), LTPAQ-SCI (p=0.668), LEMS (p=0.111), number of co-morbidities (p=0.179), age (p=0.221), waist circumference (p=0.830) and absolute VO 2 peak (p=0.088) were all similar. The medians or means for each factor are summarized in Appendix H, Table 1. Time post injury (p=0.001) was greater in individuals with a complete injury, whereas LEMS (p<0.0001) and SCIM-III (p=0.003) were greater in the incomplete cohort. BMI (p=0.352), LTPAQ-SCI (p=0.106), number of co-morbidities (p=0.622), age (p=0.393), waist circumference (p=0.720), relative VO 2 peak (p=0.496), absolute VO 2 peak (p=0.232) and peak heart rate (p=0.705), were all the similar. The medians or means for each factor are summarized in Appendix H, Table 2. There were no differences in the HRV-related categorical factors based on level of injury (Appendix H, Table 3) or severity of injury (Appendix H, Table 4). The HRV-related factors were also compared based on level and severity of injury. LTPAQ-SCI (Figure 9), LEMS (Figure 10) and SCIM-III (Figure 11) all revealed significant differences between the cohorts. Individuals with a complete injury below T5 reported less leisure time activity than individuals with an incomplete injury below T5 (p=0.019). Individuals with a complete injury equal to/above T5 displayed a lower LEMS than individuals with an incomplete injury equal to/above T5 (p<0.001) and incomplete and below T5 (p<0.001). Also individuals with a complete injury below T5 revealed a lower LEMS than individuals with an incomplete injury equal to/above T5 (p=0.001) and incomplete and below T5 (p=0.008). Finally, individuals with a complete injury equal to or above T5 had a lower SCIM-III score than individuals with an incomplete injury below T5. On the other hand, time post injury (p=0.016 but was not significant for each between group comparison), BMI (p=0.798), number of co-morbidities (p=0.583), age (p=0.271), waist circumference (p=0.958), relative VO 2 peak (p=0.302), absolute VO 2 peak (p=0.254 and p=0.154) and peak heart rate (p=0.077 and p=0.096) were similar across the four cohorts. The medians and means for each factor are summarized in Appendix H, Table 5 and the

49 36 follow-up analysis is in Appendix H, Table 6. There were no differences in the HRV-related categorical factors based on level and severity of injury (Appendix H, Table 7). Figure 9. Boxplot representation of LTPAQ-SCI based on level and severity of injury. The complete and below T5 cohort has a lower LTPAQ-SCI score than the incomplete and below T5 cohort (p=0.019).

50 37 Figure 10. Boxplot representation of LEMS based on level and severity of injury. The complete and equal to/above T5 cohort had a lower LEMS than the incomplete and equal to/above T5 (p<0.001) and incomplete and below T5 (p<0.001) cohorts. Also, the complete and below T5 cohort had a lower LEMS than the incomplete and equal to/above T5 (p=0.001) and incomplete and below T5 (p=0.008) cohorts. Figure 11. Boxplot representation of SCIM-III based on level and severity of injury. The complete and equal to/above T5 cohort had a lower SCIM-III score than the incomplete and below T5 cohort (p=0.010).

51 Assessing the heart rate variability frequency domain indices: LF, HF and LF:HF Relationship between LF and HF A high positive correlation was found between LF and HF for the entire sample (r=0.708, p<0.0001) (Figure 12) and in each of the cohorts (Table 9 and Table 10). The scatter plots of LF and HF based on level and/or severity of injury can be found in Appendix I. Figure 12. The relationship between LF and HF for total sample. There was a positive linear correlation between LF and HF in individuals with a chronic and traumatic SCI. The coefficient of determination (R 2 ) indicated that 50.8% of the variation in LF was shared by HF.

52 39 Table 9. Relationship of the LF and HFindices based on level or severity of injury Level of Injury Severity of Injury Below T5 Above/Equal to T5 Complete Injury Incomplete Injury Spearman s Rho p=0.002* p<0.0001* p<0.0001* p<0.0001* Spearman s correlation test; *p 0.05 Table 10. Relationship of the LF and HF indices based on level and severity of injury Complete and Complete and Incomplete Incomplete equal below T5 and equal and below T5 to/above T5 to/above T5 Spearman s Rho p<0.0001* Spearman s correlation test; *p 0.05 p=0.029* p=0.001* p=0.010* Relationship between LF, HF, LF:HF and influencing factors The relationships between LF, HF, LF:HF and the HRV-related factors were examined in the entire sample (Table 11). LF was negatively correlated with age (r=-0.366, p=0.006) and time post injury (r=-0.384, p=0.003). Similarly, HF was negatively correlated with age (r=-0.317, p=0.017) and time post injury (r=-0.344, p=0.009) and was positively correlated with SCIM-III (r=0.299, p=0.025). LF:HF did not reveal a significant relationship with any of the HRV indices. The scatter plots of the significant correlations listed in Table 13 can be found in Appendix J. Table 11. The relationship between LF, HF indices and the scalar HRV-related factors LF HF LF:HF Age

53 40 p=0.006* p=0.017* p=0.930 Time post injury p=0.003* p=0.009* p=0.750 BMI p= p= p=0.750 WC p= p= p=0.259 Absolute VO 2 peak p= p= p=0.162 Relative VO 2 peak p= p= p=0.311 Peak heart rate p= p= p=0.564 LTPAQ-SCI p= p= p=0.180 LEMS p= p= p=0.442 SCIM-III p= p=0.025* p=0.330 Number of comorbidities p= p= p=0.539 Spearman s correlation test; *p 0.05 Abbreviations: BMI, body mass index; WC, waist circumference; LTPAQ-SCI, leisure time physical activity questionnaire-spinal cord injury; LEMS, lower extremity motor score; SCIM- III, spinal cord independence measure

54 41 There was no significant relationship between age at injury, resting systolic blood pressure, LF or HF when examined in the entire sample (Range of values: Spearman s rho (r= r=0.073; p>0.05). Similarly, in individuals with a complete injury at or above T5 (hypothesized to be the cohort at greatest risk for CVD) also showed no significant relationship between age at injury, resting systolic blood pressure, LF or HF (Range of values: Spearman s rho (r= r=0.170; p>0.05) Predicting LF and HF from heart rate variability-related factors The HRV related factors, age, waist circumference, peak heart which are also CVD risk factors, were assessed using multiple linear regressions to determine whether they can predict LF and/or HF. Table 12 and Table 13 depict that there were no significant relationships when assessed in the entire sample. Similarly, when assessed in individuals with a complete injury equal to/above T5 (expected to be more vulnerable to CVD) there was still no significant relationship; results are summarized in Appendix K. Table 12. Multiple linear regression analysis to predict LF for the entire sample (R 2 =0.039) Parameter Regression Coefficient 95% CI p-value Intercept , Age , WC , Peak heart rate , *p 0.05 Abbreviations: WC, waist circumference; CI, confidence interval

55 42 Table 13. Multiple linear regression analysis to predict HF for the entire sample (R 2 =0.009) Parameter Regression Coefficient 95% CI p-value Intercept , Age , WC , Peak heart rate , *p 0.05 Abbreviations: WC, waist circumference; CI, confidence interval

56 43 Chapter 5: 5 Discussion This is the first study to examine the HRV characteristics in a large and representative sample of individuals with chronic traumatic SCI. The primary and secondary HRV indices were not normally distributed and revealed high inter-individual variability in HRV which is similar to what has been reported in the able-bodied population. Nevertheless, it was unclear why some subjects (n=4) were outliers when LF:HF was examined in the entire sample. Also, the primary and secondary HRV indices were not significantly different when compared across cohorts. It is possible that the lack of a relationship between HRV and level and/or severity of injury is due to other unaccounted factors influencing the ANS, or because between-subject comparisons were made instead of within-subjects. In addition, even though some of the HRV-related factors were different across cohorts, the HRV results were still similar when compared based on level and/or severity of injury. Therefore, the HRV-related factors may have little influence on HRV in individuals with chronic traumatic SCI in this study, despite being reported to influence HRV in the able-bodied population. In addition, a strong positive linear relationship was found between LF and HF and thus the LF:HF ratio may remain unchanged in individuals with chronic traumatic SCI. Finally, the bivariate and multivariate analysis between LF, HF and the potential factors that may influence HRV did not display any significant relationships. There are many biophysiological changes that occur after a SCI and thus it may be challenging to determine which factors may be influencing the HRV results. The frequency distributions of the primary and secondary HRV indices were all positively skewed indicating that the HRV values in this SCI sample were mainly low. The observed high inter-individual variability in HRV aligns with the able-bodied population. Nunan et al. 37 conducted a systematic review to determine the normal values of HRV in healthy adults. The systematic review was comprised of studies that measured short term HRV, in accordance with the Task Force guidelines, in healthy adult participants (n 30). Nunan and colleagues 37 found large inter-individual variations (up to 260, 000% of variation was reported) between the studies, especially for the frequency domain measures. Nevertheless, according to Malliani et al. 44 it should not be surprising that there are no HRV normative values within the healthy population

57 44 since the cardiac ANS is a dynamic system, consists of a large range of values, and is influenced by a number of internal and external factors. Table 14 depicts the mean and median of the LF:HF, LF and HF that were reported in healthy subjects based on the systematic review 37 and in chronic traumatic SCI based on the thesis findings. Both the mean and median values depict a large standard deviation and interquartile range, respectively. Consequently, even though the HRV values cannot be generalized to the SCI population, the values do indicate that, similar to able-bodied subjects, there are inter-individual differences in cardiac ANS function within individuals with chronic traumatic SCI. In addition, the large variations in HRV values emphasize the importance of reporting HRV as a median as opposed to a mean. Table 14. Comparison of inter-individual variations in HRV between healthy subjects and chronic traumatic SCI Mean ±SD Median (Lower, Upper quartile) HRV Indices Healthy Subjects (Systematic review) 37 Chronic Traumatic SCI (Current thesis findings) LF:HF 2.8± (1.1, 11.6) LF ms 2 519± (193, 1009) HF ms 2 657± ± (0.63, 2.85) 886± (208,1266) 834± (82, 3630) 362 (143,1087) Abbreviations: SD, standard deviation; LF:HF, low frequency to high frequency ratio; LF, low frequency; HF, high frequency

58 45 The LF:HF is a primary measure of HRV as it is reported to examine the level of sympathetic to parasympathetic activity and thus assesses the modulation of the cardiac ANS. After the LF:HF outliers were examined, the characteristics of the subjects who were considered outliers did not provide any further insights. The subjects did not demonstrate any consistent factors to account for the high LF:HF as they varied in terms of both level and severity of injury, and displayed diverse HRV-related factors (e.g. differences in sex, time post injury, smoking status, and physical fitness). In addition, when the subjects were compared to the median of the rest of the study sample, each subject displayed different factors that were outside the median range. Based on the data available it was not possible to determine why they were outliers leading one to expect that perhaps more sensitive biophysiological differences, for instance arterial stiffness and/or high levels of inflammatory cytokines, may be responsible. Given that both ANS dysfunction and CVD have been reported to be linked with level and severity of SCI, 4-6,22 it was hypothesized that HRV will differ across cohorts. However, in this study there were no differences and thus the results suggest that HRV does not depend on type of SCI (injury level and completeness). Nevertheless, cardiac autonomic regulation is part of two larger complex systems: the cardiovascular system and the autonomic nervous system. The cardiovascular component includes peripheral circulation, often altered in SCI, and influences cardiac function. 63 In addition, heart rate is not only modulated by the autonomic nervous system, but also by the intrinsic cardiac system, baroreflex function, respiration and humoural factors. 63 The ANS controls many other body functions that may also be disrupted in individuals with a SCI depending mainly on the level and completeness of pathology 44 and thus cardiac autonomic function may be influenced by other intrinsic ANS dysfunctions. Consequently, the differences in pathophysiology of cardiac autonomic function in individuals with a chronic traumatic SCI may not be ideal to examine in isolation without considering other cardiovascular abnormalities. In addition, despite the HRV differences reported in the literature (described in Chapter 1, Section 1.3.1) some investigators have emphasized that HRV is not a direct measure of the parasympathetic and sympathetic nerve activity, but instead quantifies cardiac autonomic responsiveness. 7,45 The interpretation of HRV values raises the question if discrete measurement of HRV at a single point in time is indicative of cardiac ANS modulation and whether comparing discrete HRV values between subjects has biological merit.

59 46 Based on only level of injury, the results showed that relative VO 2 peak, peak heart rate, and SCIM-III scores, were greater in the subgroup of individuals with an injury below T5. In agreement with the literature, Simmons and colleagues 64 classified level of SCI as a major determinant of relative VO 2 peak. The established general reference value for cardiorespiratory fitness is significantly higher in people with paraplegia (median: 16.0 ml/kg/min) than those with tetraplegia (median: 8.8 ml/kg/min). 64 Furthermore, in accordance with the literature, Ravensbergen and colleagues 25 reported that a person with a SCI at the level of T5 or above attains a reduced peak heart rate during exercise due to diminished sympathetic control. Hagen et al. 17 reported that individuals with complete tetraplegia are unable to raise their peak heart rate to more than 125 bpm. The increased heart rate observed during exercise has been thought to be the result of vagal withdrawal. 4,25 As SCIM-III provides insight regarding physical capacity, it was not surprising that SCIM-III was related to the level of injury. For instance, an increase in physical capacity signifies that the individual has greater voluntary functional muscle mass. 64 Even though the results indicate greater cardiorespiratory fitness (relative VO 2 peak and peak heart rate) and greater physical capacity (SCIM-III) in individuals with an injury below T5, cardiorespiratory fitness and physical capacity appeared to have a minimal impact on the HRV results as they failed to contribute to the expected higher HRV values. From our analysis we would have to conclude that, cardiorespiratory fitness level and physical capacity may not have a major influence on HRV when assessed based on level of SCI alone. Physical capacity, measured by LEMS and SCIM-III, was higher in incomplete injuries and the time post injury was longer in complete injuries. Previous findings have shown that level and severity of SCI may influence HRV results, 2 but other SCI characteristics have not been examined. For instance, it is unknown if and how time post SCI influences HRV. Based on this study, however, time post injury in addition to physical capacity may not have an influence on HRV when assessed based on severity of injury since there were no differences observed between the cohorts. As expected LTPAQ-SCI, LEMS and SCIM-III, differed based on both level and severity of injury. Bucholz and colleagues 65 found that LTPAQ-SCI has been associated with a decrease of

60 47 CVD risk factors in individuals with chronic traumatic SCI. Previous studies have shown that individuals with a complete and cervical injury report less leisure time physical activity than those with an incomplete and lower level of injury In addition, the differences in physical activity are probably because individuals with complete tetraplegia are limited in terms of in which exercises they can participate and require greater assistance with exercise protocols Also, individuals with high lesions, especially cervical, often have bradycardia and thus their low level of cardiac sympathetic function makes it difficult to participate in physical activity. 25 The results did show that individuals with complete injury engaged in less leisure time physical activity than those with an incomplete injury, but these findings were only observed in individuals with injuries below the level of T5. A possible reason as to why, contrary to the literature, severity of injury did not influence the physical activity results in individuals with high level injuries, could be the method of measuring leisure time physical activity. Most studies use the Physical Activity Recall Assessment for People with Spinal Cord Injury (PARA-SCI) questionnaire which involves recording both activities of daily living in addition to leisure time physical activity and is collected over three days as opposed to over a week. The present study, however, used the LTPAQ-SCI which recorded the number of minutes of physical activity per week and did not include activities of daily living. Expected and consistent with the literature, LEMS were lower in individuals with a complete injury and/or with an injury above the level of T5 illustrating less voluntary muscle function in the lower limbs. Similarly, SCIM-III revealed lower scores for individuals with complete and equal to/above T5 injuries than those with in incomplete and below T5 injuries. Lower physical capacity and physical activity can be used to determine whether the individual is likely to be sedentary 64 and thus has been reported to contribute to lower HRV values. 25 However, in this study, despite the differences in the physical activity observed amongst the cohorts, the HRV indices were still similar when compared based on both level and severity of injury. Therefore, physical activity and capacity may not have a substantial impact on HRV in a chronic traumatic SCI population. When revisiting the theoretical framework illustrating the link between SCI and CVD for chronic traumatic SCI, it is important to include impaired cardiac autonomic modulation as a component of the disrupted autonomic nervous system (Figure 13). Many studies have reported a diminished

61 48 LF in individuals with a cervical and/or thoracic SCI. 2,29,42,48 A reduced LF, a purported cardiac sympathetic marker, indicates a greater challenge to participate in physical activity 25 and thus contributes to sedentary behavior. A decrease in HF, a parasympathetic marker, is problematic for this population since parasympathetic activity decreases the amount of work on the heart and thus has been linked to restoring and protecting the cardiovascular system in other populations. 67 Therefore, if LF does indeed decrease after a SCI, the positive relationship between LF and HF may indicate increased risk of CVD development in individuals with a chronic and traumatic SCI. Since the primary study excluded any subjects with a cardiac disease, including arrhythmias, we have no indication of how arrhythmias interact with cardiac autonomic modulation. Furthermore, with the non-modifiable factors of age, sex, genetic history, sedentary lifestyle, smoking status and obesity being accounted for in this study albeit considering the collinearity but demonstrating a lack of effect on the HRV indices suggests that other biological contributors to CVD development will need to be examined in this chronic population. Figure 13. Possible contributors to greater CVD risk in individuals with chronic traumatic SCI. The theoretical framework represents the relationship between SCI and CVD while also considering the findings from the thesis. Impaired cardiac autonomic modulation has been added as a subgroup of disrupted autonomic nervous system to indicate that impairment also contributes to CVD development in chronic traumatic SCI. The arrow between the sympathetic

62 49 and parasympathetic cardiac activity is bidirectional since the ANS represents a shifting balance between the two systems (Modified from Figure 1.0). There is a debate in the literature regarding the relationship between LF and HF in individuals with a SCI. In this study, LF and HF displayed a positive linear relationship when assessed in the entire sample and based on level and/or severity of injury. After a cervical SCI, Claydon et al. 2 observed lower LF and higher HF, whereas Grimm et al. 30 and Wang et al. 29 reported both lower LF and HF. As for the thoracic group, Claydon et al. 2 reported no change in LF and a reduced HF while Bunten et al. 42 and Castiglioni et al. 48 reported reduced LF but no change in the HF values. Although the findings from the current study did not show a decrease in any of the HRV parameters, it did show that in the entire sample, 50.8% of the variation in LF was shared by HF and thus a decrease or even an increase in both components could result in a similar LF:HF ratio. The LF and HF relationship was strongest in individuals with an incomplete injury and equal to/above T5, as 84.3% of the variability in LF was shared by HF. In incomplete injuries the cord is not completely disconnected from the brain 30,42 and with an injury that occurs at or above T5 the sympathetic activity is disrupted, 25 thus it is possible that the modulation of ANS system was altered to a greater extent in this group. There is low resting sympathetic tone in individuals with a SCI in comparison with able-bodied subjects. 26 Consequently, most investigators indicate that a change in HF is required to align with the low levels of LF since the ANS re-balances to maintain homeostasis. 2,15,29-30 However, the physiological basis of how the ANS re-balances remains unclear. However, Billman 68 challenged the presumption of the ANS re-balancing and argued that the LF index is not more indicative of sympathetic function but is rather a complex combination between the two ANS branches along with other unidentified factors. Furthermore, the correlation between the LF and HF indices does not meet the nine Bradford-Hill criteria 69 for causation and thus it is not certain that a change in LF caused a change in HF. Therefore, the positive correlation between the LF and HF indices may not necessarily represent a re-balanced ANS system. Despite the indeterminate physiological reasons for the positive correlation observed between the LF and HF indices, overall this finding questions whether the LF:HF, the most common HRV measure, is an appropriate marker of the cardiac sympatho-vagal balance in individuals with chronic traumatic SCI.

63 50 None of the HRV related factors that were tested displayed a significant correlation with the LF:HF even though some variables did display a significant correlation with the LF and HF indices. After examining the scatter plots of LF, HF and the significantly correlated HRV-related factors, it appeared that the variation in LF and HF were only minimally shared by age, SCIM- III, and time post injury (ranging from almost 1% to 15%). The mean age (46.75±12.44) reported in the SCI and resting HRV literature is similar to the mean age reported in this study. It was expected that as age increases sympathetic activity increases, represented by higher LF, and parasympathetic activity decreases, represented by lower HF. 32 Surprisingly, however, an inverse relationship was observed between LF and age, but only 4.1% of variation in LF was shared by age. Also, unexpectedly, HF displayed a weak negative relationship with age and only 1% of variation in HF was shared by age. A higher degree of physical capacity usually indicates capability to be physically active which in turn has been linked to the predominance of the parasympathetic function. 31,67,70-71 As a result, it was surprising that the HF was only slightly positively correlated with SCIM-III; where only 0.8% of the variability in HF was shared by SCIM-III. However, SCIM-III may not be a good indicator of physical capacity. The relationship between HRV and time post injury has not yet been established for SCI, but the findings revealed that time post injury was negatively correlated with both LF and HF; 14.7% of the variability in LF was shared by time post injury, and 12.8% of variability in HF was shared by time post injury. Therefore, the results might be interpreted that as time post injury increased, both sympathetic and parasympathetic function decreased illustrating the persistence of CVD risk (for the same reasons mentioned earlier). The strengths of the relationships between LF, age and time post injury, and between HF, age, time post injury and SCIM-III were all of fair magnitude at best and thus over interpretation without further analysis is not warranted. Similarly, the multiple linear regression analysis did not reveal any significant relationships between the CVD risk factors (age, waist circumference, peak heart rate) and LF or HF when examined in the entire sample. The multivariate relationship was also assessed in individuals with a complete injury at or above T5, the cohort that is most likely to develop CVD, but again no relationships were observed. Consequently, according to our results, the CVD risk factors, which are also HRV-related factors, do not have a substantial impact on LF and HF in

64 51 individuals with a traumatic and chronic SCI. The presence of a SCI, associated with various physiological and functional changes, may minimize the impact of the HRV-related factors that have been observed in the able bodied population. When age at injury and resting systolic blood pressure were examined to determine if they were contributors to HRV in both the entire sample and in the cohort with a complete injury equal to/above T5, age at injury and LF or HF both appeared to have no relationship. Therefore, the cardiac ANS does not behave differently if a SCI occurred at a younger or older age our range being 37to 54 years. Resting systolic blood pressure has been reported to be linked with level and severity of SCI; higher level and complete injuries display lower resting systolic blood pressure 5 and the reduction in blood pressure has been reported to occur due to the reduction in sympathetic activity after a SCI. 3 Again we could not demonstrate these expected findings in the entire sample or in individuals with a complete injury at or above T5. Consequently, in individuals with a chronic and traumatic SCI, it appears that the sympathetic and parasympathetic cardiac activity is not influenced by sympathetic reduced resting blood pressure. The ANS is a complex biological system and therefore it is difficult to ascertain what and how other changing ANS activity could be influencing the HRV parameters without conducting physiological experiments that challenge the ANS system over time. 5.1 Implications and future directions Cardiac autonomic disturbances are believed to be a major contributor to the development of CVD within the SCI population. Therefore, quantifying the cardiac parasympathetic and sympathetic modulation of the heart, via a non-invasive measure, is important for diagnostic, prognostic and/or rehabilitative purposes. However, HRV values for SCI have not yet been established and the lack of HRV differences observed in this study indicate that HRV does not directly reflect the anatomical sympathetic and parasympathetic autonomic innervations and response of the heart in individuals with a chronic and traumatic SCI. The proposed HRV-related factors, in addition to age at injury and resting systolic blood pressure did not have an impact on the HRV results, which further emphasizes the complexity of the ANS. The findings from this data set suggests limited potential for assessing HRV at a single point in time in individuals with a chronic traumatic SCI to measure autonomic cardiac function..

65 52 There is disagreement in the literature as to whether the subjects should be divided into three levels i.e. cervical, high thoracic, low thoracic and lumbar, or two levels i.e. above a level of injury and below a level of injury. Consequently, to check whether there are any HRV differences between different types of SCI, in addition to a larger sample size, this work could be repeated with a different cohort selection: cervical (C1-C8), high thoracic (T1-T5), and low thoracic (T6-T12). The presence of a strictly cervical cohort allows for the assessment of completely disconnected cardiac sympathetic innervation. In addition, West et al. 5 have reported that in individuals with a chronic SCI, autonomic completeness of the injury, which can be estimated via catecholamine concentrations as well as blood pressure variability, is more closely related to the function of the cardiovascular ANS than the neurological completeness of injury. 5 Therefore, it may be more important to measure concomitantly the autonomic completeness of injury and additional autonomic dysfunctions such as orthostatic hypotension and autonomic dysreflexia to gain further insights. Also, provided that HRV represents the modulation of the cardiac ANS, it may be a valuable tool to test the responsive of the cardiac ANS to different testing conditions. 45 The testing conditions will eliminate the problem of high HRV inter-subject variability since the HRV comparisons will be made within-subjects. It may also be useful to combine HRV assessments with other cardiac measurements to assess the risk of cardiac disease in individuals with a SCI such as combining left ventricular ejection fractions with HRV assessments in order to identify cardiac patients as suggested by Kleiger and colleages 41. The International Standards to document remaining Autonomic Function after Spinal Cord Injury (ISAFSCI) 6 have been recently considered the gold standard for ANS assessment in SCI and includes measures of the heart rate, blood pressure, sweating, temperature regulation and the broncho-pulmonary system etc. and could add value to future studies using different testing paradigms to assist with interpreting the results. 5.2 Study limitations A major study limitation was that HRV was compared across cohorts and subjects that were highly variable. Intra subject comparisons using HRV may be a far better paradigm. Also, contrary to the literature, no differences were observed in HRV when compared based on level and severity of injury. The total sample size divided into four cohorts may have been too small to detect significant differences. Post-hoc power analysis revealed that based on the mean per

66 53 cohort of the primary HRV index, LF:HF, a total sample size of 72 would be required to detect a difference. Also the completeness of injury, as assessed by the International Standards for Neurological and Functional Classification of Spinal Cord Injury indicates whether there is sensory or motor function preserved in the lowest sacral segments (S4-S5). 13 Therefore neurological completeness which was used in this study, provided little information regarding the severity of autonomic dysfunction after a SCI. A final study limitation was that the breathing pattern was not monitored or recorded simultaneously with the ECG data collection in the primary study so the data were not available for analysis. Respiratory sinus arrhythmia is the natural variation of the heart rate and is driven by the breathing pattern via vagal influence of the heart. 31 The HF bandwidth has been linked to the respiratory sinus arrhythmia and thus the breathing pattern during data collection may alter the results of HRV ,35 Nunan and colleages 37 showed that the parasympathetic activity was elevated when testing was done in a resting supine position along with paced breathing. Billman 68 also suggested that all subjects must engage in paced breathing to ensure precise measurement of HRV. Consequently, depending on whether the subjects in this study engaged in controlled or spontaneous breathing, the HF component may have disproportionally represented the parasympathetic modulation of the heart.

67 54 Chapter 6: 6 Conclusions Traditional CVD risk factors, such as age, sex, obesity, and lifestyle, in addition to SCI-related changes pose an increased risk of CVD development among individuals with a SCI. Autonomic dysfunction, particularly of the cardiovascular ANS has been recently classified as a major CVD contributor in SCI and thus requires further investigation. HRV analysis was examined since it has been hypothesized to have the potential to non-invasively measure of cardiac autonomic disruption and thus assess cardiac risk in individuals with a SCI. The findings illustrated that there was an extremely wide range of HRV values in a chronic cross-sectional population thus making it difficult to develop HRV reference values for this population of SCI. Nonetheless, the inter-subject variability has also been observed in the able-bodied population which may indicate that, likewise, individuals with a SCI also experience diverse cardiac ANS function. Furthermore, despite the fact that cardiac autonomic dysfunction has been shown to be related to the level and severity of injury, our results revealed no differences across the selected cohorts. The disparity with the literature might have been due to a number of reasons: 1. Between subject comparisons were made in spite of the fact that HRV has high inter-subject variability; 2. HRV is not exclusively linked to level and severity of SCI, and 3. Cardiac autonomic function has multiple biological complexities that cannot be measured exclusively by heart rate parameters. Given that the LF:HF, is the most commonly used HRV measure of cardiac sympatho-vagal balance and the physiological interpretation of the positive relationship between LF and HF remains undetermined, HRV indices may not be applicable in individuals with a traumatic and chronic SCI. Further understanding of the biological interpretation of the HRV indices is required before routinely using HRV in SCI to monitor and/or manage CVD progression.

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74 Appendices 61

75 Appendix A 62

76 63

77 64

78 Appendix B 65

79 66

80 67 Appendix C The table below summarizes the average percentage of noise, ectopic, and artifact for each five minute segment, and the possible reasons for displaying ectopic islands. The Poincaré plot was examined after the subjects were selected and therefore a total sample of 56 subjects. Table 1. Summary of the subjects who displayed ectopic islands in their Poincaré plots before the filter was applied Subject ID Complete (C)/Incomplete (I) injury Level of injury Normal (%) Ectopic (%) Artifact (%) Possible Reason 2 C T Unclear- Normal ECG &sinus rhythm 5 I T R peak 0.001V (very small compared to others) 11 I C4 12 I C Unstable baseline; unifocal/premature ventricular beat # 35, 16, 157, 163, 206, 522 Unclear- Normal ECG &sinus rhythm 23 C C Unstable baseline 26 I T Unstable baseline 30 C T Unclear- Normal ECG &sinus rhythm

81 68 31 C C Unclear-Normal ECG &sinus rhythm 34 I T Read two R peaks for each one 39 C T4 45 I C Unclear- Normal ECG &sinus rhythm Unclear-Normal ECG &sinus rhythm 71 I C High T waves (at times larger than R wave due to unstable baseline) 72 I C Unstable baseline 88 I T Read two R peaks for each one 91 I T Read two R peaks for each one

82 69 Appendix D The following appendix examines the outliers present when the primary HRV index (LF:HF) was plotted. Table 1 describes the characteristics of the subjects who were outliers when the data was examined for the entire sample and based on level and severity of injury. Figure 1 is the frequency distribution after the outliers were omitted from the entire sample. Table 1. The characteristics of the subjects who were outliers when LF:HF was assessed in the entire sample and based on level and severity of injury, in comparison to the rest of the study sample Shaded areas indicate that the value is outside the median range Factors Study sample (without the outliers) Subject 82 Subject 17 Subject 40 Subject 19 Subject 45 Subject 61 Type of SCI Level of Injury NA C6 T6 T7 C7 C6 C2 Severity of Injury NA Complete Complete Complete Complete Incomplete Incomplete Time post injury (years) Mean: 13.78± Median: (5.00, 21.25) HRV Frequency domain measures LF (ms 2 ) Mean: ± Median: (202.78,

83 70 HF (ms 2 ) LF:HF ) Mean: ± Median: (177.14, ) Mean: 1.49±1.24 Median: 1.03 (0.57, 2.15) Demographics Age (years) Mean: 46.04± Median: (37.00, 54.25) Sex (male/female) 30/12 Male Male Male Male Male Male Cardiovascular status BMI (kg/m 2 ) Mean: 25.83± Median: (21.97, 29.08) Waist Circumference (cm) Mean: 94.25±14.22 Median:

84 71 Absolute VO 2 peak (L/min) Relative VO 2 peak (ml/kg/min) Peak Heart rate (bpm) Resting systolic blood pressure (mmhg) Resting diastolic blood pressure (mmhg) (83.05, ) Mean: 1.29±0.61 Median: 1.14 (0.82, 1.59) Mean: 16.71±7.62 Median: (11.38, 22.34) Mean: ±29.08 Median: (105.00, ) Mean: ±16.70 Median: (98.00, ) Mean: 71.02±13.37 Median: (59.50, 80.50) NT NT NT

85 72 Resting heart rate (bpm) Mean: 61.44±8.90 Median: (55.51, 67.67) Family history of heart disease (yes/no) 26/24 Yes Yes No Yes Yes Yes Smoker (yes/no) 13/37 Yes No No No No No Smoking history (yes/no) 31/19 Yes No Yes No No No Number of comorbidities Mean: 1.74± Median: 2.00 (1.00, 2.25) Functional status LTPAQ-SCI (min/week) Mean: ± NT 180 Median: (0, ) LEMS (/50) Mean: 10.84± Median: 0 (0, 22.75)

86 73 SCIM-III (/100) Mean: 61.08±21.28 Median: (50.00, 74.25) General Sleep apnea (yes/no) 11/39 No No Yes No No No Height (cm) Weight (kg) Mean: ±23.96 Median: (167.91, ) Mean: 79.24± Median: (65.77, 89.10) NA: not applicable; NT: not tested due to subject s choice

87 74 LF:HF Figure 1. Frequency distribution of the LF:HF plotted without the outliers for the entire sample.

88 75 Appendix E The following appendix includes all the frequency distributions and boxplots for the secondary HRV indices when examined in the entire sample. The numbers in the boxplots correspond with the SPSS cell numbers and not the subject numbers.

89 76 LF (ms 2 ) LF (ms 2 ) HF (ms 2 ) HF (ms 2 )

90 77 pnn50 (%) pnn50 (%) RMSSD (ms) RMSSD (ms)

91 78 Appendix F The following appendix includes the descriptive statistics of total power in the entire sample (Table 1) and comparisons of total power based on level and/or severity of injury (Tables 2, 3 and 4). Table 1. Descriptive statistics of total power in the entire sample (N=56) Total Power Mean ± SD ± Median (Lower, Upper quartile) ( , ) IQR Skewness Kurtosis p-value p<0.0001* Kolmogorov-Smirnov (K-S) test; p 0.05* Abbreviations: SD, standard deviation; IQR interquartile range Table 2. Comparison of median total power based on level of injury Below T5 (N=19) Above/Equal to T5 (N=37) p Value Total Power (ms 2 ) ( , ) Mann-Whitney test; p 0.05* ( , ) 0.789

92 79 Table 3. Comparison of median total power based on severity of injury Complete (N=38) Incomplete (N=18) p Value Total Power (ms 2 ) ( , ) Mann-Whitney test; p 0.05* ( , ) Table 4. Comparison of median total power based on level and severity of injury Complete and Complete and Incomplete and Incomplete and p equal to/above below T5 (N=11) equal to/above T5 below T5 (N=8) Value T5 (N=27) (N=10) Total Power (922.97, (ms 2 ) ) Kruskal-Wallis test; p 0.05* ( , ) ( , ) (876.03, ) 0.805

93 LF:HF 80 Appendix G The following appendix includes all the boxplots representing the primary and secondary HRV indices based on level and severity of injury. The numbers in the boxplots correspond with the SPSS cell numbers and not the subject numbers.

94 HF (ms 2 ) LF (ms 2 ) 81

95 pnn50 (%) RMSSD (ms) 82

96 83 Appendix H The following appendix includes a total of seven tables: six tables comparing the scalar and categorical HRV-related factor based on level and/or severity on injury, and one table summarizing the multiple comparisons of the significant factors; p 0.05 indicates that there was significant difference across the cohorts. Table 1. Comparison of the HRV-related factors based on level of injury Median (Lower, Upper quartile) or Mean ± Standard deviation Below T5 Above/equal p Value to T5 Time post injury (6.00,22.00) (5.00,22.00) BMI (22.71,29.05) LTPAQ-SCI (30.00,420.00) LEMS 7.00 (27.00,0.00) (21.99,29.46) (0.00,405.00) 0.00 (0.00, SCIM-III (77.00,64.00) (32.00,72.00) * Number of Comorbidities (0.00,2.00) (1.00,2.50)

97 84 Age (years) Waist circumference (cm) Relative VO2 peak (ml/kg/min) Absolute VO2 peak (L/min) Peak heart rate (bpm) ± ± ± ± ± ± ± ± ± ± * * Mann-Whitney test; Independent t-test; p 0.05* Abbreviations: BMI, body mass index; LTPAQ-SCI, leisure time physical activity questionnaire-spinal cord injury; LEMS, lower extremity motor score; SCIM-III, spinal cord independence measure Table 2. Comparison of the HRV-related factors based on severity of injury Median (Lower, Upper quartile) or Mean ± Standard deviation Complete Incomplete p Value Injury Injury Time post *** injury (6.75,25.00) (3.00,11.50) BMI (21.97,28.91) (23.98,30.17) 0.352

98 85 LTPAQ-SCI (0.00,382.50) (40.00,471.25) LEMS 0.00 (0.00,0.00) (20.75,41.25) < *** SCIM-III (43.50,71.00) (55.25,91.00) ** Number of Comorbidities Age (years) Waist circumference (cm) Relative VO2 peak (ml/kg/min) Absolute VO2 peak (L/min) Peak heart rate (bpm) 2.00 (1.00,2.00) 1.50 (0.75,3.00) ± ± ± ± ± ± ± ± ± ± Mann-Whitney test; Independent t-test; p 0.05*; p 0.01**; p 0.001*** Abbreviations: BMI, body mass index; LTPAQ-SCI, leisure time physical activity questionnaire-spinal cord injury; LEMS, lower extremity motor score; SCIM-III, spinal cord independence measure

99 86 Table 3. Comparison of HRV-related categorical characteristics based on level of injury Below T5 Above/equal to T5 p-value Sex (males/females) 14/5 30/7 p= /6 29/8 p=0.518 Current smoker (no/yes) 7/12 16/21 p=0.645 Smoking history (no/yes) 8/11 17/20 p=0.784 Family history of heart disease (no/yes) 14/5 30/7 p=0.516 Sleep apnea (no/yes) Fisher exact test; Pearson chi-square; p 0.05* Table 4. Comparison of HRV-related categorical characteristics based on severity of injury Complete Injury Incomplete Injury p-value Sex (males/females) Current Smoker (no/yes) 31/7 13/5 p= /7 11/7 p=0.133

100 87 15/23 8/10 p=0.724 Smoking history (no/yes) 19/19 6/12 p=0.241 Family history of heart disease (no/yes) 32/6 12/6 p=0.171 Sleep apnea (no/yes) Fisher exact test; Pearson chi-square; p 0.05* Table 5. Comparison of the HRV-related factors based on level and severity of injury Median (Lower, Upper quartile) or Mean ± Standard deviation Complete and Complete and Incomplete Incomplete p Value equal to/above below T5 and equal and below T5 T5 to/above T5 Time post * injury (6.00,26.00) (7.00,24.00) (3.75,17.00) (3.00,9.00) BMI (21.98,28.86) (20.53,29.05) (23.25,34.54) (23.43,29.27) LTPAQ-SCI * (0.00,420.00) (0.00,120.00) (0.00,317.50) (217.50, ) LEMS < *** (0.00,0.00) (0.00,0.00) (23.00,42.50) (15.25,39.25)

101 88 SCIM-III ** (25.00,70.00) (61.00,71.00) (39.00,81.75) (66.75,95.25) Number of Comorbidities (1.00,2.00) (0.00,2.00) (1.00,3.00) (0.00,3.50) Age (years) ± ± ± ± Waist circumference (cm) Relative VO2 peak (ml/kg/min) Absolute VO2 peak (L/min) Peak heart rate (bpm) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± (Welch) Forsythe) (Welch) (Brown- (Brown- Forsythe) Kruskal-Wallis test; One way ANOVA test; p 0.05*; p 0.01**; p 0.001*** Abbreviations: BMI, body mass index; LTPAQ-SCI, leisure time physical activity questionnaire-spinal cord injury; LEMS, lower extremity motor score; SCIM-III, spinal cord independence measure

102 89 Table 6. Follow up analysis on the HRV-related factors that were different based on level and severity of injury Time Post Injury LTPAQ-SCI LEMS SCIM-III Adjusted Sig. Adjusted Sig. Adjusted Sig. Adjusted Sig. Complete and equal to/above T5 vs. Complete and below T5 Complete and equal to/above T5 vs. Incomplete and below T5 Complete and equal to/above T5 vs. Incomplete and equal to/above T5 Complete and below T5 vs. Incomplete and below T5 Complete and below T5 vs. Incomplete and equal to/above T5 Incomplete and below T5 vs *** 0.010** *** * 0.008** ***

103 90 Incomplete and equal to/above T5 Kruskal-Wallis multiple comparison follow up test; p 0.05*; p 0.01**; p 0.001*** Table 7. Comparison of HRV-related categorical subject characteristics based on level and severity of injury Complete and Complete and Incomplete Incomplete p-value equal below T5 and equal and below T5 to/above T5 to/above T5 Sex (males/females) 22/5 9/2 8/2 5/3 p= /3 7/4 5/5 6/2 p=0.055 Current smoker (no/yes) 12/15 3/8 4/6 4/4 p=0.761 Smoking history (no/yes) 14/13 5/6 3/7 3/5 p=0.694 Family history of heart disease (no/yes) 23/4 9/2 7/3 5/3 p=0.441 Sleep apnea (no/yes) Fisher exact test; Pearson chi-square; p 0.05*

104 LF (ms 2 ) LF (ms 2 ) 91 Appendix I The following appendix includes all the scatter plot of the LF and HF based on level and/or severity of injury. HF (ms 2 ) HF (ms 2 )

105 LF (ms 2 ) LF (ms 2 ) LF (ms 2 ) LF (ms 2 ) 92 HF (ms 2 ) HF (ms 2 ) HF (ms 2 ) HF (ms 2 )

106 LF (ms 2 ) LF (ms 2 ) 93 HF (ms 2 ) HF (ms 2 )

107 LF (ms 2 ) HF (ms 2 ) 94 Appendix J The following appendix includes the scatter plots of the significant correlations between LF or HF and other selected HRV-related factors. The factors include: age, time post injury and SCIM-III.

108 LF (ms 2 ) HF (ms 2 ) 95