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The effects of intermittent hypoxia on the vasculature and the carotid baroreflex regulation of arterial… Tremblay, Joshua Christopher 2015

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The Effects of Intermittent Hypoxia on the Vasculature and the Carotid Baroreflex Regulation of Arterial Pressure  by  Joshua Christopher Tremblay  B.HSc., The University of Guelph, 2013  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  THE COLLEGE OF GRADUATE STUDIES  (Interdisciplinary Studies)  THE UNIVERSITY OF BRITISH COLUMBIA  (Okanagan)  November 2015   © Joshua Christopher Tremblay, 2015  ii Abstract Intermittent hypoxia (IH) occurs in association with obstructive sleep apnea and is thought to contribute to the pathogenesis of hypertension. The proposed mechanisms involved include endothelial dysfunction, arterial stiffness, increased vascular resistance and impaired arterial baroreflex. We sought to examine the effects of a single 6-hour bout of IH on the global vascular, and carotid baroreflex regulation of mean arterial pressure (MAP). We hypothesized that 6 hours of IH would increase 24-hour MAP, reduce endothelium-dependent vasodilation, impair vascular strain, induce oscillatory conduit artery shear patterns and shift the carotid baroreflex to operate at higher MAP while eliciting blunted control of leg vascular conductance (LVC). Ten young, normotensive  men free of sleep apnea were exposed to two 6-hour conditions: 1) breathing one-minute of room air followed by one-minute of hypoxia (IH; mean minimum oxygen saturation = 80.7±0.4% [mean±SEM]), and 2) breathing room air (SHAM). Arterial blood pressure was recorded using 24-hour ambulatory blood pressure monitoring. Brachial artery flow-mediated dilation and reactive hyperemia were measured before and immediately after each condition 6-hour protocol. Vascular strain was measured using two-dimensional speckle-tracking in the common carotid and femoral artery, in addition to upper and lower limb hemodynamics during each testing condition. Carotid baroreflex control of MAP and LVC was assessed after each testing condition using the variable pressure neck chamber technique. Six hours of IH elevated 24-hour MAP (2.6±0.8 mmHg, P=0.008). Reactive hyperemia and flow-mediated dilation were unaffected by IH. Common carotid artery strain was reduced during IH compared to SHAM (room air cycle, P=0.012; hypoxia cycle, P<0.001) and a trend towards IH-impaired common femoral artery strain was observed (P=0.055). The lower limb exhibited enhanced oscillatory shear only during IH. Intermittent hypoxia displaced the carotid baroreflex control of MAP curve to higher arterial blood pressures (P=0.045) and blunted LVC responses to hypertensive stimuli. Oscillatory shear patterns in the lower limbs, impaired vascular strain and blunted carotid baroreflex control of LVC may be involved in IH-induced hypertension, providing insight on the early pathogenic processes.    iii Preface This thesis contains original data collected and analyzed for partial fulfillment of the author’s Master of Science degree. All protocols were approved by the Clinical Research Ethics Board (UBC number: H14-01151) at the University of British Columbia. This thesis consists of a review of the literature (Chapter 1), and four additional chapters pertaining to the research questions: Research Questions and Hypotheses (Chapter 2), Materials and Methods (Chapter 3), Results (Chapter 4), and Discussion (Chapter 5).   iv Table of Contents  Abstract .................................................................................................................................... ii Preface ..................................................................................................................................... iii Table of Contents ................................................................................................................... iv List of Tables ........................................................................................................................ viii List of Figures ........................................................................................................................ xii List of Symbols, Abbreviations or Other ........................................................................... xiii Acknowledgements ............................................................................................................... xv 1    Chapter: Literature Review ............................................................................................. 1  Obstructive Sleep Apnea Definition and Epidemiology ....................................................... 1 1.1 Obstructive Sleep Apnea and Hypertension ......................................................................... 3 1.21.2.1 Pathogenesis ..................................................................................................................... 4  Sustained Hypoxia versus Intermittent Hypoxia ................................................................... 6 1.3 Intermittent Hypoxia and Arterial Blood Pressure ................................................................ 7 1.41.4.1 Intermittent Hypoxia and the Sympathetic Nervous System ............................................ 7 1.4.1.1 Chemoreflex ............................................................................................................. 9 1.4.1.2 Baroreflex .............................................................................................................. 11 1.4.1.2.1 Baroreflex Assessment Techniques .................................................................. 11 1.4.1.2.2 Intermittent Hypoxia and the Baroreflex .......................................................... 15  Intermittent Hypoxia and the Peripheral Vasculature ......................................................... 17 1.51.5.1 The Endothelium ............................................................................................................ 18 1.5.1.1 Flow-Mediated Dilation ......................................................................................... 18 1.5.1.2 Intermittent Hypoxia and Endothelial Dysfunction ............................................... 21 1.5.1.2.1 Impairment in Vasodilation............................................................................... 22 1.5.1.2.2 Enhanced Vasoconstriction ............................................................................... 23 1.5.1.3 The case for retrograde flow .................................................................................. 24 1.5.2 Arterial Stiffness ............................................................................................................. 27 1.5.2.1 Vascular Strain ....................................................................................................... 27  Renin-Angiotensin System ................................................................................................. 28 1.6 v 1.6.1 An Independent Role for Angiotensin II?....................................................................... 29  Metabolism ......................................................................................................................... 30 1.71.7.1 Insulin and the Sympathetic Nervous System ................................................................ 31  Therapeutic Intermittent Hypoxia ....................................................................................... 31 1.8 Summary ............................................................................................................................. 32 1.92    Chapter: Research Questions and Hypotheses ................. Error! Bookmark not defined.  Background ......................................................................................................................... 34 2.1 Macro and Microvascular Reactivity .................................................................................. 35 2.2 Vascular Strain .................................................................................................................... 36 2.3 Upper and Lower Limb Hemodynamics ............................................................................. 37 2.4 Carotid Baroreflex............................................................................................................... 37 2.5 Research Questions and Hypotheses ................................................................................... 38 2.63    Chapter: Materials and Methods .................................................................................. 39  Ethical Approval ................................................................................................................. 39 3.1 Participants .......................................................................................................................... 39 3.2 Screening ............................................................................................................................. 39 3.33.3.1 Epworth Sleepiness Scale ............................................................................................... 40 3.3.2 Pulmonary Function Testing ........................................................................................... 40 3.3.3 Carotid Intima-Media Thickness .................................................................................... 41 3.3.4 Nocturnal Pulse Oximetry .............................................................................................. 42  Experimental Protocols ....................................................................................................... 42 3.4 Experimental Techniques .................................................................................................... 45 3.53.5.1 Intermittent Hypoxia ....................................................................................................... 45 3.5.2 Ambulatory Blood Pressure Monitor .............................................................................. 46 3.5.3 Reactive Hyperemia Flow-Mediated Dilation ................................................................ 47 3.5.4 Vascular Ultrasound ....................................................................................................... 48 3.5.4.1 Strain Measurements .............................................................................................. 49 3.5.4.2 Hemodynamic Measurements ................................................................................ 50 3.5.5 Baroreflex Protocol ......................................................................................................... 50  Sample Size Justification .................................................................................................... 54 3.6 Statistics .............................................................................................................................. 54 3.74    Chapter: Results.............................................................................................................. 56  Participants .......................................................................................................................... 56 4.1 vi  Intermittent Hypoxia and SHAM Exposures ...................................................................... 58 4.2 24-hour Ambulatory Monitoring ........................................................................................ 60 4.3 Flow-Mediated Dilation ...................................................................................................... 62 4.4 Vascular Strain .................................................................................................................... 68 4.5 Limb Hemodynamics .......................................................................................................... 71 4.6 Carotid Baroreflex Control of Mean Arterial Pressure and Heart Rate .............................. 74 4.7 Carotid Baroreflex Control of Leg Vascular Conductance ................................................. 79 4.85    Chapter: Discussion ........................................................................................................ 81  Main Findings ..................................................................................................................... 81 5.1 Intermittent Hypoxia Elevates Arterial Blood Pressure ...................................................... 81 5.2 Flow-Mediated Dilation and Reactive Hyperemia ............................................................. 83 5.3 Common Carotid and Common Femoral Artery Vascular Strain ....................................... 84 5.4 Upper and Lower Limb Hemodynamics ............................................................................. 86 5.5 Carotid Baroreflex Control of Mean Arterial Pressure and Heart Rate .............................. 88 5.6 Carotid Baroreflex Control of Leg Vascular Conductance ................................................. 89 5.7 Methodological Considerations .......................................................................................... 91 5.85.8.1 Intermittent Hypoxia ....................................................................................................... 91 5.8.2 Carotid Baroreflex Assessment ...................................................................................... 92 5.8.3 Ultrasound Measurements .............................................................................................. 93  Perspectives and Significance ............................................................................................. 94 5.9 Future Directions................................................................................................................. 95 5.10References .............................................................................................................................. 97 Appendices ........................................................................................................................... 130 Appendix A : Forms ....................................................................................................................... 130 A.1 University of British Columbia Ethics Certificate of Full Board Approval ................. 130 A.2 Health Questionnaire .................................................................................................... 132 A.3 Epworth Sleepiness Scale ............................................................................................. 133 A.4 Diet and Sleep Log ....................................................................................................... 134 A.5 Activity Log .................................................................................................................. 135 Appendix B : Individual Raw Data Tables .................................................................................... 136 B.1 Participant Characteristics ............................................................................................ 136 B.2 Intermittent Hypoxia and SHAM Exposures ................................................................ 138 B.3 Ambulatory Blood Pressure Monitoring....................................................................... 140  vii B.4 Flow-Mediated Dilation................................................................................................ 145 B.5 Vascular Strain ............................................................................................................. 157 B.6 Limb Blood Flow and Shear ......................................................................................... 160 B.7 Carotid Baroreflex ........................................................................................................ 164 Appendix C : Reliability ................................................................................................................ 178 C.1 Carotid intima-media thickness .................................................................................... 178 C.2 Flow-Mediated Dilation Measurements ....................................................................... 179 C.3 Vascular Strain Measures ............................................................................................. 182   viii List of Tables Table 4.1:   Participant characteristics and pulmonary screening data. .................................. 57 Table 4.2:   Oxygen desaturation characteristics during SHAM and IH exposure. ................ 59 Table 4.3:   Time of day and 24-hour blood pressures. .......................................................... 61 Table 4.4:   Effect of IH on flow-mediated dilation responses to reactive                      hyperemia. ........................................................................................................... 63 Table 4.5:   Effect of IH on brachial artery microvascular reactivity following                     cuff release. .......................................................................................................... 66 Table 4.6:   Effect of IH on upper and lower limb hemodynamics. ....................................... 72 Table 4.7:   Effect of IH on upper and lower limb shear rate. ................................................ 73 Table 4.8:   Effect of IH on carotid MAP-baroreflex four-parameter modelling                     variables. .............................................................................................................. 77 Table 4.9:   Effect of IH on the heart rate-carotid baroreflex four-parameter                     modelling variables. ............................................................................................. 78 Table B.1:   Baseline common carotid intima-media thickness. ........................................... 136 Table B.2:   Baseline pulmonary function test individual data. ............................................ 137 Table B.3:   Mean end-tidal gases throughout SHAM and IH individual data. .................... 138 Table B.4:   Mean individual hypoxemia parameters throughout SHAM and IH. ............... 139 Table B.5:   Individual systolic blood pressure data throughout SHAM and IH. ................. 140 Table B.6:   Individual diastolic blood pressure data throughout SHAM and IH. ............... 141 Table B.7:   Individual mean arterial pressure data throughout SHAM and IH. .................. 142 Table B.8:   Individual heart rate data throughout SHAM and IH. ...................................... 143 Table B.9:   Individual pulse pressure data throughout SHAM and IH. ............................... 144 Table B.10: Individual baseline brachial artery diameter throughout SHAM and                      IH in the morning (AM) and afternoon (PM). .................................................. 145 Table B.11: Flow-mediated dilation individual peak response brachial artery                      diameter throughout SHAM and IH in the morning (AM) and                      afternoon (PM). ................................................................................................. 146 Table B.12: Individual absolute change in brachial artery diameter throughout                      SHAM and IH in the morning (AM) and afternoon (PM). ............................... 147 Table B.13: Individual percent flow-mediated dilation throughout SHAM and   ix                     IH in the morning (AM) and afternoon (PM). .................................................. 148 Table B.14: Individual allometrically scaled percent flow-mediated dilation                      throughout SHAM and IH in the morning (AM) and afternoon (PM). ............ 149 Table B.15: Individual flow-mediated dilation total shear rate response (SRAUC)                      until peak diameter throughout SHAM and IH throughout SHAM and                      IH in the morning (AM) and afternoon (PM). .................................................. 150 Table B.16: Flow-mediated dilation normalized to shear rate area under the curve                      throughout SHAM and IH in the morning (AM) and afternoon (PM). ............ 151 Table B.17: Time from cuff release to peak vasodilation throughout SHAM and                      IH in the morning (AM) and afternoon (PM). .................................................. 152 Table B.18: Reactive hyperemia velocity envelope throughout SHAM and IH in                      the morning (AM) and afternoon (PM). ........................................................... 153 Table B.19: Reactive hyperemia area under the curve envelope flow throughout                      SHAM and IH in the morning (AM) and afternoon (PM). ............................... 154 Table B.20: Reactive hyperemia shear rate area under the curve throughout                      SHAM and IH in the morning (AM) and afternoon (PM). ............................... 155 Table B.21: Reactive hyperemia peak blood flow response throughout SHAM and                      IH in the morning (AM) and afternoon (PM). .................................................. 156 Table B.22: Peak systolic strain individual data during morning and afternoon                      bouts of SHAM and IH. .................................................................................... 157 Table B.23: Early systolic strain rate individual data during morning and                      afternoon bouts of SHAM and IH. .................................................................... 158 Table B.24: Late systolic strain rate individual data during morning and afternoon                      bouts of SHAM and IH. .................................................................................... 159 Table B.25: Brachial and common femoral artery diameter individual data during                      morning and afternoon bouts of SHAM and IH. .............................................. 160 Table B.26: Brachial and common femoral artery mean shear rate individual data                      during morning and afternoon bouts of SHAM and IH. ................................... 161 Table B.27: Brachial and common femoral artery oscillatory shear index                      individual data during morning and afternoon bouts of SHAM and                      IH. ..................................................................................................................... 162  x Table B.28: Brachial and common femoral artery mean blood flow individual                      data during morning and afternoon bouts of SHAM and IH. ........................... 163 Table B.29: The coefficient of determination (r2) of baroreflex modelling                      following SHAM and IH. ................................................................................. 164 Table B.30: SHAM baroreflex parameters for mean arterial pressure. ................................ 165 Table B.31: Intermittent hypoxia baroreflex parameters for mean arterial pressure. ........... 166 Table B.32: SHAM baroreflex parameters for change in mean arterial pressure. ................ 167 Table B.33: Intermittent hypoxia baroreflex parameters for change in mean                      arterial pressure. ................................................................................................ 168 Table B.34: SHAM baroreflex parameters for heart rate. .................................................... 169 Table B.35: Intermittent hypoxia baroreflex parameters for heart rate. ............................... 170 Table B.36: SHAM baroreflex parameters for change in heart rate. .................................... 171 Table B.37: Intermittent hypoxia baroreflex parameters for change in heart rate. ............... 172 Table B.38: Baseline measures prior to carotid baroreflex testing following                      SHAM and intermittent hypoxia. ...................................................................... 173 Table B.39: Carotid baroreflex-mediated absolute changes in leg blood flow                      following SHAM and intermittent hypoxia. ..................................................... 174 Table B.40: Carotid baroreflex-mediated percent changes in leg blood flow                      following SHAM and intermittent hypoxia. ..................................................... 175 Table B.41: Carotid baroreflex-mediated absolute changes in leg vascular                      conductance following SHAM and intermittent hypoxia. ................................ 176 Table B.42: Carotid baroreflex-mediated percent change in leg vascular                      conductance following SHAM and intermittent hypoxia. ................................ 177 Table C.1: Reliability testing for carotid intima-media thickness. ....................................... 178 Table C.2: Unpublished intraobserver flow-mediated dilation............................................. 179 Table C.3: Reliability testing for baseline flow-mediated dilation in the current                    study. ................................................................................................................... 180 Table C.4: Baseline measurements of flow-mediated dilation measurements from                    the current study. ................................................................................................. 181 Table C.5: Reliability testing for peak systolic strain measurements. .................................. 182 Table C.6: Reliability testing for early systolic strain rate measurements. .......................... 183  xi Table C.7: Reliability testing for late systolic strain rate measurements. ............................. 184   xii List of Figures Figure 1.1:   Baroreflex curve schematic. ............................................................................... 14 Figure 1.2:   Flow-mediated dilation shear rate and diameter illustration. ............................. 20 Figure 1.3:   Demonstration of two shear profiles over the course of a cardiac                      cycle .................................................................................................................... 26 Figure 1.4:   Summary of the hypertensive effects of intermittent hypoxia. .......................... 33 Figure 3.1:   Schematic representation of the experimental protocol. .................................... 45 Figure 3.2:   The neck chamber .............................................................................................. 52 Figure 4.1:   Oxygen saturation profile of an individual participant ...................................... 58 Figure 4.2:   Individual and mean flow-mediated dilation responses ..................................... 64 Figure 4.3:   Relationship between the total shear rate response and flow-mediated                      dilation. ............................................................................................................... 65 Figure 4.4:   Reactive hyperemic response ............................................................................. 67 Figure 4.5:   Common carotid artery (CCA) circumferential strain profiles across                      the cardiac cycle for all subjects during SHAM and IH in the morning                     (AM) and afternoon (PM). ................................................................................... 69 Figure 4.6:   Common femoral artery (CFA) circumferential strain profiles across                      the cardiac cycle for all subjects during SHAM and IH in the morning                      (AM) and afternoon (PM). .................................................................................. 70 Figure 4.7:   Carotid baroreflex control of mean arterial pressure and heart rate ................... 76 Figure 4.8:   Carotid baroreflex control of the leg vasculature following SHAM                      and IH.................................................................................................................. 80   xiii List of Symbols, Abbreviations or Other  AHI Apnea-hypopnea index ANOVA Analysis of variance BA Brachial artery BMI Body mass index CCA Common carotid artery CFA Common femoral artery CIMT Common carotid artery intima-media thickness COV Coefficient of variation CP Centering point CPAP Continuous positive airway pressure CRP C-reactive protein DBP Diastolic blood pressure DLCO Diffusing capacity of the lung for carbon monoxide ECSP Estimated carotid sinus pressure ESS Epworth sleepiness scale ET-1 Endothelin-1 FEV1 Forced expiratory volume in 1 second FMD Flow-mediated dilation FVC Forced vital capacity G Gain Gmax Maximal gain Gop Gain at operating point HIF Hypoxia-inducible factor HR Heart rate HX Hypoxic breathing LBF Leg blood flow LTF Long-term facilitation LVC Leg vascular conductance MAP Mean arterial pressure MSNA Muscle sympathetic nerve activity  xiv                       NX Room air breathing ODI Oxygen desaturation index OP Operating point OSI Oscillatory shear index PA Popliteal artery PETCO2 End-tidal partial pressure of carbon dioxide   PETO2 End-tidal partial pressure of oxygen PP Pulse pressure RAS Renin-angiotensin system RH Reactive hyperemia RMS Root-mean squared ROI Region of interest ROS Reactive oxygen species SBP Systolic blood pressure SFA Superficial femoral artery SEM Standard error of the mean SNA Sympathetic nerve activity SpO2 Peripheral oxyhemoglobin saturation SRAUC Shear rate area under the curve SV Stroke volume TLC Total lung capacity VA Alveolar gas volume   E Ventilation VEGF Vascular endothelial growth factor          xv  Acknowledgements I would like to acknowledge my supervisor, Dr Glen Foster, and the members of my supervisory committee, Drs Philip Ainslie and Neil Eves for their guidance and scholarly support throughout my studies. I would like to acknowledge Dr Victoria Claydon and Matthew Lloyd for their assistance in the design development of the baroreflex neck chamber.   1 1    Chapter: Literature Review In this chapter, the reader is given an introduction to the pathogenesis of obstructive sleep apnea (OSA), its cardiovascular consequences and the proposed mechanisms linking OSA with cardiovascular disease. Focusing on clinical and experimental investigations, an up-to-date description of the proposed mechanisms linking OSA and hypertension is explored. Attention is drawn to the role of blood pressure regulation and the peripheral vasculature in the progression of intermittent hypoxia-induced hypertension.    Obstructive Sleep Apnea Definition and Epidemiology 1.1Preceding any medical definition or acknowledgement, a case study of exceptional drowsiness by Canton (1889) included insightful commentary: “When in sound sleep a very peculiar state of the glottis is observed, a spasmodic closure entirely suspending respiration. The thorax and abdomen are seen to heave from fruitless contractions of inspiratory and expiratory muscles; their efforts increase in violence for about a minute or a minute and a half, the skin meantime becoming more and more cyanosed, until at last, when the condition to the onlooker is most alarming, the glottic obstruction yields, a series of long inspirations and expirations follows, and cyanosis disappears. This acute dyspnoeic attack does not awaken the patient. ... If in the midst of dyspnoeic attack he is forcibly aroused, the glottic spasm at once relaxes. The night nurse states that these attacks go on through the night.” Described as having narcolepsy, it appears without a doubt that the aforementioned patient suffered from OSA, a condition that would continue to be misdiagnosed and ignored until three quarters of a century later. In a nocturnal polygraphic study of an obese patient suffering from somnolence, Gastaut et al. (1966) identified that “the tongue moves back and causes the obstructive apnoea responsible for the rapid hypoxia which rouses the subject, who a little later dozes off again.”    2 Obstructive sleep apnea manifests from compromised pharyngeal anatomy, rendering the soft-tissue organ located in the upper airway susceptible to collapse. During wakefulness, the pharynx is reflexively dilated, but at the onset of sleep, this protective reflex can fail leading to a narrowing or collapse of the airway (Dempsey et al., 2010). The cessation of airflow is eventually overcome, and the patient resumes breathing until the subsequent obstruction. Polysomnography is the gold standard to diagnose OSA and measures sleep state, snoring, body movements, heart rate (HR), respiratory efforts, air flow and oxygen saturation during sleep (Berry et al., 2012). The comprehensive sleep assessment allows quantification of the apnea-hypopnea index (AHI) and the oxygen desaturation index (ODI). An apnea is defined as a greater than 90% reduction in air flow for 10 seconds, and a hypopnea is a greater than 30% reduction in flow associated with a 3% decrease in oxyhemoglobin saturation or arousal (Berry et al., 2012). The AHI is the number apneas and hypopneas per hour, and the ODI is the number of desaturations (typically ≥3 or 4% oxyhemoglobin desaturation) associated with a reduction in respiratory effort per hour (Choi et al., 2000; Berry et al., 2012). The threshold for clinical diagnosis of OSA is an AHI or ODI of ≥15 events/hour, or ≥5 events/hour with documented symptoms of excessive daytime sleepiness (Epstein et al., 2009). Obstructive sleep apnea can be subdivided based on severity into mild (AHI=5-14 events/hour), moderate (AHI=14-29 events/hour) and severe (AHI ≥30 events/hour) (Epstein et al., 2009). Early reports, including the Wisconsin Sleep Cohort Study and the Penn State Cohort, established a moderate prevalence of OSA (AHI ≥15 events/hour; 2-4% in women, 7-9% in men) (Young et al., 1993; Quan et al., 1997; Bixler et al., 2001). More recent investigations suggest much higher prevalence. For example, a follow-up of the Wisconsin Sleep Cohort Study estimates that 13% of men and 5.6% of women aged 30-70 have an AHI of ≥15 events/hour (Peppard et al., 2013). The Sleep Heart Health Study, a multi-cohort study (n=5615), identified 25% of community-dwelling adult men and 11% of women demonstrate an AHI of ≥15 events/hour (Young et al., 2002). The HypnoLaus population-based study (Lausanne, Switzerland) performed at-home polysomnography on 2121 adults between the ages of 40 and 85 observed an AHI ≥15 events/hour in 23.4% of women and 49.7% of men (Heinzer et al., 2015). Such high prevalence is not reflected clinically, as 70-80% of patients suffering from OSA remain undiagnosed (Young et al., 1997; Kapur et al., 2002).  3   Obstructive Sleep Apnea and Hypertension 1.2Obstructive sleep apnea has been suggested as an independent risk factor for cardiovascular complications, a suspicion that has been established from the onset of its clinical acknowledgment. Tilkian et al. (1976) reported that the blockage and subsequent clearage of the upper airway during sleep causes cyclical arterial hypoxemia, hypercapnia and marked surges in arterial blood pressure. While not examined, it is interesting to note that of the 12 patients studied, 7 had systemic hypertension. The high-prevalence of hypertensive patients may have been overlooked since most of the patients were overweight, although symptoms of snoring and OSA preceded obesity in all patients (Tilkian et al., 1976). Several studies in the 1980s explored the relationship between hypertension and OSA, finding that 60-80% of severe sleep apnea patients have hypertension (Guilleminault et al., 1980; Guilleminault et al., 1981). In a study of 50 hypertensive patients and 50 age-matched controls, 30% of the hypertensive patients had OSA, and not a single case reported in the controls (Kales et al., 1984). Additional clinical investigations demonstrated a 22-30% prevalence of OSA in patients with essential hypertension (Lavie et al., 1984; Fletcher et al., 1985; Williams et al., 1985). Early evidence suggested a bidirectional relationship between OSA and hypertension. Affirming early findings, a trio of follow-up investigations in 2000 from the aforementioned Wisconsin Sleep Cohort Study, the Sleep Health Study and the Penn State Cohort demonstrated a 58-64% prevalence of hypertension in adults with an AHI ≥15 events/hour (Bixler et al., 2000; Nieto et al., 2000; Peppard et al., 2000). Perhaps most fascinating and insightful was the unanimous finding that the relationship persisted even after accounting for confounding factors (demographics, anthropometric variables, smoking and alcohol consumption) and was related to the severity of OSA. In support, Lavie et al. (2000) determined that an increase in AHI of 1 increased the odds of hypertension by 1%, and each 10% decrease in minimum oxyhemoglobin saturation increased the odds of hypertension by 13%.   The primary treatment intervention for OSA is continuous positive airway pressure (CPAP), which provides a pneumatic splint for the nasopharyngeal airway. By delivering a constant flow of air through the nares, CPAP therapy abolishes cyclical oxyhemoglobin desaturations  4 and reduces the frequency of arousals by preventing collapse of the airway (Sullivan et al., 1981; Younes, 2004). Investigation of CPAP therapy has received considerable clinical interest in its efficacy in reducing blood pressure. A meta-analysis, including 28 studies, identified mean reductions in systolic (SBP) and diastolic blood pressure (DBP) of 2.58 mmHg and 2.01 mmHg respectively (Montesi et al., 2012). Continuous positive airway pressure treatment is most effective in lowering arterial blood pressure in younger patients (<50 years) with a greater degree of daytime hypersomnolence, severe OSA, and who exhibit greater CPAP adherence (Montesi et al., 2012). Therefore, CPAP can contribute to an improvement in arterial blood pressure.   1.2.1 Pathogenesis To fully understand the association of OSA and hypertension, it is critical to decipher which factors, or combination thereof, could contribute to an augmented arterial blood pressure. Patients with OSA suffer from poor sleep quality, repeated arousals, large intrathoracic pressures swings and cyclical arterial hypoxemia and reoxygenation (intermittent hypoxia; IH) and arterial hypercapnia. Seminal work by Eugene Fletcher thoroughly investigated the effects of IH in an experimental rat model. Rats were housed in chambers and exposed to rapid changes in ambient oxygen to induce changes in oxyhemoglobin saturation. The fraction of inspired oxygen in the chamber was gradually reduced to 3-5% over 12-15 seconds and returned to 20.9% over 15-18 seconds. The 30-second duty cycle was repeated for 6-8 hours per day. An initial investigation, exposing rats to 7 hours of IH for 35 days, produced a 13.7 mmHg increase in mean arterial blood pressure (MAP) (Fletcher et al., 1992c). The drastic hypertensive effect of IH was originally attributed to the activation of the peripheral chemoreflex. The carotid chemoreceptors, located in the carotid body, respond to decreases in arterial partial pressure of oxygen (PaO2) by sending signals via the carotid sinus nerve to the rostral ventrolateral medulla in the brainstem leading to increases in sympathetic nerve activity (SNA) and stimulating the adrenal medulla to secrete catecholamines thereby increasing arterial blood pressure (see Intermittent Hypoxia and the Sympathetic Nervous System, page 7). A series of follow-up studies by Fletcher and colleagues removed or blocked key components of this pathway in order to assess the influence of repeated peripheral chemoreceptor activation by IH on arterial blood pressure. An identical 35-day IH  5 protocol produced a 13 mmHg MAP increase in carotid sinus nerve-intact rats, whereas rats that had undergone surgical carotid body denervation showed no increase in MAP (Fletcher et al., 1992a), demonstrating the necessity of intact carotid chemoreceptors to observe the IH-induced rise in MAP. Subsequently, rats who had undergone chemical sympathetic denervation did not show an increase in MAP, suggesting that peripheral sympathetic nerves controlling vascular tone may facilitate the development of IH-induced hypertension (Fletcher et al., 1992b). Intact adrenal medulla and renal artery sympathetic nerves are also essential, as both adrenal medullectomy and renal denervation eliminated any increase in diurnal MAP (Bao et al., 1997a).  Next, the influence of the renin-angiotensin system (RAS) was examined (see Renin-Angiotensin System, page 28). Chronic IH increased plasma renin activity four-fold while administration of an AT1R blocker (losartan) prevented any rise in MAP (Fletcher et al., 1999). Finally, impaired vascular reactivity following chronic IH due to reduced nitric oxide (NO) release and/or production and increased peripheral resistance were suggested as contributors to increased MAP (Tahawi et al., 2001) (see Intermittent Hypoxia and the Peripheral Vasculature, page 17).  The carotid chemoreceptors respond not only to decreases in PaO2, but also the arterial partial pressure of carbon dioxide (PaCO2). While OSA patients demonstrate increases in PaCO2 during apneas, experimental models of IH demonstrate a reduction in PaCO2 during the hypoxic stimulus due to increased ventilation (  E). Fletcher et al. (1995) compared the effects of hypocapnic, eucapnic and hypercapnic IH, 8 hours a day for 35 days, on arterial blood pressure. The magnitude of MAP increase was similar in each condition, implicating oxygen as the key driver in IH-induced hypertension. Acutely, however, both eucapnic and hypercapnic IH evoke much greater increases in MAP compared to hypocapnic IH and these rises are attributed to elevated SNA (Bao et al., 1997b). Investigating another symptom of OSA, Bao et al. (1999) did not find a sustained rise in MAP when subjecting rats to repeated acoustic arousals.   To summarize the early work of Fletcher and colleagues, rats exposed to chronic IH display marked increases in MAP. These sustained increases in MAP are caused in part due to increased SNA from recurrent carotid chemoreceptor stimulation and occur independent of  6 PaCO2. The RAS and NO appear to contribute to the increased vascular tone responsible for elevated MAP. The primary stimulus for OSA-induced hypertension appears to be IH.   Sustained Hypoxia versus Intermittent Hypoxia 1.3The response to sustained hypoxia represents an adaptive mechanism designed to restore homeostasis. Consider the response to severe hemorrhage. Blood loss leads to tissue hypoxia due to decreased O2-carrying capacity and the resultant erythropoeitic response serves to restore O2-carrying capacity, improving O2 delivery (Prabhakar & Semenza, 2012). The response is not always beneficial, however, as inhabitancy at high altitude (>3000m) can cause excessive erythrocytosis, increasing blood viscosity and leading to chronic mountain sickness (Monge, 1943). Likewise, IH similar to that observed in OSA promotes maladaptation, likely owing to its recent pathological inception, manifesting most commonly from obesity-related transient upper airway obstruction during sleep (Prabhakar & Semenza, 2012). The maladaptive IH observed in OSA is characterized by high-frequency oxyhemoglobin desaturation and rapid resaturation over the course of several hours, although IH can be therapeutic when the hypoxia is mild, cycles are low frequency and the exposure is brief (see Therapeutic Intermittent Hypoxia, page 31). The stark distinction in the physiological responses to IH compared to sustained hypoxia appears to originate at the molecular level. Hypoxia-inducible factors 1α and 2α (HIF-1α, HIF-2α) are stabilized under hypoxic conditions and activate the transcription of hundreds of target genes, encoding proteins (i.e., erythropoietin and vascular endothelial growth factor, VEGF) that mediate responses to hypoxia (erythrocytosis and angiogenesis). Importantly, they are essential in the carotid body response to hypoxia. Mice partially deficient in HIF-1α display marked impairments in ventilatory responses to hypoxia (Kline et al., 2002), meanwhile mice partially deficient in HIF-2α display exaggerated hypoxic sensitivity (Peng et al., 2011a). Sustained hypoxia activates both HIF-1α and HIF-2α. On the other hand, IH increases HIF-1α expression (Yuan et al., 2008) and degrades HIF-2α in the carotid body (Nanduri et al., 2009) by increased reactive oxygen species (ROS) produced by xanthine oxidase (Nanduri et al., 2013; Nanduri et al., 2015). Promoting a pathogenic positive feedback loop, elevated HIF-1α levels increase ROS production via increased expression of the pro-oxidant, nicotinamide adenine dinucleotide phosphate-oxidase 2 (NOX2) (Yuan et al., 2011) and  7 reduced HIF-2α transcription of the anti-oxidant, superoxide dismutase 2, mitochondrial (Nanduri et al., 2009). The shift towards a pro-oxidant state in the carotid body contributes to the elevation in carotid body sensory activity that persists beyond IH exposure, termed long-term facilitation (LTF). Indeed, mice treated with a ROS-scavenger do not exhibit LTF (Peng et al., 2003). Therefore, at a molecular level, IH differs from sustained hypoxia in that it produces and propagates oxidative stress. Enhanced carotid body sensitivity may contribute to the tonic elevations in SNA and pathogenesis of hypertension in OSA.   Intermittent Hypoxia and Arterial Blood Pressure 1.4Knowledge on the elevation of blood pressure with IH is based largely upon two fields of research: 1) studies on OSA patients and 2) experimental models. The former is ideally investigated with age- and body mass index (BMI)-matched controls or randomized, placebo-controlled, blinded CPAP therapy on newly diagnosed patients. The latter focuses on either animal (see Pathogenesis, page 4) or human models. Given the scope of the current thesis, emphasis will be placed on human experimental models, with supplementary support when appropriate from clinical and animal model studies.   1.4.1 Intermittent Hypoxia and the Sympathetic Nervous System Direct recordings of SNA in humans are achieved using a technique called microneurography, where a tungsten-tip needle electrode is inserted percutaneously into a peripheral nerve (usually the peroneal nerve) (Vallbo et al., 2004). Once in the nerve, the needle is carefully maneuvered until the electrode is in close enough proximity to a bundle of sympathetic nerves destined for muscle to record muscle sympathetic nerve activity (MSNA) (Vallbo et al., 2004). Muscle sympathetic nerve activity correlates well with both cardiac and renal norepinephrine spillover, measures of sympathetic activity in nerves to the heart and kidneys (Wallin et al., 1992; Wallin et al., 1996). In healthy humans, MSNA is dependent upon sleep stage (Somers et al., 1993), whereas in OSA, MSNA is dictated by duration and frequency of apneas (Somers et al., 1995). Each apnea is accompanied by an increase in sympathetic traffic (Hedner et al., 1988). Of particular interest, the augmented MSNA persists into waking hours (Carlson et al., 1993) and the higher tonic MSNA occurs independent of the common comorbidity, obesity (Narkiewicz et al., 1998b). Administration  8 of 100% O2 (Leuenberger et al., 1995) and CPAP (Somers et al., 1995; Waradekar et al., 1996; Imadojemu et al., 2007) decreases MSNA, suggesting an integral role of the carotid chemoreceptor in the propagation of tonic elevations in MSNA. Indeed, acute hypoxia produces an elevation of MSNA that outlasts exposure (LTF) (Morgan et al., 1995). Subsequent investigations interested in deciphering whether hypoxia or hypercapnia alone evokes such LTF revealed that hypoxia produced sustained elevations in MSNA while hypercapnia did not (Xie et al., 2001). Patients with OSA present increased daytime MSNA that is thought to be caused by IH.   Experimental human studies aiming to simulate the IH experienced by OSA patients support the findings that IH is the primary perpetrator in augmenting resting MSNA. Sympathetic activity remained elevated 20 minutes after a 20-minute bout of 20 seconds apnea and 40 seconds room-air breathing (Xie et al., 2000). Employing a stronger stimulus, Cutler et al. (2004) primed participants with two breaths of 95-100% nitrogen prior to a 20 second apnea followed by 30 seconds of room air breathing. The one-minute cycle was repeated for 20 minutes and MSNA remained elevated three hours beyond the cyclical hypoxic apneas (Cutler et al., 2004). A similar protocol administering 20 seconds of hypoxic gas (10% O2) prior to a 20 second apnea and 20 second room air recovery for 30 minutes caused a 50% increase in MSNA 30 minutes post-experimental protocol (Leuenberger et al., 2005). To assess the effects of experimental IH during sleep, Gilmartin et al. (2010) had participants sleep in altitude tents set to 13% O2. Boluses of O2 were administered every 2.5 to 4 minutes throughout the duration of the night to induce oxyhemoglobin reoxygenation. After spending 28 consecutive nights sleeping inside the tent, the participants displayed elevated daytime MSNA. In a similar study, Tamisier et al. (2011) delivered O2 via nasal cannula for 15 seconds every 2 minutes and demonstrated similar rises in MSNA following 14 nights. In healthy humans, both brief (20-30 minutes) and chronic (14-28 nights) IH augment MSNA beyond the exposure.   Patients with OSA have elevated daytime MSNA, which can be lowered upon removal of IH by administration of 100% O2 or CPAP treatment (Narkiewicz & Somers, 2003). Studies aimed to mimic sleep apnea in humans, while varied in methodology, display a clear increase  9 in MSNA that outlasts exposure and appears to be dependent on hypoxia. Next, the impact of OSA and specifically IH on two reflexes that are primary mediators of sympathetic tone, the chemoreflex and baroreflex, will be examined. 1.4.1.1 Chemoreflex The carotid body is a sensory organ located at the bifurcation of the common carotid artery (CCA). Receiving greater than 10 times the amount of blood flow per unit mass than the cerebral vasculature, the carotid body is preferentially situated to continuously sample and monitor arterial blood gases (Kumar & Prabhakar, 2012). Primarily responsible for O2 sensing, the carotid body responds to arterial hypoxemia by increasing carotid sinus nerve activity, reflexively increasing   E, MSNA and arterial blood pressure (Kumar & Prabhakar, 2012). Aortic bodies serve a similar function and collectively, the carotid and aortic bodies are referred to as peripheral chemoreceptors.   As mentioned above, hypoxia evokes sympathoexcitation (see Intermittent Hypoxia and the Sympathetic Nervous System, page 7). Two methods to assess peripheral chemoreceptor sensitivity are commonly employed. Firstly, cardiorespiratory responses to hypoxia can be measured (Duffin, 2007). Larger increases in   E, arterial blood pressure or MSNA in response to a hypoxic stimulus are indicative of heightened peripheral chemosensitivity. Secondly, removal of peripheral chemoreceptor input via 100% O2 administration causes marked decrease in   E, arterial blood pressure and MSNA in individuals with greater tonic peripheral chemoreceptor activity (Seals et al., 1991; Narkiewicz et al., 1998c). Patients with OSA usually demonstrate enhanced peripheral chemosensitivity, displaying enhanced ventilatory, pressor and MSNA responses to acute hypoxia (Hedner et al., 1992; Narkiewicz et al., 1999; Khodadadeh et al., 2006) and greater ventilatory, MAP and MSNA reductions in response to hyperoxic administration (Tafil-Klawe et al., 1991b; Narkiewicz et al., 1998c). However, absent confounding complications, Foster et al. (2008) did not find any difference in the acute hypoxic ventilatory response in patients with OSA. Conversely, Osanai et al. (1999) found depressed peripheral chemosensitivity in OSA patients by assessing ventilatory response to two breaths of 100% O2 under hypercapnic hypoxic conditions. Finally, Imadojemu et al. (2007) found an increased MSNA response to acute hypoxia compared to  10 controls, and one month of CPAP therapy partly normalizes the heightened sympathetic responses to hypoxia. These observations infer a contribution of tonic peripheral chemoreceptor activation for the augmented daytime sympathetic nerve activity in patients with OSA.   To isolate the influences of IH on peripheral chemosensitivity, several studies on healthy humans have been conducted. A chronic IH protocol (14 nights), using altitude simulation tents set to 13% O2 with boluses of O2 delivered to resaturate oxyhemoglobin by 10%, doubled the ventilatory response to hypoxia (Tamisier et al., 2009). To determine whether the increased peripheral chemosensitivity occurs more acutely, Pialoux et al. (2009), using a hypoxic chamber, cycled end-tidal partial pressures of O2 (PETO2) from 45 mmHg for 2 minutes to 88 mmHg for 2 minutes, 6 hours a day for 4 days. The acute hypoxic ventilatory response was approximately 50% greater than baseline after one day and rose to 84% greater following the fourth day. The response remained significantly elevated even 4 days after the final IH exposure. Plasma levels of 8-hydroxy-2’deoxyguanosine, a marker of oxidative stress, increased following IH exposure and oxidative stress positively correlated with the acute hypoxic ventilatory response, suggesting a role for oxidative stress in enhancing peripheral chemosensitivity. Employing the same protocol, with 1 minute hypoxia and 1 minute reoxygenation instead of 2, and maintaining end-tidal partial pressure of CO2 (PETCO2) isocapnic, Foster et al. (2010) failed to observe any change in the acute hypoxic ventilatory response, though did demonstrate an augmented pressor response (MAP response to isocapnic hypoxia) that was abolished when participants were administered the AT1R blocker, losartan, prior to exposure. In a follow-up study, Pialoux et al. (2011) abolished oxidative stress with losartan administration, implicating angiotensin II (Ang II) as a possible source of ROS production. Using a similar IH protocol, Beaudin et al. (2015) found an increase in the acute hypoxic ventilatory response that was unaffected by nonsteroidal anti-inflammatory drug administration, suggesting that ROS production is not related to inflammation through the cyclooxygenase pathway. Experimental human investigations have suggested that IH may enhance peripheral chemosensitivity via increases in oxidative stress that may partially be due to increased Ang II and its action on the AT1R.   11 The peripheral chemoreflex appears to be involved in the augmented SNA seen in OSA. Although not a unanimous finding, enhanced peripheral chemosensitivity seems to manifest from IH. While the chemoreflex increases SNA, the baroreflex acts to suppress it. The following section will investigate the influence that arterial blood pressure regulation via the arterial baroreflex has on SNA following IH exposure.         1.4.1.2 Baroreflex Unlike the chemoreflex, which serves to regulate arterial blood gases, the arterial baroreflex sustains short-term and long-term arterial blood pressure homeostasis (Lohmeier & Iliescu, 2015). Arterial baroreceptors are mechanoreceptors located at the carotid sinus bifurcations and aortic arch (Mancia & Mark, 1983). Increases in arterial blood pressure stretch the baroreceptors, sending afferent signals centrally to the nucleus tractus solitarii resulting in a reflex-mediated increase in parasympathetic nerve activity and decrease in SNA (Mancia & Mark, 1983). This acts to induce bradycardia and peripheral vasodilation, therefore effectively and quickly lowering arterial blood pressure (Sagawa, 1983). Decreases in arterial blood pressure unload the baroreceptors, decrease afferent neuronal firing, thus decreasing parasympathetic nerve activity and removing inhibition of SNA (Mancia & Mark, 1983). The arterial baroreflex is, therefore, a classic negative feedback system affecting both the heart and vasculature in a manner to restore arterial blood pressure. A number of means of baroreflex assessment exist, each with their own strengths and weaknesses. The most commonly used techniques will be discussed briefly below. 1.4.1.2.1 Baroreflex Assessment Techniques Firstly, we will consider the least invasive methods. Spontaneous baroreflex sensitivity (BRS) can be assessed using sequence and spectral analyses. The sequence technique selects a series of three or more consecutive cardiac cycles where systolic blood pressure (SBP) or R-R intervals progressively increase or decrease. Subsequently, a linear regression is applied to each of the sequences and the average regression slope is termed cardiac BRS and is measured in ms/mmHg (Bertinieri et al., 1988; Parati et al., 1988). Where the sequence method is concerned with the time domain, the spectral analysis deals with the frequency domain. After mathematical transformations, two power spectrum density bands can be  12 quantified: low frequency and high frequency. Low frequency bands reflect a combination of sympathetic and parasympathetic influences and high frequency bands reflect parasympathetic activity (Pagani et al., 1986). Both techniques are noninvasive and can be measured spontaneously, however they fail to quantify the full sigmoidal baroreflex as they do not evoke large enough pressure responses to determine either threshold or saturation points.  In order to elucidate baroreflex threshold and saturation, greater perturbations in arterial blood pressure must be evaluated. A popular means to achieve large swings in arterial blood pressure is through pharmacological intervention. Termed the modified Oxford technique, a bolus of sodium nitroprusside is administered intravenously followed a minute later by administration of phenylephrine hydrochloride (Rudas et al., 1999). The sequence takes approximately 3 minutes and is often repeated 2-3 times. Sodium nitroprusside, a depressor, reduces MAP by approximately 15 mmHg, while phenylephrine hydrochloride, a pressor, increases MAP equivocally above baseline levels (Rudas et al., 1999). If MSNA recordings are obtained simultaneously, the sympathetic baroreflex gain can be assessed as the relationship between integrated MSNA and DBP (Ebert & Cowley, 1992). Cardiac baroreflex gain, on the other hand, can be characterized by the slope of R-R interval and SBP (Eckberg & Eckberg, 1982). Increased baroreflex gain implies greater sensitivity and responsiveness. Due to the systemic effects of the aforementioned pharmacological infusions, both the aortic and cardiac baroreflexes are affected. Further, the arterial vasculature is manipulated by the vasoactive substances and therefore does not permit observation of the vascular effects of the baroreflex (Cooper & Hainsworth, 2009).   To avoid pharmacological consequences that may cloud interpretation, external transmural stimulation of the carotid baroreceptors in isolation can be applied. Ernsting and Parry (1957) first describe stimulating the carotid arterial stretch receptors by enclosing the neck in a Perspex box maintained at subatmospheric pressures. The carotid sinus stretch simulated an increase in blood pressure, resulting in a decrease in HR and fall in MAP, and Ernsting and Parry (1957) noted a linear relationship between the magnitude of suction and subsequent drop in MAP. In contrast, application of increased pressure within the neck chamber,  13 decreasing transmural pressure and compressing the carotid artery, reflexively increases MAP (Thron et al., 1967). Several advantages of the neck chamber technique were described in a review of the technique by Fadel (2008): 1) The ability to precisely control the rate, intensity, timing and duration of the pressure stimulus 2) Nonpharmacological 3) A full carotid baroreflex curve can be constructed 4) It can be applied under various experimental conditions 5) Permits the assessment of baroreflex-mediated vascular responses  The pressure applied is brief (5 seconds) to avoid any adaptation of the carotid baroreceptors or counteraction from the aortic and cardiopulmonary baroreceptors. Multiple trials of varying pressure (often -80, -60, -40, -20, +20 and +40 mmHg) are delivered in random sequence and peak HR and MAP averaged for each level of pressure and suction. The averages of each trial are plotted against the estimated carotid sinus pressure (ECSP; pressure inside chamber minus MAP) and fitted to a four-parameter logistic function (see Figure 1.1, page 14). Upon construction of the stimulus-response curve, numerous parameters can be obtained and compared (see Baroreflex Protocol, page 50). Limitations of the neck chamber are largely methodological and include: achieving an air-tight seal, anatomical access to the carotid sinus and mitigating any emotional distress caused by collar discomfort.    14  Figure 1.1: Baroreflex curve schematic. Panel A: An illustration of a carotid baroreflex curve derived from the neck chamber technique. The estimated carotid sinus pressure (ECSP) is the pressure exerted on the carotid sinus, where negative pressure implies suction. It is calculated by subtracting the pressure within the neck chamber from the mean arterial pressure (MAP) from the 5 cardiac cycles preceding the stimulus. The operating point (OP) is the baseline MAP or HR. The OP does not always align with the centering point (CP), the inflection point, where there is equal depressor and pressor response to a given change in carotid sinus pressure. The threshold and saturation points represent the carotid sinus pressure at which MAP or HR is within 5% of the upper or lower plateau of the sigmoid function. The operating range is the difference between saturation and threshold points. Both the maximal slope (i.e., slope at CP) and slope at OP are of interest and represent carotid baroreflex sensitivity. Panel B: The neck chamber protocol permits the construction of a complete stimulus-response curve. As a result, both the baroreflex sensitivity and setpoint can be compared. The solid black line represents the “baseline” carotid baroreflex curve. The long broken lines demonstrate an upwards shift in the set-point, where saturation, threshold, OP and CP are greater, but sensitivity is unaltered. By contrast, the smaller broken lines represent a reduced baroreflex sensitivity, as evident by a flatter slope.    15 1.4.1.2.2 Intermittent Hypoxia and the Baroreflex Several groups have investigated BRS in OSA patients, offering inconsistent findings probably influenced by the variability of methods of assessment. Researchers from Poland and Germany were the first to investigate baroreflex function in OSA patients and are interestingly the only ones to employ the neck chamber technique. Tafil-Klawe et al. (1991a) applied a range of neck suction (-20, -40, -60 mmHg) and pressure (20, 40, 60 mmHg) in 25 borderline hypertensive OSA patients (SBP: 138±21 mmHg, DBP: 89±1.6 mmHg), finding decreased R-R interval responsiveness to hypertensive and hypotensive stimuli, and in turn reduced gain (i.e., BRS), compared to 20 healthy, age-matched, normotensive controls. Follow-up investigations expand on such findings, adding that the cardiac response to carotid baroreceptor activation (simulated hypertension) is further impaired in hypertensive OSA patients compared to normotensive OSA patients (Klawe et al., 1997) and responses to neck pressure and suction improve with a month of CPAP treatment (Klawe et al., 1999). These under cited investigations suggest that the cardiac baroreflex is impaired in OSA patients, especially with hypertension, and may relate to IH.  Using the Valsalva maneuver to derive an index of BRS, Cortelli et al. (1994) demonstrated an impairment in OSA patients. Expanding on these findings over a decade later, Noda et al. (2007) applied multiple regression analyses uncovering a significant inverse relationship between time spent hypoxemic and BRS. Supporting the role of hypoxemia, CPAP treatment abolished the time spent hypoxemic and compliance for 3 months significantly improved the BRS index (Noda et al., 2007). Spontaneous BRS, too, appears to be reduced in OSA (Resta et al., 1996; Bonsignore et al., 2002) and improves with long-term CPAP therapy (Bonsignore et al., 2002). Pharmacological interventions have been less clear. Originally, Ziegler et al. (1995) observed impaired baroreflex control of HR during hypoxia (15% O2), but enhanced pressor response to phenylephrine while breathing room air. Conversely, Carlson et al. (1996) administered only sodium nitroprusside and found that both cardiac (R-R interval vs MAP) and sympathetic (MSNA vs DBP) baroreflexes were impaired in OSA patients. The responses to such reductions in MAP evoked by nitroprusside remained significant after accounting for age and BMI, however the OSA patients and controls were not matched for blood pressure (OSA: SBP: 158±5 mmHg, DBP: 82±5; Controls: SBP:  16 135±3 mmHg, DBP: 62±4 mmHg) (Carlson et al., 1996). Narkiewicz et al. (1998a) undertook a similar investigation with matched-controls and found that normotensive, non-obese OSA patients have similar cardiac and sympathetic baroreflex responses to a hypertensive stimulus (phenylephrine) and similar cardiac, but decreased sympathetic baroreflex response to a hypotensive stimulus (nitroprusside). In summary, OSA patients appear to have some degree of baroreflex dysregulation that can be improved with CPAP therapy. Human experimental models have been utilized to elucidate the contribution of IH, hypercapnia and inspiratory resistance on BRS.  To discern the contributions of hypoxia, hypercapnia and inspiratory resistance on the carotid barorelex, Cooper et al. (2004) outfitted healthy volunteers with a neck chamber and constructed stimulus-response curves while breathing asphyxic air (12% O2, 5% CO2), with inspiratory resistance (-10 mmHg) and in combination. Twenty seconds of sustained delivery of -40, -20, -10, 10, 20, 40 and 60 mmHg occurred 10 minutes after breathing room air, 10 minutes following each of the above interventions and again after resuming normal breathing for 10 minutes. Asphyxia shifted the MAP set point upwards (to higher MAP). Inspiratory resistance reduced vascular resistance gain (calculated from MAP/brachial blood flow), asphyxia increased the set-point of vascular resistance and in combination, gain was depressed and set-point elevated. To separate the effects of hypoxia and hypercapnia, Cooper et al. (2005) performed the same study on subjects breathing 12% O2 or hyperoxic hypercapnia (95% O2, 5% CO2). Hypoxia reduced the sensitivity of baroreflex control of vascular resistance while the hyperoxic hypercapnia, utilized to assess the contribution of central chemoreceptors, increased the set-point of vascular resistance. While insightful, neither study investigated IH, which evokes appreciably different physiological responses (see 1.3, page 6). To induce cyclical desaturation/resaturation, Monahan et al. (2006) had subjects perform an end-expiratory apnea for 20 seconds every minute for 30 minutes. To ensure adequate desaturation, hypoxic gas was titrated during the 40 seconds of free breathing so that oxyhemoglobin saturation reached the low-to-mid 80s (%). Cardiovagal and sympathetic BRS were determined 7, 30 and 50 minutes post-intervention using the modified Oxford technique. Neither were impaired, however, both were shifted to higher levels of arterial blood pressure, adjusting the set-point beyond the duration of the exposure. Lastly, in  17 their assessment of 14 nights of IH, Tamisier et al. (2011) observed increased cardiovagal BRS gain and decreased sympathetic BRS gain using the modified Oxford technique. Experimental studies on humans do not display impaired cardiac baroreflex, but rather suggest a greater importance of vascular resistance, in agreement with the tonic increases in SNA in OSA patients. The impact of IH on baroreflex control of the vasculature remains unexplored and may provide insight on the initial resetting of the baroreflex to operate at higher pressures, facilitating an increase in MAP.  The augmented chemoreflex and attenuated baroreflex offer attractive hypotheses regarding the elevated SNA that persists following IH. The proceeding sections focus on the influence of IH and increased SNA on the peripheral vasculature, and the role it plays in promoting a hypertensive environment.   Intermittent Hypoxia and the Peripheral Vasculature 1.5When considering an increasingly complex series of physiological responses, it is worth taking a step back to focus on the bigger picture. Arterial blood pressure is determined from vascular resistance and cardiac output (stroke volume [SV] x HR). Patients with OSA display augmented MAP, but similar HR to controls (Hla et al., 1994). Fourteen nights of IH significantly increases MAP without altering SV or HR (Tamisier et al., 2011), highlighting the importance of increased vascular resistance in propagating and sustaining the rise in MAP associated with IH exposure.  Let’s first consider the vascular response to hypoxia. During acute, sustained hypoxia, the peripheral vasculature experiences a net vasodilation despite increased norepinephrine spillover (a marker of SNA), due in part to β-receptor activation (Blauw et al., 1995) and local control of vascular tone (NO and prostaglandins) (Markwald et al., 2011). Patients with OSA display an enhanced pressor response to sustained hypoxia, but similar tachycardiac responses to healthy controls, thus showing increased vascular resistance (or impaired vasodilation) (Hedner et al., 1992). Remsburg et al. (1999) measured forearm blood flow by venous occlusion plethysmography and observed impaired hypoxic vasodilation (increased forearm vascular resistance) in OSA patients compared to younger, non-obese controls.  18 Despite the unmatched control group, the altered response raises the specific importance of vascular tone on MAP in OSA patients. More recently, Reichmuth et al. (2009) observed impaired forearm vasodilation (increase in vascular conductance per unit decrease in oxyhemoglobin saturation) that was enhanced following six weeks of CPAP, however, they too compared obese OSA patients to non-obese controls. Using Doppler ultrasound to measure lower limb blood flow, Moradkhan et al. (2010) did not observe impairment in hypoxic vasodilation despite elevated resting MSNA and increased sympathetic response to hypoxia. Interestingly, regional α-adrenergic block (phentolamine) increased hypoxic vasodilation equally in OSA patients and controls, leading the authors to postulate upregulated metabolic vasodilator mechanisms in OSA patients (Moradkhan et al., 2010).   Obstructive sleep apnea appears to promote increased vascular resistance and altered hypoxic vasodilation. The following section will consider the evidence that OSA promotes endothelial dysfunction and impairs vascular regulation and reactivity.  1.5.1 The Endothelium The vascular endothelium forms the single-cell innermost lining of blood vessels. Preferentially located in direct contact with blood, the endothelium mediates vascular homeostasis via the synthesis and release of vasoactive substances. A healthy endothelium favours vasodilation and inhibition of coagulation, proliferation and inflammation (Widlansky et al., 2003). Endothelial dysfunction can be defined as the inverse; promoting vasoconstriction, coagulation, smooth muscle proliferation and inflammation (Widlansky et al., 2003) and precedes atherosclerosis (Celermajer et al., 1992). Endothelial function has been implicated as “an excellent “barometer” of underlying vascular health as it represents an orchestrated response to the many known and unknown processes that contribute to the development, progression, and clinical expression of atherosclerosis” (Vita & Keaney, 2002). Unsurprisingly, given the importance of vascular health, an extension can be made to overall cardiovascular risk.  1.5.1.1 Flow-Mediated Dilation  19 Flow-mediated dilation (FMD) is a common assessment of endothelial function where a hyperemic stimulus increases shear stress causing endothelial-dependent arterial vasodilation that is at least partially mediated by NO (Pyke & Tschakovsky, 2005; Green et al., 2014). Briefly, a cuff is placed downstream of the conduit artery of interest (usually the brachial artery, BA). Using B-mode and Doppler ultrasonography to simultaneously record vessel diameter and blood velocity, a baseline measure is recorded for one minute before inflating the cuff to a suprasystolic pressure (220-250 mmHg) for 5 minutes. Rapid cuff deflation results in a surge of blood flow (reactive hyperemia, RH), and consequently shear stress, evoking a vasodilatory response (see Figure 1.2, page 20) (Thijssen et al., 2011a). The RH, quantified as the peak blood flow response, is a measure of microvascular reactivity whereas FMD is a measure of macrovascular reactivity (Anderson et al., 2011; Thijssen et al., 2011a). Shear stress represents the frictional or drag force in a vessel and is related to blood velocity and viscosity and inversely related to diameter (Davies & Tripathi, 1993; Gnasso et al., 2001). Shear rate is often substituted for shear stress when viscosity measures are not performed and is an adequate surrogate measure (Pyke et al., 2004).  Blood flow, by contrast, is a measure of blood passing through a vessel over time and relates to blood velocity and vessel diameter. The magnitude of RH is dependent upon resistance artery vasodilation and can be used as a surrogate of microvascular reactivity (Anderson et al., 2011). Diameter and blood velocity are recorded for 3 minutes post-occlusion and FMD is calculated as the percent change in artery diameter from pre-cuff inflation baseline to post-cuff release peak vasodilation (Thijssen et al., 2011a). The calculation of FMD is corrected for baseline diameter (as smaller diameters yield greater percent increases) (Atkinson & Batterham, 2013) and the total shear rate response (shear rate area under the curve, SRAUC) (Pyke & Tschakovsky, 2007; Padilla et al., 2008). The SRAUC is thought to be the primary stimulus for the peak diameter response. The increased shear stress signals vasodilator production in the endothelium, including nitric oxide (NO), prostaglandins and endothelial-derived hyperpolarizing factors which diffuse into the smooth muscle causing vasodilation (Pyke & Tschakovsky, 2005; Thijssen et al., 2011a). In a meta-analysis of studies investigating FMD as a predictor of prognosis for future cardiovascular disease, Green et al. (2011) concluded “that a 1% increase in FMD was associated with a relative risk of 0.91, that is, a 9% (95% CI: 4% to 13%) decrease in the future risk of cardiovascular events.” The vascular  20 endothelium plays an essential role in vascular regulation. Obstructive sleep apnea is associated with augmented vascular tone in part due to tonic elevations in SNA and is reflected by increased vascular resistance and ultimately hypertension. The following will discuss the implications of IH on endothelial function.     Figure 1.2: Flow-mediated dilation shear rate and diameter illustration. Diameter (line) and shear rate (shaded area) profiles during a reactive hyperemia flow-mediated dilation protocol. The baseline period occurs for 1 minute prior to occlusion by cuff inflation. The occlusion persists for 5 minutes before the cuff is released, evoking a reactive hyperemic response that increases shear rate. Measurements are continued for 3 minutes post-occlusion. The shear rate area under the curve (SRAUC) is believed to be the main stimulus for peak diameter (identified by the asterisk, *). Shear stress mechanotransduction initiates a signaling cascade resulting in vasodilator production, including nitric oxide, prostaglandins and endothelial-derived hyperpolarization factor. Flow-mediated dilation is calculated as the percent increase in diameter from baseline to peak vasodilation and is corrected for baseline diameter and SRAUC. Definition of abbreviations: SR, shear rate.   21 1.5.1.2 Intermittent Hypoxia and Endothelial Dysfunction  Intermittent hypoxia lays the foundation for endothelial dysfunction. The increase in ROS initiates a positive feedback loop where increased stabilization of HIF-1α and degradation of HIF-2α promotes a pro-oxidant environment that further increases ROS (Semenza & Prabhakar, 2015). The resulting oxidative stress decreases NO availability and alters endothelial nitric oxide synthase (eNOS) enzymatic activity (Chen et al., 2010). Indeed, eNOS begins to overproduce superoxide and consequently decrease NO production, a dysfunction termed eNOS uncoupling (De Pascali et al., 2014). In OSA patients with low cardiovascular risk, eNOS uncoupling appears to be attributed to decreased tetrahydrobiopterin availability and contributes to endothelial dysfunction (Varadharaj et al., 2015). Reduced markers of NO availability are consistently found in OSA and usually appear to be ameliorated with CPAP (Ip et al., 2000; Schulz et al., 2000; Lavie et al., 2003; Noda et al., 2007; Alonso-Fernandez et al., 2009; Varadharaj et al., 2015). Pialoux et al. (2011) demonstrated a reduction in NO metabolism end-products following a 6-hour IH paradigm that was prevented when participants were administered AT1R blockade (losartan) beforehand, highlighting the involvement of the RAS in augmenting oxidative stress. Functionally, antioxidant treatment with ascorbic acid improves FMD in OSA patients, providing evidence for oxidative stress-mediated endothelial dysfunction (Grebe et al., 2006). Interestingly, oxidative stress alone does not account for endothelial dysfunction (Jurado-Gamez et al., 2011). The promotion of circulating adhesion molecules (Ohga et al., 1999; Zamarron-Sanz et al., 2006), hypercoagulability (Phillips et al., 2012), increased damaging microparticles (Priou et al., 2010; Ayers et al., 2013) and decrease in endothelial progenitor cells (Berger & Lavie, 2011) extend the OSA-induced endothelial dysfunction beyond NO availability.  Systemic inflammation is present in OSA (Ryan et al., 2009; Lavie, 2012). Of particular interest is C-reactive protein (CRP), synthesized in the liver and regulated by interleukin-6, an inflammatory cytokine (Castell et al., 1990), which directly induces adhesion molecule expression on endothelial cells (Pasceri et al., 2000). Serum CRP measurements serve as a marker of vascular wall inflammation and are strong independent predictors of future vascular risk (Blake & Ridker, 2001). The prevalence of obesity in OSA, itself an  22 inflammatory disease (Gregor & Hotamisligil, 2011), often complicates the assessment of inflammation in OSA. However, the majority of studies show that patients with OSA display higher serum CRP than age and BMI-matched controls that positively correlates with AHI (Nadeem et al., 2013). Intermittent hypoxia-induced systemic inflammation did not manifest following 14 nights of IH exposure in healthy humans (Tamisier et al., 2011).   OSA appears to promote endothelial dysfunction, evidenced from increased pro-atherogenic circulating markers. Next, the functional vasodilatory and vasoconstrictor implications will be examined.        1.5.1.2.1 Impairment in Vasodilation The association between OSA and endothelial function has received considerable attention. Hoyos et al. (2015) highlight that 23 of 29 studies comparing endothelium-dependent vasodilation between OSA patients and controls show significant impairment in the former. Furthermore, out of 16 CPAP interventional studies, only two did not observe improved endothelium-dependent vasodilation. A systematic review analyzing the relationship between OSA and markers of subclinical cardiovascular disease found that each of the fourteen studies that examined FMD showed a positive correlation between OSA severity and FMD impairment (Ali et al., 2014). Finally, meta-analyses of the effects of CPAP treatment on FMD have revealed absolute improvements of 2.92% (95% confidence interval: 2.21-3.63) (Xu et al., 2015) and 3.87% (95% confidence interval: 1.93-5.80) (Schwarz et al., 2015). Despite seemingly unanimous findings of the aforementioned systematic reviews and meta-analyses, they must be interpreted critically. In one of the most cited investigations of endothelial function in OSA (approaching 500 citations), Ip et al. (2004) demonstrate reduced FMD in patients with OSA compared to controls, which is improved markedly (nearly doubled) following four weeks of CPAP and decreases following a week of CPAP withdrawal. Unfortunately, FMD was assessed as the percentage increase in BA diameter from baseline to 60 seconds post-occlusion. Calculating FMD based on the 60 second value can result in a 25-40% underestimation of true FMD (Black et al., 2008) and individuals with even moderate cardiovascular risk demonstrate delayed time-to-peak FMD in response to RH (Padilla et al., 2009a). Another potential source of error comes from comparing groups with  23 different baseline diameters. When considering a very small change in diameter, the percent FMD is amplified in smaller arteries. Therefore, to compare groups of different baseline diameters, allometric scaling is recommended where FMD = (Peak Diameter – Baseline Diameter)/Baseline Diameter0.89 (Atkinson & Batterham, 2013). Accounting for baseline diameter is especially important in the case of OSA, where patients with moderate-to-severe OSA have significantly larger BA diameters compared to those without OSA (Chami et al., 2009). Namtvedt et al. (2013) compared FMD in obese OSA and non-obese OSA with obese controls and concluded that “OSA is associated with endothelial dysfunction independently of obesity” however the obese subjects had significantly smaller BA diameters. If allometric scaling is applied to the reported means of diameter, the obese subjects display an FMD of 9.6%, compared to 8.7% in OSA, reducing the difference from the 10.1% and 6.4% presented (Namtvedt et al., 2013).   Two experimental human studies have assessed RH (i.e. microvascular reactivity), using venous occlusion plethysmography. An increase in forearm blood flow following 15 minutes of ischemia was observed following 2 weeks of nocturnal IH, but after 4 weeks both peak and total blood flow responses (2.5 minutes following release of occlusion) were impaired (Gilmartin et al., 2010). Tamisier et al. (2011) found a decreased peak calf blood flow following five minutes of ischemia, but unchanged blood flow response following peak RH after 2 weeks of nocturnal IH. Combined, there appears to be heterogeneous limb microvascular responses to IH. While investigations in OSA patients are plentiful, endothelial function in response to IH is scant in healthy humans. Given the widespread manifestation of endothelial dysfunction in OSA, mechanistic insight from in vivo human models is warranted.  1.5.1.2.2 Enhanced Vasoconstriction With endothelial dysfunction, instead of promoting vasodilation, the endothelium shifts to a state of vasoconstriction. Endothelin-1 (ET-1), a circulating vasoconstrictive neuropeptide, has been found to be increased in OSA patients, with reductions following CPAP (Phillips et al., 1999; Gjorup et al., 2007), although neither group controlled for BMI. Conversely,  24 Grimpen et al. (2000) found no effect of long-term CPAP treatment (>1 year) on ET-1 levels on normotensive, non-obese OSA patients, nor did Saarelainen et al. (1997) following 3 months of CPAP treatment. Jordan et al. (2005) found increased precursor to ET-1, but not ET-1 in OSA patients. Following 6 hours of IH, Foster et al. (2010) did not find an increase in ET-1 production in healthy humans. While Moller et al. (2003) did not observe elevated plasma ET-1, they did find an increase in plasma Ang II, another vasoconstricting hormone, in OSA patients. Thromboxane A2, a vasoconstricting prostanoid, is elevated in OSA patients (Krieger et al., 1991) and in healthy volunteers following 6 hours of IH (Beaudin et al., 2014). Furthermore, catecholamines, when acting on α-receptors, cause vasoconstriction. Thus, the augmented SNA in OSA promotes vasoconstriction. Indeed, elevated circulating catecholamines are observed in OSA patients (Fletcher et al., 1987; Carlson et al., 1993; Marrone et al., 1993). An increase in circulating vasoconstrictors contributes to the state of vasoconstriction present in OSA.  In addition to increasing circulating vasoconstrictors, IH may evoke enhanced vasoconstrictor sensitivity. While the vasoconstriction response to norepinephrine appears to be diminished in OSA patients (Grote et al., 2000), other vasoactive substances may produce heightened responses. Ten normotensive, non-obese OSA patients displayed increased forearm vasoconstrictor response to Ang II infusion compared to well-matched controls (Kraiczi et al., 2000). The hypothesis of enhanced vasoconstrictor sensitivity is supported by animal studies. Exposing rats to IH at a rate of 20 cycles per hour (nadir 5% O2, 5% CO2: peak 21% O2, 0% CO2), 7 hours per day for two weeks not only elevates ET-1 levels, but increases in ET-1 receptor and increased vasoconstrictor sensitivity to ET-1 (Allahdadi et al., 2005).   Endothelial dysfunction manifests from the multifactorial consequences of IH. The perpetuating increases in oxidative stress, inflammation, SNA and RAS activity promote vasoconstriction and inhibit vasodilation.  1.5.1.3 The case for retrograde flow  25 Shear stress is the frictional force acting on the endothelial cell surface as a result of blood flow and is directly related to blood velocity and viscosity and inversely related to vessel diameter (Malek et al., 1999). Laminar blood flow exerts high antegrade shear on the endothelium and promotes a vasoprotective endothelial cell phenotype. Vascular curvature, branches, bifurcations and downstream resistance disturb the unidirectional flow, causing oscillations that reduce antegrade flow and increase retrograde flow, upregulating genes and proteins that promote atherogenesis and damaging the endothelium (Chiu & Chien, 2011; Jenkins et al., 2013; Padilla et al., 2014). The relationship between antegrade and retrograde shear can be assessed as the oscillatory shear index [OSI; |retrograde shear|/(|antegrade shear| + |retrograde shear|)], where purely oscillatory shear would produce an OSI of 0.5 (Totosy de Zepetnek et al., 2014). Figure 1.3 demonstrates the shear pattern over a single cardiac cycle during a low and high oscillatory shear situation. Increased SNA by lower-body negative pressure increases retrograde shear and OSI in the BA (Padilla et al., 2010). Acute blood flow disturbances causing increases in retrograde shear and OSI consistently impair FMD in upper and lower limbs of healthy humans (Thijssen et al., 2009; Tinken et al., 2009; Schreuder et al., 2014; Totosy de Zepetnek et al., 2014). In a fortuitous moment of happenstance, Millar et al. (2011) were conducting a study investigating MSNA in hypertension when a subject dozed off. Already measuring MSNA, and BA diameter and blood velocity with Doppler ultrasound, the subject began to develop spontaneous obstructive apneas. As expected, the apneas caused marked increases in MSNA, though more intriguing, apneas increased retrograde blood flow, hypothetically via hypoxia-induced sympathetic activation causing arteriolar vasoconstriction. Unfortunately, this unique case report has yet to be expanded upon, although repetitive increases in retrograde flow may provide real contributions to the noted vascular impairment induced by IH.  During sleep, OSA patients experience marked peripheral vasoconstriction, as evidenced by surges in peripheral resistance following each apnea (Anand et al., 2001; Imadojemu et al., 2002). The surge in post-apneic peripheral resistance is augmented by hypoxia, indicating that patients with more severe OSA may experience more pronounced pressor responses (Leuenberger et al., 2001). Therefore, patients with OSA can experience hundreds of surges in peripheral resistance, possibly associated with retrograde flow, exerting damaging effects  26 on the endothelium. Patients with OSA display increased blood viscosity (Toraldo et al., 2013), amplifying the shear stress exerted on the endothelium.  Disturbed blood flow represents a potent stimulus for endothelial dysfunction. Obstructive sleep apnea has widespread effects on the vasculature, in which cyclical bouts of retrograde shear stress may accompany IH.     Figure 1.3: Demonstration of two shear profiles over the course of a cardiac cycle. The oscillatory shear index (OSI) describes the relationship between antegrade and retrograde shear [|retrograde shear| / (antegrade shear| + |retrograde shear|)]. Purely oscillatory shear would produce an OSI of 0.5. Larger OSI impairs endothelial function, promoting a pro-atherogenic milieu. The anti-atherogenic shear pattern is demonstrated by the broken line, where there is large antegrade shear and minimal retrograde shear, compared to a pro-atherogenic pattern, the solid line, which has reduced antegrade shear, large retrograde shear and an OSI approaching 0.5.   27 1.5.2 Arterial Stiffness Given the substantial evidence linking IH and endothelial dysfunction, it is unsurprising that OSA patients free of cardiovascular disease display early signs of atherosclerosis, and such vascular abnormalities correlate with OSA severity (Drager et al., 2005) and improve after four months of CPAP treatment (Drager et al., 2007). Arterial stiffness can be estimated from pulse wave velocity measurements and is an independent risk factor for cardiovascular events (Mancia et al., 2013). Patients with OSA consistently display elevated subclinical markers of cardiovascular disease, including increased central pulse wave velocity (Ali et al., 2014). Experimental models identify IH as a potential cause, as rats exposed to one minute of 10% O2 every 4 minutes, 12 hours per day for 2 weeks develop increased arterial wall stiffness (Phillips et al., 2006; Philippi et al., 2010). Jelic et al. (2002) observed acute elevations in arterial augmentation index, an estimate of arterial stiffness, during nocturnal obstructive apneas and suggest that these functional impairments may precede structural remodelling. Furthermore, the elevations occurred independent of surges in arterial blood pressure. Structural remodelling, as assessed by common carotid artery intima-media thickness (CIMT), does eventually occur in OSA and correlates with hypoxemia (time <90% oxyhemoglobin saturation and mean nadir oxyhemoglobin saturation) (Suzuki et al., 2004). The structure and function of arteries appears to be impaired in OSA, due in part to IH, and may contribute to, or result from, endothelial dysfunction and hypertension. 1.5.2.1 Vascular Strain A novel ultrasonographic technique permits the evaluation of arterial stiffness in the large arteries by assessing two-dimensional vascular wall tissue motion and deformation across the cardiac cycle, termed vascular strain. Strain describes the deformation of an object normalized to its original shape and size, and strain rate describes the rate of deformation. Vascular strain primarily provides insight on the responses of the vessel to systole.  Peak systolic strain represents the percent increase in vessel diameter during systole. Early systolic strain rate is the result of the blood pressure rise associated with ventricular ejection, and late systolic strain rate is the vessel recoil that occurs during ventricular relaxation (Bjallmark et al., 2010). Vascular strain can be quantified using speckle-tracking echocardiography, where specific acoustic signals are tracked frame-by-frame (Leitman et al., 2004). Originally  28 utilized for cardiac mechanics, the technique has been used to assess common carotid artery (CCA) strain and strain rate and shown to be superior to conventional measures in the detection of age-dependent changes in the mechanical properties of the vessel (Bjallmark et al., 2010; Rosenberg et al., 2014) and effectively characterizes cardiovascular risk (Catalano et al., 2011). Recently, evaluation has been expanded to the common femoral artery (CFA), demonstrating feasibility and reproducibility in the muscular artery (Charwat-Resl et al., 2015). To date this technique has not been applied in the context of OSA and IH. Given the noted vascular impairments in OSA, investigations employing this novel technique are warranted.   Renin-Angiotensin System 1.6An intimate relationship between SNA, RAS and MAP exists, where increased renal SNA leads to increased renin production in the kidney, initiating a cascade of conversions resulting in the vasoactive mediator angiotensin II. Circulating angiotensin II raises MAP by 1) causing vasoconstriction and 2) stimulating adrenal secretion of aldosterone secretion, which increases blood volume by reducing sodium excretion, causing water retention (Laragh & Sealey, 2010). The RAS has been implicated in the process of IH-induced elevations in MAP. In rats, 8 hours of IH per day for 35 days increases plasma renin activity four-fold and renal denervation or pharmacological blockade of the AT1R (losartan) prevent the rise in MAP (Fletcher et al., 1999). Confirming the importance of RAS in IH-mediated hypertension, suppression of RAS via salt-loading blocked the rise of MAP in the same model (Fletcher et al., 2002). A role of RAS in IH-induced endothelial dysfunction has been proposed, as AT1R blockade prevents the impaired endothelium-dependent vasodilation in rats exposed to IH (10% O2 for 1 minute and 45 seconds, 21% O2 for 2 minutes and 15 seconds) for 12 hours per day for 28 (Marcus et al., 2012). An extension to humans has been established, as patients with OSA display increased circulating Ang II and aldosterone (Moller et al., 2003), while treatment with valsartan (AT1R blocker) causes a fourfold greater decrease in MAP than CPAP in hypertensive OSA patients (Pepin et al., 2010). In experimental human models, losartan completely abolished a 7.9 mmHg increase in MAP following 6 hours of IH in 6 of 9 participants, while the remaining 3 displayed an attenuated increase (Foster et al., 2010). Expanding upon this observation, Pialoux et al. (2011) were  29 able to show that AT1R blockade prevents the rise in oxidative stress caused by IH, proposing the RAS as one of the mechanisms involved in upregulating ROS production during IH.  1.6.1 An Independent Role for Angiotensin II? Angiotensin II has profound effects on blood pressure control, extending far beyond its cardiorenal activities. Through stimulation of AT1R, Ang II promotes sympathetic outflow in the central nervous system (Reid, 1992) in a process mediated by ROS (Zimmerman et al., 2002). Components of RAS (angiotensinogen, angiotensin converting enzyme and AT1R) are expressed locally at the carotid body, important given the influence of Ang II on activating the carotid chemoreceptors (Fung, 2014).  In addition to hypoxia, hypercapnia and acidosis, the carotid chemoreceptor responds to Ang II (Fung, 2014). The carotid bodies are also able to synthesize Ang II locally (Lam & Leung, 2002). Similar to IH, repetitive application of Ang II to the carotid body ex vivo increases oxidative stress (via NOX2) leading to carotid body sensory LTF, which is abolished with AT1R blockade (Peng et al., 2011b). One week of eight hours of intermittent hypoxia per day induces upregulation of RAS in rat carotid body (Lam et al., 2014). Furthermore, Ang II, via ROS production, causes vascular inflammation mediated by AT1R (Griendling et al., 1994) and increases endothelial oxidative stress (Landmesser et al., 2007). Therefore, Ang II must be included when considering the ROS-mediated positive feedback loop as it works in parallel with IH (see Sustained Hypoxia versus Intermittent Hypoxia, page 6). Carotid body RAS upregulation may contribute to the enhanced peripheral chemosensitivity and endothelial dysfunction induced by IH.    Angiotensin II may play a direct role in the development of vascular inflammation by increasing vascular permeability, leukocyte infiltration and tissue remodelling (Cheng et al., 2005). Vascular endothelial growth factor is a potent angiogenic cytokine that stimulates vascular smooth muscle proliferation, contributing to the development of atherosclerosis (Celletti et al., 2001). Angiotensin II induces expression of VEGF, and interestingly Ang II infusion increases local HIF-1α expression in mice vascular wall cells (Zhao et al., 2004).  30 Hyoxia-inducible factor-1α plays a major role in the expression of VEGF, making the finding logical, however in the case of IH, this offers a specific target for the repetitive hypoxic provocations. Hyoxia-inducible factor-1α is not the only transcriptional factor elevated by Ang II. Nuclear transcription factor kappa-B (NF-ĸB) is responsible for the regulation of inflammatory genes and is activated in the vasculature by Ang II (Tummala et al., 1999).  In a small study (n=16), Takahashi et al. (2005) found elevated Ang II and VEGF in male patients with moderate-to-severe OSA compared to age and BMI-matched controls. Another small investigation found increased VEGF only in patients with severe nocturnal hypoxemia, finding a correlation between time spent <90% oxyhemoglobin saturation and VEGF (r=0.67, P<0.01) (Schulz et al., 2002). As is the case with inflammatory markers in OSA, the findings have not been uniform. Maeder et al. (2015) found no difference in serum VEGF between 65 patients with moderate-to-severe OSA compared to 33 with mild or without OSA. Venous endothelial expression of NF-ĸB was greater in patients with OSA and decreased after four weeks of CPAP treatment (Jelic et al., 2010). Interestingly, another alternative mechanism of NF-ĸB activation has been demonstrated in OSA, where IH triggers an inflammatory immune response evidenced by increased toll-like receptor activity (Akinnusi et al., 2013).  It is important to reiterate that Ang II is more than just a vasoconstrictive peptide. Angiotensin II is triggered by increased ROS, and contributes to ROS generation via NOX2. Furthermore, Ang II contributes to peripherally- (carotid body) and centrally-mediated elevation in SNA. Finally, Ang II increases vascular inflammation. Clearly, Ang II promotes a hypertensive environment and the efficacy of AT1R blockers in lowering or preventing the rise in MAP with IH highlights its importance in the pathogenesis of IH-induced hypertension.   Metabolism 1.7Sleep-disordered breathing is independently associated with type II diabetes, glucose intolerance and insulin resistance (Punjabi et al., 2004; Reichmuth et al., 2005). Risk of type II diabetes is associated with nocturnal IH (Muraki et al., 2010) and metabolic function has been shown to improve with CPAP treatment (Harsch et al., 2004; Dorkova et al., 2008). In  31 healthy humans, 25 deoxygenation/reoxygenation cycles (85-95% oxyhemoglobin saturation) per hour for 8 hours decreased insulin sensitivity and secretion, and glucose effectiveness (Louis & Punjabi, 2009). A cross-sectional assessment from the Wisconsin Sleep Cohort Study identified that the combination of metabolic syndrome and OSA have a synergistic effect on endothelial dysfunction (Korcarz et al., 2014). Due to the high prevalence of obesity in OSA, the interpretation of investigations on adipokines in OSA remain cloudy, though may link metabolic disturbances with the development of atherosclerosis (Levy et al., 2009; Lam et al., 2015).   1.7.1 Insulin and the Sympathetic Nervous System Similar to hypoxia, insulin causes peripheral vasodilation. To preserve MAP following a meal, hyperinsulinemia evokes sympathoexcitation (Scherrer & Sartori, 1997) and LTF (Anderson et al., 1992). Centrally, circulating insulin crosses the blood-brain barrier and acts within the arcuate nucleus to increase SNA (Luckett et al., 2013), however, recently peripheral action on the carotid chemoreceptors has been suggested (Limberg et al., 2014). In mice, insulin triggered the carotid chemoreceptors and carotid sinus nerve resection prevented diet-induced insulin resistance and hypertension (Ribeiro et al., 2013). Evidence of improved insulin sensitivity following CPAP treatment and hyperoxia support an involvement of the carotid chemoreflex (Jakobsson & Jorfeldt, 2006; Dorkova et al., 2008). The metabolic syndrome is associated with disturbances in glucose and lipid metabolism can produce ROS, leading the NO-deficiencies and subsequent endothelial dysfunction. Endothelial dysfunction impairs insulin delivery to target tissues, leading to impaired insulin-stimulated glucose and lipid metabolism (Cersosimo & DeFronzo, 2006). The metabolic consequences of IH exacerbate and stem from endothelial dysfunction, elevations in SNA, inflammation and oxidative stress.   Therapeutic Intermittent Hypoxia 1.8Based on the above evidence, it may seem paradoxical to propose IH as a potential treatment for hypertension. However, low frequency, short duration cycles of moderate hypoxia (10-15% O2) for 10-20 days have profound antihypertensive effects (Serebrovskaya et al., 2008). The severe desaturation:reoxygenation IH profile in OSA induces massive ROS generation,  32 while the mild therapeutic IH produces low levels of ROS that function as signalling molecules inducing the positive cardiovascular outcomes.   Consequently, therapeutic IH does not cause inflammation, but rather suppresses pro-inflammatory mediator production (Serebrovskaya et al., 2011) and increases NO synthesis in hypertensive patients (Lyamina et al., 2011). Therapeutic IH, achieved by modest bouts of hypoxia (9-16%) that are short in duration (15 seconds to 4 minutes), small in number (~10 cycles) and have a brief length of repetition (<1 hour) leads to beneficial cardiovascular outcomes (Serebrovskaya et al., 2008; Navarrete-Opazo & Mitchell, 2014; Mateika et al., 2015).   Summary 1.9The independent association between OSA and hypertension has sparked interest in the mechanisms responsible. Work in the 1990s on rodent models implicated IH as causal, elevating SNA via peripheral chemoreceptor activation, increasing RAS activation and impairing vasodilation. Later confirmed in OSA patients and healthy humans, IH mediates the increase in MAP via increased vascular resistance. The increase in ROS production evokes a cascade of events in the carotid body, vasculature and kidney that further exacerbates ROS production, propagating the vascular impairment even in the absence of IH. The above review can be summarized in our working model illustrated in Figure 1.4.     33   Figure 1.4: Summary of the hypertensive effects of intermittent hypoxia. Intermittent hypoxia causes oxidative stress contributing to increased carotid chemoreflex and impaired carotid baroreflex, collectively causing increased sympathetic nerve activity via the brain stem that outlasts intermittent hypoxia exposure (1). The elevations in sympathetic activity and oxidative stress cause endothelial dysfunction and increase circulating angiotensin II from the kidneys. Decreased nitric oxide bioavailability, increased circulating vasoconstrictors and increased sensitivity to vasoconstrictors shift the vasculature to a pro-vasoconstriction state, causing increased vascular resistance. Increased downstream vascular resistance increases retrograde shear stress, further damaging the endothelium. Eventually, systemic inflammation and atherosclerosis occur (2). Endothelial dysfunction contributes to insulin resistance, which in concert with the vasculature, renal system and carotid body, further increase oxidative stress, producing a harmful positive feedback cycle where increased oxidative stress begets reactive oxygen species production (3).   34 2    Chapter: Research Questions and Hypotheses This chapter introduces the specific scientific literature pertaining to this thesis and establishes the five research questions and hypotheses that were the investigated in this thesis.    Background 2.1Brief, intermittent high-frequency hypoxemia, similar to OSA, causes an increase in oxidative stress and SNA. The increased risk for morbidity and mortality in patients with OSA has been attributed to IH (Dewan et al., 2015). Nocturnal IH is well recognized for its cardiovascular comorbidities, specifically hypertension. Community-based studies have established that 60% of adults with an AHI ≥15 events/hour are hypertensive (Bixler et al., 2000; Nieto et al., 2000; Peppard et al., 2000). This is particularly alarming when considering the prevalence of OSA. Two population-based studies using up-to-date polysomnography standards have shown between 40-50% of men and approximately 25% of women have OSA (Tufik et al., 2010; Heinzer et al., 2015). Experimental studies exposing healthy humans to IH equivalent to moderate-to-severe OSA increases arterial blood pressure beyond exposure (Foster et al., 2009; Foster et al., 2010; Tamisier et al., 2011). The hypertensive effect manifests even after a single bout; Foster et al. (2010) demonstrated an 8-mmHg increase in MAP fifteen minutes following 6 hours isocapnic IH. The increase is thought to be mediated by increased SNA and oxidative stress and can be prevented with AT1R blockade (Foster et al., 2010; Pialoux et al., 2011). We sought to expand on the findings of Foster et al. (2010) by measuring 24-hour blood pressure with ambulatory blood pressure monitoring.  Arterial blood pressure is the product of cardiac output (SV x HR) and vascular resistance. Fourteen nights of IH did not alter cardiac output, but increased daytime SBP and DBP by 8 mmHg and 5 mmHg respectively, suggesting the increased blood pressure owed to increased vascular resistance (Tamisier et al., 2011). Muscle sympathetic nerve activity is positively correlated with vascular resistance; therefore the IH-mediated sympathoexcitation may be responsible for the acute hypertensive response (Charkoudian et al., 2005). Additionally, the oxidative stress caused by IH promotes an increase in carotid body SNA that outlasts the IH  35 stimulus (Semenza & Prabhakar, 2015) while simultaneously damaging the endothelium (Jelic et al., 2008). The precise mechanisms whereby IH promotes hypertension remain incompletely understood, but initially there appears to be an increase in vascular resistance. We were particularly interested in the effects of acute IH on endothelium-dependent macro and microvascular vasodilation, elastic and muscular large artery mechanics, upper and lower limb hemodynamics, and the baroreflex control of MAP and leg vascular conductance (LVC) in the initial facilitation of IH-induced hypertension.   Macro and Microvascular Reactivity 2.2Vascular reactivity can be assessed at the macro and microvascular level by BA FMD and RH. Both FMD (macrovascular reactivity) and RH (microvascular reactivity) are somewhat predictive of cardiovascular risk in general populations (Anderson et al., 2011; Green et al., 2011). Macrovascular endothelial function, FMD, is assessed as the percent-increase in BA diameter following a 5-minute ischemic stimulus (cuff inflation), whereas forearm microvascular function can be determined from the peak RH evoked by the removal of the ischemic stimulus. Cuff release evokes a surge in shear rate, the frictional or drag force in a vessel, and is related to blood velocity and inversely related to diameter (Pyke et al., 2004). The increased shear rate signals vasodilator production in the endothelium, including NO, prostaglandins and endothelial-derived hyperpolarizing factors which diffuse into the smooth muscle causing vasodilation (Pyke & Tschakovsky, 2005; Thijssen et al., 2011a). The total shear rate response is thought to be the primary stimulus for the peak diameter response (Pyke & Tschakovsky, 2007; Padilla et al., 2008). Reductions in FMD are near-ubiquitously observed in patients with OSA, and improvement with CPAP treatment infers IH as at least partially responsible (Hoyos et al., 2015). IH is thought to cause endothelial dysfunction by reduced NO bioavailability due to oxidant-mediated eNOS uncoupling, impaired repair capacity and inflammation (Atkeson et al., 2009; Varadharaj et al., 2015).   Reactive hyperemia occurs as a result of myogenic and local vasodilators in resistance arteries, specifically via hyperpolarization of endothelial and vascular smooth muscle cells by activation of inwardly rectifying potassium channels and Na+/K+-ATPase (Crecelius et al., 2013). Small and resistance arteries and arterioles contribute to peripheral resistance  36 (Thijssen et al., 2011c), important given the relationship between augmented SNA and vascular resistance in young men (Charkoudian et al., 2005). Patients with OSA display blunted forearm RH (Kato et al., 2000). A compensatory increase in forearm RH has been observed following 2 weeks of nighttime IH, followed by an impairment after 4 weeks (Gilmartin et al., 2010). The same compensation does not appear to be present in the lower limb, as peak calf RH was impaired following 2 weeks of nightly IH (Tamisier et al., 2011). This suggests that eventually both upper and lower limb microvascular reactivity are impaired by IH, but the reduction in the lower limb reactivity precedes upper limb microvascular impairment.   Vascular Strain 2.3Obstructive sleep apnea patients free of cardiovascular disease display early signs of atherosclerosis, including increased arterial stiffness, which positively correlate with OSA severity (Drager et al., 2005). Elevations in SNA cause a decrease in arterial distensibility (Parati & Salvi, 2014), leading to impaired micro- and macrovascular reactivity (Malik et al., 2008). The assessment of large artery mechanics has recently been proposed as a surrogate measurement of arterial stiffness (Bjallmark et al., 2010). Specifically, vascular strain is the deformation of the vessel walls normalized to its original shape and size, whereas strain rate is the rate of deformation. Large artery circumferential strain and strain rate can be assessed during a cardiac cycle using two-dimensional speckle tracking ultrasonography. Peak systolic strain (%) represents the vessel distension during systole. Early systolic strain rate (1/s) is the result of the blood pressure rise associated with ventricular ejection, and late systolic strain rate (1/s) is the vessel recoil that occurs during ventricular relaxation (Bjallmark et al., 2010). Common carotid artery peak systolic strain is a marker of local arterial stiffness that effectively distinguishes between young and old and low and high cardiovascular risk (Bjallmark et al., 2010; Catalano et al., 2011; Oishi et al., 2013; Rosenberg et al., 2014). Recently, the feasibility and reproducibility of CFA strain (a muscular artery) measurements were confirmed (Charwat-Resl et al., 2015). This novel technique has never been used in the context of OSA or IH. We sought to examine whether a 6-hour IH exposure reduces vascular strain or strain rate in an elastic artery (CCA) and muscular artery (CFA).   37  Upper and Lower Limb Hemodynamics 2.4Periods of disturbed blood flow, characterized by increased retrograde shear, promotes a pro-atherogenic endothelial cell phenotype (Padilla et al., 2014). The pattern of oscillatory shear is characterized by the oscillatory shear index (OSI), where OSI=|retrograde shear| / (|antegrade shear| + |retrograde shear|). Hemodynamic alterations, including disturbed shear patterns, have been proposed in OSA (Millar et al., 2011), but have yet to be investigated. The surges in vascular resistance accompanying each nocturnal apnea (Imadojemu et al., 2002) certainly suggest the possibility of increases in retrograde shear due to increased downstream resistance. The primary cause for the hemodynamic alterations may be increases in SNA and/or oxidative stress. Elevations in SNA cause increases in OSI, which lead to reductions in endothelium-dependent vasodilation in the upper limb (Padilla et al., 2010; Thijssen et al., 2014). Administration of vitamin C prior to experimental increases in OSI prevents the decline in FMD, suggesting an oxidative stress mediated reduction in NO bioavailability (Johnson et al., 2013). Alterations in shear patterns may be a particularly vulnerable target site for the consequences of IH. Since periods of disturbed blood flow precede functional impairment (i.e., FMD), increased OSI may be an early mechanism linking OSA and endothelial dysfunction and subsequently hypertension.   Carotid Baroreflex 2.5The carotid baroreflex regulates arterial blood pressure by initiating a negative feedback loop. An acute elevation in arterial blood pressure is sensed as increased transmural pressure at the aortic arch and carotid sinus, evoking a reflex reduction in SNA and increase in parasympathetic nerve activity, causing a passive vasodilation and decrease in HR to reduce vascular resistance and blood flow, rapidly reducing arterial blood pressure (Mancia & Mark, 1983). Baroreflex impairment has been noted in OSA, where an augmented chemoreflex and attenuated baroreflex facilitate persistent sympathetic excitation (Prabhakar & Kumar, 2010). In healthy humans, Monahan et al. (2006) effectively elevated baroreflex set-point after only 30 minutes of IH, demonstrating the IH-induced shift of the baroreflex to operate at a higher arterial blood pressure. Asphyxia resets the carotid baroreflex-vascular resistance curve to higher pressures, and sustained hypoxia reduces its sensitivity (Cooper et al., 2004; Cooper et al., 2005). The carotid baroreflex controls blood pressure primarily through changes in  38 vascular conductance, as opposed to cardiac output (Ogoh et al., 2002). We postulate that the increased setpoint owes to an increased peripheral resistance, where baroreflex activation, and subsequent sympathoinhibition, produces a lesser degree of passive vasodilation, reflected as blunted carotid baroreflex control of vascular conductance.    Research Questions and Hypotheses 2.6Employing a human experimental model, the present investigation sought to identify the initial mechanisms involved in the pathogenesis of IH-induced hypertension. The global vascular and carotid baroreflex responses to acute IH were investigated with the following research questions: 1) Is 24-hour arterial blood pressure elevated by IH? IH will elevate 24-hour arterial blood pressure. 2) Are upper limb endothelium-dependent macro and microvascular vasodilation blunted following IH? Brachial artery FMD will be reduced, reflecting impaired macrovascular reactivity. Peak RH, a measure of microvascular reactivity, will be blunted due to the increased peripheral resistance following IH. 3) Are CCA and CFA vascular mechanics impaired by IH? Common carotid artery and CFA peak systolic strain, early systolic strain rate and late systolic strain rate will be impaired by IH. 4) Does IH alter upper or lower limb conduit artery hemodynamics? Intermittent hypoxia will increase OSI in both upper and lower limbs, reflecting increased downstream resistance and promoting endothelial dysfunction. 5) Does IH influence carotid baroreflex control of MAP, HR or LVC?   The IH exposure will not affect carotid baroreflex sensitivity, however the curve will be shifted upwards, reflecting an increased arterial blood pressure set-point. Carotid baroreflex control of lower limb vascular conductance will be impaired and control of HR will not be altered   39 3    Chapter: Materials and Methods This chapter provides an overview of the experimental methods used to systematically answer our research questions. Details are provided on the recruited participants, screening measurements, the experimental protocols and procedures, variables collected, and data and statistical analyses.   Ethical Approval 3.1All experimental procedures and protocols were reviewed and approved by the Clinical Research Ethics Board at the University of British Columbia and conform to the Declaration of Helsinki (see University of British Columbia Ethics Certificate of Full Board Approval, page 130). All participants provided written informed consent prior to participation in this study.    Participants 3.2Ten healthy male volunteers were recruited from the University of British Columbia – Okanagan study body. Volunteers were required to complete a health history questionnaire and were excluded if they had a history of cardiovascular, respiratory or renal disease (see Health Questionnaire, page 132). Females were not invited to participate in the study given the variation in vascular function and arterial blood pressure across the menstrual cycle (Adkisson et al., 2010). Inclusion criteria required volunteers to be free from cardiovascular, respiratory and kidney disease, non-smokers, normotensive (SBP: <140 mmHg, DBP: <90 mmHg), not taking any medications, have a BMI<30 kg/m2 and to be free of sleep apnea.    Screening 3.3To ensure adequate health and absence of undiagnosed disease that may confound study outcomes, a simple series of screening procedures were performed. Before any screening, height and weight were recorded from a calibrated scale. Automatic blood pressure measurements were made in triplicate (HEM-775CAN, Omron Healthcare, Cannockburn, IL, USA), and volunteers excluded if they displayed pressures greater than 140 mmHg for SBP or 90 mmHg for DBP. Volunteers were subsequently screened for daytime sleepiness (Epworth Sleepiness Scale), pulmonary function (spirometry), atherosclerotic disease  40 (CIMT) and nocturnal hypoxemia (nocturnal pulse oximetry). All volunteers were then given an experimental walk-through and the opportunity to try on the neck chamber.  3.3.1 Epworth Sleepiness Scale Volunteers filled out the Epworth Sleepiness Scale as an index of somnolence and participants were excluded if they scored >16 (Johns, 1991) (see Epworth Sleepiness Scale, page 133). The simple, self-administered questionnaire assess daytime sleepiness by asking how likely one is to doze off in eight different situations commonly encountered in daily life. Epworth Sleepiness Scale scores have been found to correlate with ODI and minimum oxyhemoglobin saturation (Johns, 1991).   3.3.2 Pulmonary Function Testing In order to determine whether the participants had healthy lung function, pulmonary function was assessed using spirometry (forced vital capacity; FVC) (Miller et al., 2005), lung volumes by whole body plethysmography (Wanger et al., 2005), and a single breath carbon monoxide test to quantify diffusion capacity (diffusing capacity of the lung for carbon monoxide, DLCO) (Macintyre et al., 2005). For each of these tests, participants sat within the body plethysmography box (V6200, Vmax Sensormedics, Yorba Linda, CA, USA) with a rigid upright posture and their feet flat on the ground, whilst breathing through a spirometer and bacteriological filter with nose clamped. A calibration check of volume using a 3 litre syringe (5530, Hans Rudolph, Shawnee, KS, USA) was performed and box leak constant was obtained by applying 3 cmH2O constant pressure for 1 minute.   Forced vital capacity was obtained as the maximal volume of air exhaled with maximally forced effort from a maximal inspiration (Miller et al., 2005). The forced expiratory volume in 1 second (FEV1) was the volume of air exhaled during the first second of the FVC. The ratio of FEV1/FVC was assessed to rule out any obstructive lung disease.   Total lung capacity was obtained as follows. Firstly, the plethysmography door was closed. Secondly, the volunteer breathed quietly until a stable end-expiratory level was achieved. Thirdly, a shutter was closed at end-expiration for 2-3 seconds and the volunteer was  41 instructed to pant at a frequency of 1 Hz. This permits determination of thoracic gas volume based on Boyle’s Law and denotes functional residual capacity. Finally, the shutter was opened and the volunteer performed a maximal expiration before executing a slow inspiratory vital capacity maneuver. Total lung capacity was obtained as the sum of functional residual capacity and inspiratory capacity (Wanger et al., 2005).   The DLCO measures the exchange of carbon monoxide from the alveoli to the red blood cells in the pulmonary capillaries. Before the maneuver, the gas-analyzer was zeroed. The DLCO maneuver began with an exhalation followed by a rapid full inhalation held for 10 seconds before exhalation. The breath contained 0.3% carbon monoxide and methane, as a tracer gas, to determine the initial alveolar carbon monoxide and the alveolar volume from which carbon monoxide uptake occurred. The DLCO was calculated as the carbon monoxide uptake multiplied by the alveolar volume divided by time and normalized for the initial partial pressure of carbon monoxide in the alveoli (Macintyre et al., 2005).  Established reference equations were used to determine predicted values for spirometry (Knudson et al., 1983), lung volumes (Crapo et al., 1982) and diffusing capacity (Burrows et al., 1961). Volunteers were excluded if any test results were <75% of their age, height, weight and race-based prediction.  3.3.3 Carotid Intima-Media Thickness Ultrasound image of the CCA wall produces two echogenic lines: the lumen-intima interface and the media-adventitia interface. Combined, the thickness of the intimal and medial layers of the CCA wall constitute the CIMT. Common carotid artery intima-media thickness greater than or equal to 75th percentile for the individual’s age, sex, and race/ethnicity is indicative of increased cardiovascular disease risk (Stein et al., 2008). After laying supine for a minimum of 20 minutes, a longitudinal image of the right CCA was optimized with the carotid bifurcation in view (ML6-15-D probe, Vivid E9, GE, Fairfield, CT, USA). With clearly defined double-lined CCA walls, 5-beat cine loops were saved in duplicate. Analysis was performed using automated caliper within EchoPAC v.13 (GE, Fairfield, CT, USA). Common carotid artery intima-media thickness was calculated based on automatic contour  42 detection of the double-wall layers on a user-defined search region along the vessel wall in the distal 1 cm of the CCA during end-diastole. Multiple CIMT measurements were made between pairs of intima and adventitia points along the wall. Posterior CIMT was recorded. A single optimal angle of incidence, always in the anterior plane, was used to assess CIMT. Volunteers were excluded if they had a posterior CIMT of >0.833 mm (75th percentile for 25-year-old Caucasian men) (Tzou et al., 2007).   3.3.4 Nocturnal Pulse Oximetry Once completing the screening session, volunteers were sent home with a wrist-worn pulse oximeter (WristOx2™, Model 3150, Nonin, Plymouth, MN, USA) and instructed to wear the pulse oximeter that night while they sleep. This data was used to exclude volunteers with nocturnal hypoxemia to ensure we did not study volunteers who might be naturally exposed to IH. A sleep log was kept to indicate the time and quality of sleep (see Diet and Sleep Log, page 134). Measures of peripheral oxyhemoglobin saturation (SpO2) were recorded at a sample rate of 1/second. Data analysis for ODI (desaturations of >4% per hour of sleep), mean and mean minimum SpO2, and percent of time <90% and <85% SpO2 was performed using nVision Software (Nonin, Plymouth, Minnesota, MN, USA). Volunteers were excluded if they displayed an ODI of ≥5 events per hour.   Experimental Protocols 3.4All volunteers who met the inclusion and exclusion criteria (n=10) (herein referred to as participants) were invited to visit the Cardiopulmonary Laboratory of Experimental and Applied Physiology on two occasions separated by one week. Participants arrived at 07:15 after abstaining from exercise, alcohol, caffeine, supplements and antioxidants (i.e., vitamin C) for 12 hours. Given the duration of the visit, participants were permitted to have a light breakfast before arrival. Participants were instructed to void their bladders before the start of testing and were then outfitted with an ambulatory blood pressure monitor (cardio(x)plore, Meditech, Budapest, Hungary) (see Ambulatory Blood Pressure Monitor, page 46). The experimental protocol is illustrated in Figure 3.1 (page 45). Participants lay supine for 20 minutes, allowing blood volume to distribute and settle evenly before beginning measurements (Thijssen et al., 2011a). Reactive hyperemia and FMD were conducted on the  43 left BA to measure micro and macrovascular endothelium-dependent vasodilation (see Reactive Hyperemia Flow-Mediated Dilation, page 46). After completion of RH and FMD, participants remained supine and were instrumented with a sealed face mask (Ultra Mirage Full Face Mask, ResMed, San Diego, CA, USA). Attached to the face mask were sample lines for mouth pressure (Pneumotach Amplifier 1 Series 1110, Hans Rudolph, Shawnee, KS, USA), O2 (Oxygen Analyzer S-3A-I, AEI Technologies, Pittsburgh, PA, USA) and CO2 (Carbon Dioxide Analyzer CD-3A, AEI Technologies, Pittsburgh, PA, USA). Mouth pressure was measured to distinguish between inhalation (negative pressure) and exhalation (positive pressure). Measures of O2 and CO2 allowed continual assessment of PO2 and PCO2. Two pulse oximeters were worn to record and measure SpO2. One was wrist-worn (WristOx2™, Model 3150, Nonin) and the other (PureSAT, Nonin, Plymouth, Minnesota, USA) was directly connected to Power Lab (16/35, ADInstruments, Colorado Springs, CO, USA) for live display (Oximeter Pod, ML320, ADInstruments, Colorado Springs, CO, USA). Following instrumentation, the participant was instructed to perform two brief (~5 seconds) end-inspiratory apneas to determine the sample delay from the facemask to the gas analyzers. Participants breathed freely for at least 5 minutes or until stable PETCO2 values were achieved. After establishing baseline PETCO2 and time delays, the facemask was connected via tubing to an automatic controller for a Sliding-TypeTM Pneumatic Directional three-way Valve (4285 Series, Hans Rudolph, Shawnee, KS, USA). Using custom-built software (LabVIEW 2013, National Instruments, Austin, TX), the valve switched every minute for the next six hours. On the SHAM day, the valve alternated between two room air sources, while on the IH day, the valve cycled between room air and hypoxia (HYP-123, Hypoxico, New York, NY, USA) (see Intermittent Hypoxia, page 45). After 10 minutes of breathing with the valve alternating on minute cycles, lights were dimmed and two dimensional speckle tracking ultrasound was performed on the CCA and CFA (see Strain Measurements, page 49). This allowed measurement of strain, the deformation of an object normalized to its original shape and size, and strain rate, the rate of deformation. Overall, CCA strain is a surrogate measurement of arterial stiffness (Bjallmark et al., 2010) and CFA strain feasibility and reliability has recently been confirmed (Charwat-Resl et al., 2015). Immediately following strain measurements, 4 minutes each of BA and CFA hemodynamics were recorded using simultaneous B-mode and Doppler ultrasonography (see Hemodynamic  44 Measurements, page 49). Hemodynamics were recorded to evaluate upper and lower limb blood flow, diameter and shear. Common carotid artery strain, CFA strain, BA blood flow and CFA blood flow were recorded over four-minute intervals each (2 minutes normoxia [NX] and 2 minutes hypoxia [HX]), always in that order. Once the ultrasound measurements were complete, the participant was assisted into a seated position at a desk where they were permitted to work or read at a computer so long as they remained seated, awake, and the facemask was sealed. The participants were permitted to have a brief snack and washroom break if needed. At the 5-hour mark, participants returned to the supine position. After 20 minutes of laying supine, the sequence of CCA strain, CFA strain, BA blood flow and CFA blood flow measurements were made similar to morning measurements. Following ultrasound measurements, the participants remained supine until the 6-hour SHAM or IH protocol had completely elapsed. At 6 hours, the automatic valve switch was turned off and tubing disconnected, but the facemask remained until PETCO2 and SpO2 became steady (~5 minutes). The facemask and pulse oximeters were then removed, and a RH FMD was performed. Following the RH FMD, the participant was permitted to use the washroom before returning and commencing the carotid baroreflex control of MAP, HR and LVC assessment using the neck chamber protocol (see Baroreflex Protocol, page 50). After the neck chamber protocol, participants were sent home, still wearing the ambulatory blood pressure monitor. Systolic and diastolic arterial blood pressure were measured at 15-minute intervals during the day (07:30-22:00) and every half hour throughout the night (22:00-06:00) before being returned the following day. The participant returned one week later to undergo the alternate condition (i.e., SHAM or IH). The order was pseudo-randomized, ensuring that half of the participants had SHAM on their first visit (n=5) and the other half started with IH (n=5).   45  Figure 3.1: Schematic representation of the experimental protocol. Participants lay supine from 07:30-09:00, seated from 09:00-13:30 and supine once again for the remainder of the visit. Each participant performed the entire protocol twice, once breathing only room air (SHAM) and once alternating between one minute of room air and one minute hypoxia (intermittent hypoxia; IH). Arrows denote where vascular strain measurements of the common carotid and common femoral artery and flow measurements of brachial and common femoral artery were made.   Experimental Techniques 3.5Specific techniques and measurements are described in the following section. Explanation of the data analysis and statistical approach for each measurement follows description of the technique.  3.5.1 Intermittent Hypoxia All respiratory parameters were acquired at 200 Hz using an analog-to-digital converter (Powerlab/16SP ML 880, ADInstruments) interfaced with a personal computer. Lab acquisition software was used to collect and analyze ventilatory and cardiovascular variables (LabChart V7.1, ADInstruments, Colorado Springs, CO, USA). Using an automatic switch valve, participants cycled between one minute of hypoxia and one minute of room air for six hours during the IH condition, with the aim of achieving an ODI (defined as number of desaturations ≥4% per hour) of 30, equivalent to severe OSA. The level of hypoxia delivered was not constant, rather a desaturation of 80% SpO2 was targeted each cycle, in accordance with previous investigations (Foster et al., 2010). All participants were exposed to the minimum O2 possible from the hypoxic generator (HYP-123, Hypoxico), and when that stimulus was insufficient to drop SpO2 to the desired level, 100% nitrogen was titrated and  46 triggered to enter the inspiratory tubing only during inspiration (negative mouth pressure) by a custom-built software (LabVIEW 2013, National Instruments). The software permitted not only controlled titration of nitrogen, but also CO2. Hypoxia causes hyperventilation, in turn causing hypocapnia. To avoid any hypocapnic influence, CO2 was added to the inspiratory tubing during inspiration in attempt to avoid fluctuations in PETCO2. Carbon dioxide was usually administered from ~25 seconds following the start of the hypoxic cycle and delivered with each inspiration for close to one minute, though modifications were made within and between participants based on PETCO2. Thus, IH was a purely hypoxic stimulus with fluctuations in PETCO2 minimized.  The PETCO2 was analyzed on a breath-by-breath basis throughout the 6-hour exposure in LabChart (V7.1, ADInstruments) by acquiring the PCO2 at end-expiration (the point at which mouth pressure changes from positive to negative). The SpO2 was measured similarly to the nocturnal pulse oximetry assessment (see Nocturnal Pulse Oximetry, page 42), ODI, mean and mean minimum SpO2, time spent <90% and <85% SpO2 were acquired from nVision software (Nonin).   3.5.2 Ambulatory Blood Pressure Monitor An ambulatory blood pressure cuff (cardio(x)plore, Meditech) was secured firmly around the right upper arm. The first recording was made at 7:30, and cuff inflation repeated every 15 minutes. Measurement interval was lengthened to once per half hour from 22:00-06:00. Each measurement included SBP, DBP and HR. Mean arterial pressure was calculated as [(2 x DBP) + SBP] / 3. Pulse pressure (PP) was calculated as the difference between SBP and DBP. The cuff was removed during the baroreflex assessment. After leaving the laboratory, participants were instructed to assume a seated position during cuff inflations. An activity log was assigned to ensure similar activity profiles between days and to notify the investigators of any periods where blood pressure measurements may be affected (see Activity Log, page 135). Recordings ceased 24-hours after cuff instrumentation.   47 Data were extracted from Cardiovisions software (Meditech). Blood pressure measurements were sub grouped into daytime (07:00-22:00) and nighttime (22:00-07:00) (Tamisier et al., 2011).   3.5.3 Reactive Hyperemia Flow-Mediated Dilation Brachial artery RH FMD, a measure of endothelium-dependent vasodilation, was conducted according to the internationally recognized guidelines (Thijssen et al., 2011a). The measure was conducted in normoxia twice per condition (SHAM and IH), immediately prior to facemask outfitting (AM) and immediately following the 6-hour exposure (PM). In each scenario, the participant had been laying supine for a minimum of 20 minutes and all external stimuli were reduced (lights dimmed, noise kept to a minimum). Assessment was coordinated so it did not coincide with an ambulatory blood pressure cuff measurement. To start, the participant extended their left arm to an angle of ~80⁰ from their torso. An arm tourniquet cuff (SC5, Hokanson, Bellevue, WA, USA) connected to an aneroid sphygmomanometer (DS400, Hokanson, Bellevue, WA, USA) was secured distal to the epicondyles. The forearm was rested on a form-fitting pillow (Versa Form, Patterson Medical, Mississauga, ON, Canada) that was molded to restrain movement using a vacuum pump (Versa Form Vacuum Pump, Patterson Medical, Mississauga, ON, Canada). Image acquisition was obtained using a 10 MHz multifrequency linear array probe (15L4, Terason t3200, Teratech, Burlington, MA, USA) attached to a high-resolution ultrasound machine (Terason t3200, Teratech, Burlington, MA, USA). The probe was placed in the distal third of the upper arm and simultaneous B-mode and Doppler ultrasound were recorded to obtain diameter and blood velocity measurements. The left BA was imaged in the distal third of the upper arm, proximal to the antecubital fossa. The image was obtained in the longitudinal plane and optimized to achieve clear vascular boundaries. Once image quality was optimized, screen recording (Camtasia Studio 8, TechSmith, Okemos, MI, USA) captured at least one minute of baseline images. The insonation angle was set to 60⁰ and the vessel image adjusted manually to accommodate. The Doppler sampler gate was extended to the walls of the vessel without ever touching them (~80-90% of luminal cross-sectional area).  The forearm cuff was then rapidly inflated to suprasystolic levels (220-250 mmHg) and remained inflated for 5 minutes. Screen capture was paused from the onset of cuff inflation until 30 seconds prior to  48 deflation. The cuff was rapidly deflated after 5 minutes, and simultaneous B-mode and Doppler ultrasound recorded 3 minutes post-deflation (Black et al., 2008). The screen capture was saved as an audio video interleave file for future analysis using an edge-detection software (Woodman et al., 2001). A region of interest (ROI) was placed around the highest quality portion of the B-mode cross-sectional image of the artery. A second ROI surrounded the Doppler strip to record blood velocity. Blood flow was automatically calculated as the product of cross-sectional area and Doppler velocity, and shear rate was calculated as 4 times the velocity divided by the diameter. The software automatically tracks the walls of the vessel and velocity trace within the ROIs and has a mean intraobserver coefficient of variation (COV) of 6.7% for BA FMD (Woodman et al., 2001). FMD was determined automatically by the software’s algorithm as the peak diameter following cuff release. Reactive hyperemia was assessed as the peak and total blood flow responses after cuff release. The sonographer had a root mean square (RMS) COV for within-participant between-day FMD measurements of 10.4% (n=6, Tremblay, JC, see Table C.2, page 179).    FMD is a measure of macrovascular reactivity and is expressed as an absolute and percent increase in diameter from baseline to peak vasodilation. Flow-mediated dilation was normalized for the total shear rate response (SRAUC) until peak diameter response (Pyke & Tschakovsky, 2007; Padilla et al., 2008, 2009a) and allometrically scaled for baseline diameter (Atkinson & Batterham, 2013). Baseline diameter, peak diameter, SRAUC and time to peak response were also reported.   Reactive hyperemia is a measure of microvascular reactivity and is expressed as peak response (peak blood flow) and total blood flow (blood flow AUC) response until diameter plateaued or returned to baseline following cuff release. To ensure the RH changes persisted independent of diameter, the velocity envelope, which is obtained directly from tracing of the Doppler strip, was assessed. The velocity envelope offers a surrogate of true mean velocity measures (Harris et al., 2010). The total shear rate response (SRAUC) from cuff release to diameter plateau or return to baseline was also measured.    3.5.4 Vascular Ultrasound  49 All ultrasound measurements were made after at least 20 minutes of laying supine. After 10 minutes of SHAM or IH and again after 5 hours and 20 minutes, a series of measurements were made in the following order: CCA strain, CFA strain, BA blood flow and CFA blood flow. Each were recorded over four minutes, so that during IH two measurements were made during room air breathing (NX) and two during hypoxic breathing (HX). All ultrasound measurements were made by the same sonographer (Tremblay, JC).   3.5.4.1 Strain Measurements Strain measurements were obtained using a ML-16S MHz high frequency linear array transducer connected to a high-resolution ultrasound machine (Vivid E9, GE). Cross-sectional images of the right CCA and CFA were optimized for clear identification of the double-lined wall (lumen-intima interface and media-adventitia interface), when possible completely encompassing the lumen. Both arteries were imaged approximately 2 cm proximal to their respective bifurcations, similar to previous investigations (Oishi et al., 2013; Charwat-Resl et al., 2015). The skin was marked for probe placement of the CFA. Strain measurements were recorded as the 3 cardiac cycles preceding the valve switch to either HX or NX.  Using the 2D strain package in the EchoPAC (GE) software, circumferential strain and strain rate were obtained by placing a ROI that encompasses the cross-sectional area of the vessel wall. The width was reduced to a minimum before manually adjusting calipers of six distinct segments to avoid the mis-tracking of extravascular tissue. Adequate tracking was verified and adjusted when required. The software detects frame-to-frame movement of the natural ultrasound reflections (speckles) on standard ultrasonic images in two dimensions based on the block-matching and autocorrelation search algorithms (Leitman et al., 2004). In accordance with Bjallmark et al. (2010), circumferential peak systolic strain, early systolic strain rate and late systolic strain rate were obtained. Peak systolic strain (%) represents the increase in vessel diameter during systole. Early systolic strain rate (1/s) occurs after the QRS-complex and is the result of the blood pressure rise associated with ventricular ejection, and late systolic strain rate (1/s) is the trough that occurs after the T-wave and occurs during  50 ventricular relaxation. Each of these parameters were obtained for a single cardiac cycle, and the average of 3 cardiac cycles represent the value for a given condition (SHAM, HX and NX) and time of day (AM and PM).  3.5.4.2 Hemodynamic Measurements Hemodynamic measurements were obtained using a 10 MHz multifrequency linear array probe (15L4, Terason t3200, Teratech) attached to a high-resolution ultrasound machine (Terason t3200, Teratech). The left BA was imaged in the same location as the FMD was performed. Common femoral artery flow measurements were made distal to the inguinal crease, ~2 cm proximal to its bifurcation (superficial and deep femoral arteries). To ensure proper probe placement, the bifurcation was first visualized during CFA flow measurements. Both arteries were measured in the longitudinal plane and optimized to achieve clear vessel boundaries. Simultaneous B-mode and Doppler ultrasound were performed and continuously recorded using screen capture software (Camtasia Studio 8, TechSmith) for four minutes (starting at the audible valve switch). The insonation angle was set to 60⁰ for each measurement and the vessel image adjusted manually to accommodate. The Doppler sample gate was extended to the walls of the vessel without ever touching them (~80-90% of luminal cross-sectional area). The location of probe placement was marked on the skin and was the same as that used for FMD measurements (BA) and baroreflex assessment (CFA).  To assess the hemodynamic and shear patterns, measures of diameter, flow, shear rate and oscillatory shear index (OSI) were acquired. The OSI was calculated as |retrograde shear| / (|antegrade shear| + |retrograde shear|), where an OSI of 0.5 indicates equal amounts of antegrade and retrograde shear. Data was excluded for 2 participants for lower limb hemodynamic measurements due to acquisition error, therefore analysis is based on n=8.  3.5.5 Baroreflex Protocol The baroreflex plays a critical role in acute and chronic arterial blood pressure regulation (Lohmeier & Iliescu, 2015). We assessed the carotid baroreflex by loading and unloading (simulating hyper- and hypotension) the baroreceptors located at the carotid sinus. A malleable lead neck chamber formed from an elliptical piece of sheet lead and rimmed with  51 sponge rubber was built based on the model described by Eckberg et al. (1975) (see Figure 3.2, page 52). The chamber was connected to a programmable pressure controller (PPC-1000, Engineering Development Laboratory, Newport News, VA, USA) (Pellinger & Halliwill, 2007). The pressure was generated by stepping motor driven bellows, as recommended by Sprenkle et al. (1986), and commands delivered by a PC-based software (LabChart V7.1, ADInstruments) set to trigger immediately after an R-wave. Pressure was recorded from within the neck chamber (Pneumotach Amplifier 1 Series 1110, Hans Rudolph).  Before outfitting the participants with the neck chamber, continuous beat-by-beat arterial blood pressure by finger photoplethysmography (Finometer Pro, Finapres Medical Systems, Biomedical Instruments, The Netherlands) and 3-lead ECG (ML132, ADInstruments, Colorado Springs CO, USA) were recorded. Reconstruction of BA pressure from the finger photoplethysmography was performed using a return-to-flow calibration (Bos et al., 1996). After return-to-flow calibration, the Physiocal procedure checks the blood pressure setpoint, adjusting it if necessary, at approximately 1 minute intervals (Imholz et al., 1998). These adjustments and recordings provide reliable measurements for beat-to-beat changes in arterial blood pressure, but do not perfectly match BA arterial blood pressure (Imholz et al., 1998). For this reason, automatic arterial blood pressure measurements were recorded on the opposite arm in triplicate to permit back-calibration (HEM-775CAN, Omron Healthcare).   A removable foam cut-out was added to the inner border of the chamber before placement and molding on the participant’s neck. The neck chamber covered the anterior two-thirds of the neck with the seal extending superiorly along the body of the mandible to the rami and inferiorly to the clavicles and sternum. To further reinforce the seal, a tensor bandage surrounded the chamber and posterior neck.   Once the neck chamber was sealed, simultaneous B-mode and Doppler ultrasound of the CFA was performed and recorded (Camtasia Studio 8, TechSmith) continuously for the duration of the baroreflex assessment. After a minimum of 5 minutes of baseline recording, Physiocal was turned off and the series of neck suctions and pressures pulses were delivered.  52 Physiocal temporarily halts arterial blood pressure recording, which would prove to be detrimental when assessing beat-by-beat changes with carotid baroreflex stimulation. A random ordered sequence of three consecutive suctions (-80, -60, -40 and -20 mmHg) followed by random order of three consecutive pressures (+40 and +20 mmHg) were executed. Each stimulus was triggered by an R-wave and was held for 5 seconds. A minimum of 45 seconds separated each stimulus to allow MAP and HR to return to baseline values. After each step (i.e., 3 x -80 mmHg), an automatic arterial blood pressure measurement was recorded, to permit back-calibration for BA arterial blood pressure (HEM-775CAN, Omron Healthcare), and Physiocal was reactivated. Participants were coached to maintain a steady respiratory rate and avoid large gasps or breath holds from 10 seconds before until 10 seconds following neck pressure/suction. To prevent leakage from the neck chamber, an investigator applied pressure to the borders of the chamber 10 seconds before stimulus delivery and without evoking any changes in MAP or HR.   Figure 3.2: The neck chamber utilized to apply neck suction and pressure, permitting carotid baroreflex assessment. The port annotated with ‘1’ was connected to a programmable pressure controller and the port identified by ‘2’ sampled pressure within the chamber.  The peak responses in MAP and HR were determined as the greatest change across 2-4 cardiac cycles from the 5 cardiac cycles preceding the stimulus. Leg blood flow (LBF)  53 measurements were taken from the same period as the peak MAP responses (typically 6-8 seconds post-stimulus) (Raven et al., 1997; Ogoh et al., 2002). Leg vascular conductance (LVC) was calculated as LBF/MAP. The transmural carotid sinus pressure was estimated (ECSP) as the pressure within chamber during the 5-second pulse subtracted from the prestimulus MAP. The prestimulus MAP was the average beat-to-beat MAP recordings from the 5 cardiac cycles preceding pulse delivery. Four-parameter logistic curves of MAP versus ECSP and HR versus ECSP were fitted in SigmaPlot 11 (Systat Software, San Jose, CA, USA) as first described by Kent et al. (1972):  Equation 1:              (            (    ) )  Where x is the response variable, HR or MAP, the minimum is the minimum HR or MAP response, range is the maximum HR or MAP response – minimum HR or MAP response, G is the gain coefficient, y is the ECSP, and CP is the centering point, or the point of inflection. The maximum response was calculated by adding the minimum response to the range. Maximal gain (Gmax) and gain about the operating point (Gop) are indexes of baroreflex sensitivity and were calculated as in Ogoh et al. (2005):  Equation 2:                    Equation 3:                                 {              ]}]   The threshold and saturation points were calculated as -2.944/G+CP and 2.944/G+CP respectively (McDowall & Dampney, 2006) and represent the carotid sinus pressure at which MAP or HR is within 5% of the upper or lower plateau of the sigmoid function. To clarify, the minimum and maximum points represent the HR or MAP responses and the threshold and saturation represent the ECSP within 5% of the upper and lower plateaus.   In total, parameters recorded include minimum, maximum, range, G, CP, Gmax, Gop, threshold and saturation. These provide measurements of the carotid baroreflex setpoint and  54 sensitivity. Each parameter was obtained from absolute MAP and HR and changes in MAP and HR from prestimulus baseline. Modelling parameters were not always physiologically acceptable and data points were excluded if they fell outside of 2 standard deviations of the mean. As a result, modelling was successful in a subset of participants. Absolute MAP modelled well in 5 participants, change in MAP from prestimulus MAP in 7 participants, absolute HR in 6 participants and the change in HR from prestimulus HR was successful in 5 participants. Absolute and changes in MAP and HR were compared between SHAM and IH at each level of pressure and suction in all participants (n=10).  Leg blood flow and LVC are not modelled well using the aforementioned Kent equation (Keller et al., 2004). Both absolute and percent changes in LBF and LVC are presented. The absolute change in LVC represents the relative contribution of the leg vascular bed to the total vascular system whereas the percent changes in LVC represent an index of vascular responsiveness (vasoconstriction or vasodilation) (Buckwalter et al., 2001).    Sample Size Justification 3.6The sample size of 10 participants was based on changes in MAP in response to IH from previously published data (Foster et al., 2010). The mean difference was 7.9 mmHg and standard deviation was 6.3 mmHg, and 10 participants were required to provide sufficiently high statistical power (80%) for two-sided tests with an alpha of 0.05.    Statistics 3.7Statistical analysis was performed in SigmaPlot 11 (Systat Software). All data are compared within-participants and significance was set at P<0.05. When equal variance tests failed, data were log-transformed. When significant F-ratios were detected, all post hoc comparisons were made using Tukey’s HSD. Data are presented in the text as means ± standard error of the mean (SEM), and exact P-values are cited.    The PETCO2, ODI, mean and mean minimum SpO2 and time spent <90% and <85% SpO2 were compared within-participants between SHAM and IH using a one-way repeated measures analysis of variance (ANOVA). Daytime, nighttime and 24-hour SBP, DBP, MAP,  55 PP and HR were compared within-participants between SHAM and IH using one-way repeated measures ANOVA. A 2x2 within-participant repeated measures ANOVA compared FMD and RH results between time of day (AM vs PM) and condition (SHAM vs IH). Linear regressions and Pearson correlation coefficients were determined within-participants for the relationship between SRAUC and FMD (%). Statistically, normalization for SRAUC should only occur if SRAUC and FMD are at least moderately correlated (Atkinson et al., 2009). Vascular strain parameters for the CCA and CFA were compared between AM and PM and SHAM, HX and NX by 2x3 repeated measure ANOVA. Similar to vascular strain, the flow and shear parameters were compared using 2x3 within-participant repeated measures ANOVA (time of day [AM vs PM] and condition [SHAM vs HX vs NX]).  Baroreflex modelling parameters were compared using within-participant, one-way repeated measures ANOVA (SHAM and IH). The absolute and change in MAP and HR and percent and absolute changes in LBF and LVC evoked by the carotid baroreflex were compared at each targeted pressure level (-80, -60, -40, -20, 0, 20, 40 mmHg) by within-participant, two-way repeated measures ANOVA (SHAM vs IH x pressure level).     56 4    Chapter: Results  Participants 4.1Participant characteristics and pulmonary function data are presented in Table 4.1 (page 57). Ten healthy, normotensive men volunteered to participate and met the inclusion/exclusion criteria. Screening measurements of MAP and PP were 84.1±1.7 mmHg and 57.7±1.8 mmHg. Participants had normal CIMT, and the intraobserver RMS COV for CIMT was 4.72% (n=8, Tremblay, JC, see Table C.1, page 178). No participant had a BMI >30 kg/m2. The participants did not display excessive daytime somnolence or nocturnal hypoxemia, as assessed by ESS, ODI and mean nocturnal SpO2, excluding the possibility of OSA. All participants had normal lung function and volumes exceeding their age, race and body size predicted values.     57 Table 4.1: Participant characteristics and pulmonary screening data. Baseline Characteristics Mean ± SEM Pulmonary Function Testing Mean ± SEM % Predicted ± SEM Age (years) 26.3±1.3 FVC (l) 6.1±0.2 114±3.3 Height (cm) 176.9±0.9 FEV1 (l) 4.7±0.2 106±3.9 Body Mass (kg) 76.5±3.0 FEV1/FVC  78±1.1  BMI (kg/m2) 24.4±0.9 TLC (l) 7.2±0.1 106±2.2 ESS Score 5.8±1.0 DLCO (ml/min/mmHg) 37.6±1.6 105±5.2 SBP (mmHg) 123±2.5 DBP (mmHg) 65±1.6 DLCO/VA (ml/min/mmHg/l) 5.3±0.1 103±3.5 ODI (events/hour) 1.5±0.4 Mean Nocturnal SpO2 (%) 95.3±0.4    CIMT (mm) 0.50±0.03    Definition of abbreviations: BMI, body mass index; CIMT, common carotid artery intima-media thickness; DBP, diastolic blood pressure; DLCO, diffusion capacity of the lung for carbon monoxide transfer; DLCO/VA, DLCO corrected for alveolar volume; ESS, Epworth Sleepiness Scale; FEV1, forced expiratory volume in one second; FVC, forced vital capacity; ODI, oxygen desaturation index; SBP, systolic blood pressure; SpO2, peripheral oxyhemoglobin saturation; TLC, total lung capacity.      58  Intermittent Hypoxia and SHAM Exposures 4.2Oxygen saturation characteristics are presented in Table 4.2 (page 59). The IH exposure was meant to simulate severe OSA and effectively produced an ODI of 29.9±0.5 events per hour. Mean nadir SpO2 was 80.7±0.04% and participants spent 44.1±1.9% of time below 90% SpO2 and 22.0±1.6% of time below an SpO2 of 85% during the IH condition. The SHAM condition, in comparison, did not produce clinically relevant hypoxemia. A representative SpO2 recording from an individual participant’s SHAM and IH conditions is displayed in Figure 4.1 (page 58). The PETCO2 was controlled during IH, but some participants became mildly hypocapnic relative to the free-breathing PETCO2 observed in SHAM. The mean breath-by-breath PETCO2 throughout the entire 6-hour exposure was 34.0±0.8 mmHg during IH compared to 35.4±0.6 mmHg during SHAM (P=0.027).   Figure 4.1: Oxygen saturation profile of an individual participant during a 6-hour SHAM (panel A) and intermittent hypoxia (panel B) trial. Definition of abbreviations: SpO2, peripheral oxyhemoglobin saturation.   59 Table 4.2: Oxygen desaturation characteristics during SHAM and IH exposure.  SHAM IH P-value Mean SpO2, % 96.7±0.4 92.0±0.4 <0.001 Mean Minimum SpO2, % N/A 80.7±0.4 N/A % of Time <90% SpO2 0.06±0.05 44.1±1.9 <0.001 % of Time <85% SpO2 0.01±0.01 22.0±1.6 <0.001 ODI, events/hour 1.6±0.5 29.9±0.5 <0.001 Data are presented as mean ± SEM. Definition of abbreviations: IH, intermittent hypoxia; ODI, oxygen desaturation index; SpO2, peripheral oxyhemoglobin saturation.     60  24-hour Ambulatory Monitoring 4.324-hour ambulatory blood pressure monitoring data is presented in (Table 4.3).  Intermittent hypoxia led to a significant increase in 24-hour and daytime MAP, SBP, and DBP.  While DBP and MAP did not remain elevated during the night, there was a trend for SBP to remain elevated (3.9±1.8 mmHg) during the sleeping hours (P=0.053). Pulse pressure was unaltered in daytime and 24-hour timeframes, but was greater during the nighttime hours following IH (P=0.036). 24-hour HR was similar between conditions (SHAM: 56.8±2.3; IH: 57.7±2.2 beats per minute, P=0.47).     61 Table 4.3: Time of day and 24-hour blood pressures.  SHAM IH Difference P-value Daytime SBP, mmHg 120.7±1.9 123.7±1.5 3.0±0.9 0.007 Daytime DBP, mmHg 65.9±1.3 68.6±1.6 2.7±1.1 0.036 Daytime MAP, mmHg 84.2±1.1 87.0±1.2 2.8±0.8 0.009 Daytime PP, mmHg 54.8±2.2 55.1±1.9 0.3±1.2 0.840 Nighttime SBP, mmHg 108.5±2.2 112.4±2.2 3.9±1.8 0.053 Nighttime DBP, mmHg 55.7±1.5 56.4±1.1 0.7±1.3 0.630 Nighttime MAP, mmHg 73.3±1.5 75.0±1.1 1.8±1.4 0.233 Nighttime PP, mmHg 52.8±1.9 56.0±2.4 3.2±1.3 0.036 24-hour SBP, mmHg 117.4±1.9 120.7±1.5 3.4±1.0 0.007 24-hour DBP, mmHg 63.1±1.2 65.4±1.3 2.2±0.9 0.034 24-hour MAP, mmHg 81.2±1.1 83.8±1.0 2.6±0.8 0.008a 24-hour PP, mmHg 54.2±2.1 55.4±2.0 1.1±1.1 0.322 Daytime hours were defined as 7:00-22:00 and nighttime hours as 22:00-7:00. The difference was calculated by subtracting SHAM from IH. Data are presented as mean ± SEM. Definition of Abbreviations: DBP, diastolic blood pressure; IH, intermittent hypoxia; MAP, mean arterial pressure; PP, pulse pressure; SBP, systolic blood pressure. afailed equal variance test, P-value was corrected using log-transformed 24-hour MAP values.       62  Flow-Mediated Dilation 4.4Brachial artery FMD measurements before and after SHAM and IH are presented in Table 4.4 (page 63). Morning FMD measurements were similar and reproducible between days. The RMS COV for baseline FMD between SHAM and IH was 12.9±7.9%. Flow-mediated dilation displayed a similar decrease from AM to PM in both conditions (see Figure 4.2, page 64). This was a consistent finding, with 7 of 10 participants demonstrating a blunted FMD following IH and 9 of 10 participants following SHAM. Baseline diameters were 3.3±1.2% larger during IH than SHAM (P=0.048). Correcting FMD for baseline diameter via allometric scaling still produced a reduction from AM to PM (P=0.031) without an effect of IH (see Table 4.4, page 63). The SRAUC (P=0.001) and time to peak (P=0.014) were reduced from AM to PM. When normalized for SRAUC, FMD increased from AM to PM (P=0.008) to a similar extent following SHAM and IH. Within-participant relationships between FMD and SRAUC are presented in Figure 4.3 (page 65) and have an overall correlation coefficient of 0.72 (r2=0.51, P<0.001), suggesting a very strong positive relationship.   Forearm microvascular reactivity data are presented in Table 4.5 (page 66). The peak blood flow, total blood flow (AUC), total shear rate (AUC) and velocity envelope across the complete duration of cuff release were less in the PM compared to AM and were unaffected by IH. Both peak RH (peak blood flow response) and total RH (blood flow AUC) were reduced from AM to PM (P<0.001 and P=0.004) (see Figure 4.4, page 67). Following IH, the peak blood flow response was reduced by 18.8±7.3% and the total blood flow response by 32.8±17.6%. The changes were similar following SHAM, with reductions of 24.4±4.5% and 33.1±8.5% for peak and total blood flow responses, respectively. To ensure the changes persisted independent of diameter, the velocity envelope was assessed and demonstrated a similar diurnal decrease (P<0.001), as did the total shear rate response (P=0.003).      63 Table 4.4: Effect of IH on flow-mediated dilation responses to reactive hyperemia.   Condition P-value  Time SHAM IH Time Condition Interaction Allometrically Scaled FMD, % AM 7.20±0.58 7.06±0.44 0.031 0.719 0.380 PM 5.90±0.57 6.28±0.54 Baseline Diameter, cm AM 0.42±0.01 0.43±0.02 0.316 0.048 0.885 PM 0.42±0.01 0.43±0.01 Peak Response Diameter, cm AM 0.45±0.01 0.47±0.01 0.041 0.053 0.890 PM 0.44±0.02 0.46±0.01 FMD, cm AM 0.033±0.002 0.033±0.002 0.020 0.356 0.467 PM 0.027±0.002 0.029±0.002 SRAUC, au AM 31466±4383 29448±3304 0.001 0.572 0.921 PM 19965±2965 18413±2053 FMD/SRAUC, (103[%/au]) AM 0.27±0.02 0.28±0.02 0.008 0.496 0.754 PM 0.36±0.04 0.39±0.03 Time to Peak, s  AM 60.1±3.8 53.5±6.0 0.014 0.775 0.141 PM 41.6±3.2 45.5±4.4 All measures were recorded from the point of peak brachial artery diameter response.  Data are presented as mean ± SEM. Definition of Abbreviations: FMD, flow-mediated dilation; IH, intermittent hypoxia; SRAUC, shear rate area under the curve.  64  Figure 4.2: Individual and mean flow-mediated dilation responses before (AM) and after (PM) six hours of SHAM and intermittent hypoxia (IH). Mean data are displayed ± SEM. The P-value denotes the time of day (AM vs PM) effect. Definition of abbreviations: FMD, flow-mediated dilation.  65  Figure 4.3: Relationship between the total shear rate response and flow-mediated dilation. Within-participant individual correlations between shear rate area under the curve (SRAUC) and percent flow-mediated dilation (FMD) for each measurement condition. Linear regression tests were performed for the 4 FMD measurements made on each participant (AM and PM, SHAM and intermittent hypoxia). Each participant is represented by a different symbol. The thick black line represents the overall relationship for all subjects (r2=0.51, P<0.001).  66 Table 4.5: Effect of IH on brachial artery microvascular reactivity following cuff release.   Condition P-value  Time SHAM IH Time Condition Interaction Velocity envelope, cm/min AM 2133±132 2347±196 <0.001 0.606 0.412 PM 1505±168 1466±123 Blood flow AUC, au AM 36791±2709 43812±4852 0.004 0.363 0.326 PM 24974±3224 24642±3159 SRAUC, au AM 40295±3732 42562±3616 0.003 0.841 0.424 PM 29861±5018 26373±3569 Peak blood flow response, ml/min AM 812±48 849±72 <0.001 0.224 0.878 PM 607±46 659±48 All measures were recorded until diameter returned to baseline or ceased to decrease (plateau), with the exception of the peak blood flow response. Data are presented as mean ± SEM.  Definition of Abbreviations: AUC, area under the curve; IH, intermittent hypoxia; SRAUC, shear rate area under the curve.     67  Figure 4.4: Reactive hyperemic response before (AM) and after (PM) 6-hour exposure to SHAM and intermittent hypoxia (IH). The peak blood flow response was significantly blunted in the PM compared to AM (P<0.001) as was the total blood flow response (area under the curve, P=0.004). The single data points at time = 0 represent the baseline (pre-cuff inflation) blood flow measures.  Data points are mean ± SEM.    68  Vascular Strain 4.5Common carotid artery strain profiles are presented in Figure 4.5 (page 69). Common carotid artery peak systolic strain, early systolic strain rate and late systolic strain rate were reduced from AM to PM. Peak systolic CCA strain was reduced from AM to PM during IH (HX, P<0.001; NX, P=0.012), but no diurnal reduction during SHAM was observed (P=0.933). Common femoral artery strain profiles are presented in Figure 4.6 (page 70). No time of day effect was observed in CFA peak systolic strain (time of day P=0.840) or early systolic strain rate (time of day P=0.863), but both showed a trend towards lower values during IH (peak systolic strain, P=0.055; early systolic strain rate, P=0.080). Late systolic strain rate was similar during AM and PM and between SHAM, HX and NX (time of day P=0.197, condition P=0.903).    69  Figure 4.5: Common carotid artery (CCA) circumferential strain profiles across the cardiac cycle for all subjects during SHAM and IH in the morning (AM) and afternoon (PM). Panels A and B are CCA strain (%) measurements made in the AM and PM respectively. Peak systolic strains for each condition (hypoxic breathing, HX; room air breathing, NX; and SHAM) are represented by circles. An interaction effect between condition and time of day identified a reduction in peak systolic strain during HX and NX from AM to PM (P<0.001 and 0.012 respectively). Panels C and D are CCA strain rate (1/s) from AM and PM. Diamonds represent early systolic strain rate and triangles represent late systolic strain rate. A time of day effect showed a decrease in early systolic strain rate and increase in late systolic strain rate from AM to PM (P=0.015 and 0.014 respectively). Data are presented as mean ± SEM.    70  Figure 4.6: Common femoral artery (CFA) circumferential strain profiles across the cardiac cycle for all subjects during SHAM and IH in the morning (AM) and afternoon (PM). Panels A and B are CCA strain (%) measurements made in the AM and PM respectively. Peak systolic strains for each condition (hypoxic breathing, HX; room air breathing, NX; and SHAM) are represented by circles. A trend towards an effect of condition was observed for peak systolic strain (SHAM vs HX vs NX, P=0.055). Panels C and D are CCA strain rate (1/s) from AM and PM. Diamonds represent early systolic strain rate and triangles represent late systolic strain rate. A trend towards an effect of condition was observed for early systolic strain rate (SHAM vs HX vs NX, P=0.080). Data are presented as mean ± SEM.    71  Limb Hemodynamics 4.6Diameter, blood flow, shear rate and OSI were assessed in the BA and the CFA and compared between SHAM, NX and HX bouts across the day (AM and PM) (see Table 4.6, page 72, and Table 4.7, page 73). Two participants were removed from the CFA flow measurements due to poor image quality (n=8). No interaction effects were observed, however BA flow and shear rate were halved in the afternoon compared to morning measurements. Coinciding with the decrease in shear rate, OSI doubled. Unlike the BA, CFA did not display a time of day effect on flow, shear rate or OSI. Common femoral artery flow was reduced during the NX and HX cycles of IH (P=0.043 and 0.022) and CFA shear rate was reduced during the HX cycles of IH compared to SHAM (P=0.047). The CFA OSI was greater during the NX bouts of IH compared to SHAM (P=0.038). Both the BA and the CFA were more constricted in the afternoon (P=0.006 and 0.027 for BA and CFA diameter). No correlations between absolute or reductions in FMD and OSI or retrograde shear were observed.     72 Table 4.6: Effect of IH on upper and lower limb hemodynamics.   Condition P-value  Time  SHAM HX NX Time  Condition  Interaction  BA diameter, cm AM 0.43±0.01 0.44±0.01 0.44±0.01 0.006 0.098 0.939 PM 0.42±0.02 0.43±0.02 0.43±0.02 CFA diameter, cm AM 1.00±0.05 1.01±0.05 1.00±0.05 0.027 0.263 0.760 PM 0.97±0.05 1.00±0.05 1.00±0.05 BA flow, ml/min AM 38.5±7.0 40.0±7.0 42.6±8.3 0.006 0.375 0.944 PM 16.4±3.0 17.5±2.0 19.5±2.4 CFA flow, ml/min AM 205.7±27.9 159.8±19.3 162.4±12.3 0.069 0.016 SHAM vs HX, 0.022 SHAM vs NX, 0.043 0.308 PM 171.3±25.3 152.5±21.1 157.2±22.3 Data are presented as mean ± SEM. Definition of Abbreviations: BA, brachial artery; CFA, common femoral artery; HX, hypoxia; NX, normoxia; OSI, oscillatory shear index.          73  Table 4.7: Effect of IH on upper and lower limb shear rate.   Condition P-value  Time  SHAM HX NX Time  Condition  Interaction  BA shear rate, 1/s AM 110.6±20.6 101.0±13.4 107.1±15.9 0.002 0.541 0.710 PM 50.5±12.9 47.3±4.7 54.6±8.2 CFA shear rate, 1/s AM 45.7±7.4 33.1±4.2 35.7±5.0 0.244 0.042 SHAM vs HX, 0.047 0.956 PM 42.9±8.0 31.3±3.1 32.8±3.5 BA antegrade  shear rate, 1/s AM 119.7±19.4 108.9±13.1 115.9±15.2 0.003 0.447 0.356 PM 68.6±12.2 67.3±6.2 75.6±8.8 CFA antegrade shear rate, 1/s AM 64.4±7.4 46.8±4.3 50.7±5.1 0.567 0.012 SHAM vs HX, 0.012 0.207 PM 58.5±8.0 47.7±4.2 51.0±4.6 BA retrograde shear rate, 1/s AM -9.2±2.6 -7.8±1.8 -9.0±2.5 0.003 0.580 0.505 PM -18.1±2.8 -20.0±3.2 -21.1±4.2 CFA retrograde  shear rate, 1/s AM -17.4±3.1 -15.6±2.0 -17.7±2.2 0.298 0.292 0.948 PM -19.2±3.7 -17.8±2.7 -20.0±3.1 BA OSI AM 0.09±0.03 0.07±0.02 0.08±0.02 <0.001 0.704 0.706 PM 0.22±0.03 0.22±0.02 0.21±0.02 CFA OSI AM 0.20±0.03 0.23±0.02 0.23±0.03 0.195 0.036 SHAM vs NX, 0.038 0.882 PM 0.21±0.03 0.24±0.02 0.25±0.02 Data are presented as mean ± SEM. Definition of Abbreviations: BA, brachial artery; CFA, common femoral artery; HX, hypoxia; NX, normoxia; OSI, oscillatory shear index.  74  Carotid Baroreflex Control of Mean Arterial Pressure and Heart Rate 4.7The absolute and change from prestimulus MAP and HR responses to carotid baroreflex activation and inhibition are displayed in Figure 4.7 (page 76). The absolute MAP response demonstrated an effect of condition, where MAP following IH was greater than SHAM (P=0.045). No interaction effect of condition and level of pressure was observed in the absolute MAP response. The carotid baroreflex-mediated change in MAP, when normalized for prestimulus MAP, was unaffected by IH at each level of neck pressure and suction. Both absolute HR and change in HR displayed similar responses following SHAM and IH at each level of neck pressure and suction. The OP was 85.9±2.4 mmHg following SHAM and 88.1±1.3 mmHg following IH (P=0.262).  Both MAP and HR were successfully fitted to a four-parameter logistic function in a subset of participants. Parameter values for MAP (absolute, n=5; change, n=7) are presented in Table 4.8 (page 77). Intermittent hypoxia led to an increase in the absolute MAP gain parameter (P=0.018), but the maximal gain and gain about the OP were unchanged (P=0.133 and 0.588). Absolute MAP threshold was shifted rightwards, to a higher ECSP following IH (SHAM=41.9±6.3 mmHg, IH=67.0±5.6 mmHg, P=0.020) and saturation points were unaltered (P=0.247). There was a trend towards increases in minimum absolute MAP responses (SHAM=77.5±1.1 mmHg, IH=82.3±1.6 mmHg, P=0.095), but the maximum MAP responses and range were similar (P=0.842 and 0.329). Neither absolute MAP OP nor CP were different between conditions in the subset of participants (P=0.407 and 0.940). Modelling was successful in a larger group of participants when expressed as the change in MAP, thereby normalizing for prestimulus MAP (n=7). The change in gain parameter observed with absolute MAP modelling was not evident when assessed as change in MAP (P=0.900). Similarly, the shifted threshold and the trend towards blunted minimum response did not persist when modelled as the change in MAP (P=0.328 and 0.498). Maximum responses, range, CP and OP were similar between conditions, as were maximal gain, gain about the OP and saturation when assessed as the change in MAP.  Carotid baroreflex control of HR parameters were unaffected by IH when expressed as absolute HR (n=6) or change in HR (n=5) (see Table 4.9, page 78). The minimum and  75 maximum responses, sensitivity, setpoints and saturation were similar between SHAM and IH in both the absolute and change in HR. Only the change in HR ECSP threshold trended towards significance (SHAM=66.6±5.0 bpm, IH=56.5±5.5 bpm, P=0.065), but was unaltered in absolute HR modelling (P=0.679). Overall, the cardiac carotid baroreflex curves were similar between conditions.    76  Figure 4.7: Carotid baroreflex control of mean arterial pressure and heart rate. Data are displayed as absolute mean arterial pressure (MAP) and heart rate (HR; panels A and B) and change in MAP and HR from baseline (C and D). Each estimated carotid sinus pressure (ECSP) target (-80, -60, -40, -20, 0, 20 and 40) was compared between SHAM and intermittent hypoxia (IH) by two-way repeated measures ANOVA. There was a significant effect of condition on the absolute MAP response, where MAP was greater following intermittent hypoxia (IH) than SHAM (P=0.045). Data points are presented as means ± SEM.    77 Table 4.8: Effect of IH on carotid MAP-baroreflex four-parameter modelling variables.   SHAM IH P-value MAP Minimum, mmHg Absolute 77.5±1.1 82.3±1.6 0.095 Change -8.1±0.8 -7.6±0.9 0.498 MAP Maximum, mmHg Absolute 95.0±1.8 95.9±2.9 0.842 Change 6.9±1.1 6.7±1.3 0.829 MAP Range, mmHg Absolute 17.6±1.9 13.6±2.0 0.329 Change 15.0±1.3 14.3±1.1 0.368 Gain, mmHg/mmHg Absolute -0.09±0.03 -0.20±0.04 0.018 Change -0.09±0.02 -0.09±0.01 0.900 CP, mmHg Absolute 82.9±6.9 83.7±4.8 0.940 Change 90.4±2.8 93.6±3.5 0.203 OP, mmHg Absolute 82.3±3.4 85.4±1.6 0.407 Change 86.3±2.8 89.3±1.0 0.179 ECSP Threshold, mmHg Absolute 41.9±6.3 67.0±5.6 0.020 Change 44.2±10.6 55.2±8.2 0.328 ECSP Saturation, mmHg Absolute 123.8±13.7 100.5±5.8 0.247 Change 136.6±7.6 132.1±7.5 0.695 Gmax, mmHg/mmHg Absolute -0.36±0.05 -0.72±0.22 0.133 Change -0.31±0.07 -0.32±0.05 0.972 Gop, mmHg/mmHg Absolute -0.32±0.04 -0.41±0.15 0.588 Change -0.26±0.04 -0.29±0.06 0.714 Absolute MAP is based on n=5 and change in MAP is based on n=7. Data are presented as means ± SEM. The mean coefficients of determination of the curves for SHAM and IH were r2 values of 0.94±0.01 vs 0.95±0.01 for absolute MAP and 0.98±0.01 vs 0.98±0.004 for change in mean arterial pressure. Definition of abbreviations: CP, centering point; Gmax, maximal gain; Gop, gain at operating point; IH, intermittent hypoxia; MAP, mean arterial pressure; OP, operating point.     78 Table 4.9: Effect of IH on the heart rate-carotid baroreflex four-parameter modelling variables.  SHAM IH P-value HR Minimum, bpm Absolute 52.9±3.8 51.5±2.9 0.351 Change -7.9±1.5 -8.4±1.2 0.324 HR Maximum, bpm Absolute 65.0±3.6 64.3±3.6 0.788 Change 4.3±0.7 5.3±0.5 0.217 HR Range, bpm Absolute 12.1±0.8 12.8±2.7 0.753 Change 12.2±1.7 13.7±1.6 0.148 Gain, bpm/mmHg Absolute -0.14±0.03 -0.12±0.03 0.627 Change -0.11±0.02 -0.08±0.02 0.205 CP, mmHg Absolute 80.2±4.6 92.4±5.2 0.185 Change 98.5±5.6 97.9±2.4 0.910 OP, mmHg Absolute 86.9±3.3 89.6±1.2 0.304 Change 90.3±2.9 89.3±1.8 0.716 ECSP Threshold, mmHg Absolute 52.6±7.1 59.8±11.1 0.679 Change 66.6±5.0 56.5±5.5 0.065 ECSP Saturation, mmHg Absolute 107.8±10.4 125.0±6.3 0.226 Change 130.3±11.1 139.4±9.6 0.380 Gmax, bpm/mmHg Absolute -0.42±0.08 -0.31±0.06 0.385 Change -0.31±0.06 -0.26±0.02 0.383 Gop, bpm/mmHg Absolute -0.35±0.10 -0.25±0.07 0.536 Change -0.27±0.05 -0.24±0.03 0.555 Absolute HR is based on n=6 and change in HR is based on n=5. Data are presented as means ± SEM. The mean coefficients of determination of the curves for SHAM and IH were r2 values of 0.98±0.01 vs 0.98±0.01 for absolute heart rate and 0.95±0.02 vs 0.97±0.02 for change in heart rate. Definition of abbreviations: CP, centering point; ECSP, estimated carotid sinus pressure; Gmax, maximal gain; Gop, gain at operating point; HR, heart rate; IH, intermittent hypoxia; OP, operating point.     79  Carotid Baroreflex Control of Leg Vascular Conductance 4.8Carotid baroreflex control of CFA blood flow and vascular conductance are displayed in Figure 4.8 (page 80). Expressed as absolute responses to neck pressure and suction, neither LBF nor LVC were altered following IH compared to SHAM. When assessed as percent change, however, a blunted response to the hypertensive stimuli (neck suction) was observed following IH. Percent LBF responses were smaller at the -60 mmHg target range (SHAM = 21.8±3.2%, IH = 12.9±2.0%, P=0.002), while the percent LVC change was less following IH at -40 mmHg (SHAM: 24.0±2.5%, IH: 18.3±2.2%, P=0.044), -60 mmHg (SHAM: 31.8±3.2%, IH: 22.0±2.3%, P=0.003) and -80 mmHg (SHAM: 40.6±3.9%, IH: 32.2±3.9%, P=0.002) target pressures. Baseline LBF and LVC were not different between SHAM and IH. Following SHAM, LBF was 152±20 ml/min, compared to 185±25 ml/min following IH (P=0.196). Baseline LVC, immediately prior to carotid baroreflex testing, was 1.8±0.3 ml/min/mmHg following SHAM and 2.1±0.3 ml/min/mmHg following IH (P= 0.293).    80  Figure 4.8: Carotid baroreflex control of the leg vasculature following SHAM and IH. Common femoral artery blood flow (LBF, ml/min) and vascular conductance (LVC, ml/min/mmHg) responses to neck pressure and suction are displayed. Responses are presented as the absolute change in LBF (panel A) and LVC (panel B) and as percent changes (LBF, panel C; LVC, panel D). Each estimated carotid sinus pressure (ECSP) target (-80, -60, -40, -20, 0, 20 and 40) was compared between SHAM and intermittent hypoxia (IH) by two-way repeated measures ANOVA, with significant differences present for percent LBF changes at -60 mmHg (P=0.002), while percent LVC responses were lower following IH at -40 mmHg (P=0.044), -60 mmHg (P=0.003) or -80 mmHg (P=0.002). Values are presented as mean ± SEM.    81 5    Chapter: Discussion This chapter describes the findings presented in the Results section (Chapter 4). In this chapter, research questions are answered, study limitations addressed and perspectives and significance are established.   Main Findings 5.1The current investigation sought to elucidate potential contributors to acute IH-induced hypertension. Specifically, focus was directed to the peripheral vasculature and the carotid baroreflex. The principle findings were: 1) a single 6-hour exposure to IH increases 24-hour arterial blood pressure, as reflected in sustained elevations of SBP and PP during the night-time hours following exposure (22:00-06:00); 2) microvascular endothelial function, assessed by peak and total RH, displayed a diurnal reduction, causing a reduced shear rate response and decrease in FMD, which was not exacerbated by IH; 3) Vascular strain in the CCA and CFA were reduced during IH while OSI was elevated in in the lower limb; and 4) IH shifted the carotid baroreflex control of MAP to higher pressures and blunted the relative LVC response to carotid baroreflex loading. The hypotheses that 6 hours of IH would increase 24-hour arterial blood pressure, reduce vascular strain parameters, shift the carotid baroreflex control of MAP to operate at higher pressures and blunt the carotid baroreflex control of LVC were confirmed. The unexpected absence of an effect of IH on macro and microvascular endothelial function and heterogeneous influence of IH on blood shear patterns in the upper and lower limbs refuted the hypotheses. Impairments in vascular strain, lower limb hemodynamic shear patterns and carotid baroreflex control develop following a single 6-hour bout of IH – together, independent of changes in upper limb vessel function (FMD), these changes may play an important role in the early pathogenesis of IH-induced hypertension.   Intermittent Hypoxia Elevates Arterial Blood Pressure 5.2The present investigation was contingent on IH producing an increase in arterial blood pressure. Indeed, 6 hours of IH was sufficient to increase 24-hour SBP, DBP and MAP by 3 mmHg compared to SHAM. Rats exposed to 12 hours of IH per day for 14 days displayed a significant rise in MAP during normoxic breathing even after the first exposure (Marcus et  82 al., 2009). The increased MAP was sustained throughout the duration of the experiment, suggesting that a single IH exposure is as effective as multiple exposures at elevating MAP (Marcus et al., 2009). In humans, the single 6-hour isocapnic IH condition adapted has previously produced similar (Beaudin et al., 2014) and larger (Foster et al., 2010) increases in DBP and MAP. Foster et al. (2010) observed a 6.6±2.1 mmHg increase in MAP, however recordings were made using finger photoplethysmography calibrated to automated arm cuff measurements. 24-hour ambulatory monitoring gives a better prediction of risk than office measurements and permits monitoring beyond the laboratory visit (Pickering et al., 2005). Exposing participants to 6 hours of 2 minute cycles of hypoxia:normoxia without controlling for PETCO2 for four days produced a 4 mmHg increase in MAP (Foster et al., 2009). Administering IH during sleep without controlling for PETCO2 caused a similar 3 mmHg increase in daytime DBP and MAP assessed by ambulatory blood pressure monitoring, but SBP was unaltered following a single night (Tamisier et al., 2011). The increase in blood pressure was more pronounced following 14 nights of IH, with daytime SBP and DBP increasing by 8 mmHg and 5 mmHg respectively (Tamisier et al., 2011). The magnitude of arterial blood pressure increase appears to be dependent on the duration of the exposure and may be affected by CO2. Administration of IH causes hyperventilation and subsequent hypocapnia. Patients with OSA experience hypercapnic IH and hypercapnia is sympathoexcitatory. Indeed, isocapnic hypoxia is associated with a greater sympathetic activation compared with poikilocapnic hypoxia (Tamisier et al., 2004). Therefore, investigations that do not control for CO2 may produce a lesser degree of sympathoexcitation and smaller increase in blood pressure.   The sustained nocturnal increase in SBP was a novel finding. Increased SBP can be the result of both increased vascular resistance and arterial stiffness (Franklin et al., 1997). Muscle sympathetic nerve activity positively correlates with both total peripheral resistance and indices of arterial stiffness in men (Hart et al., 2009; Casey et al., 2011). Our finding of increased nighttime SBP could be indicative of increased vascular resistance and stiffness due to persistent sympathoexcitation. If this is the case, the vasculature is especially vulnerable to IH and may be responsible for the hypertensive effect of even a single bout of IH. The increased SBP occurred without an increase in DBP at night following IH, producing  83 an elevated PP. Pulse pressure is influenced primarily by the status of large arteries, where impaired visco-elastic properties of the large arteries can result in an augmented PP (Safar, 1989). Additionally, a higher PP can be caused by increased SV (Safar et al., 1987). Acutely, carotid chemoreceptor stimulation increases myocardial contractility via increased cardiac sympathetic drive, and may cause an initial increase in SV (Vatner & Rutherford, 1978). Studies in mice have observed an increase in left ventricular contractility following 4 weeks of IH (Naghshin et al., 2009; Naghshin et al., 2012), however left ventricular dysfunction has also been observed following 3 and 5 weeks of IH in a more severe rat model of IH (Chen et al., 2005; Chen et al., 2008). In humans, Tamisier et al. (2011) did not observe any change in SV following 14 nights of IH. Perhaps an increase in SV develops following a single bout of IH due to increased cardiac sympathetic drive, however, based on our findings (see Common Carotid and Common Femoral Artery Vascular Strain, page 84), the increased PP appears to be due to increased vascular stiffness and resistance.   We were the first to assess the 24-hour blood pressure response to 6 hours of isocapnic IH and we confirmed the hypertensive effect of a single bout of IH. The novel finding of increased nighttime SBP and PP suggest an effect of persistent sympathoexcitation on the vasculature.    Flow-Mediated Dilation and Reactive Hyperemia 5.3In the present study there was no difference in FMD between SHAM and IH conditions. In contrast to the established attenuation of morning FMD (Etsuda et al., 1999), we found that FMD was lower in the afternoon measurements following SHAM and IH. The reduction in FMD was attributed to the large reduction in the SRAUC response following 5 minutes of forearm ischemia. Indeed, normalization for SRAUC revealed an increase in FMD from AM to PM (P=0.008). Therefore, the reduction in FMD observed following SHAM and IH was due to a blunted SRAUC response, not impaired macrovascular reactivity per se.   The attenuated RH response may be reflective of impaired microvascular reactivity (Anderson et al., 2011). The peak and total cuff-release hemodynamic response was assessed until return to baseline diameter or prolonged plateau. Following SHAM and IH, the peak  84 hyperemic flow response was reduced by 24.4±4.5% and 18.8±7.3%, respectively, and the total hyperemic flow response was reduced by 33.1±8.5% and 32.8±17.6%. Similar findings were noted when expressed as velocity, suggesting that the changes were not driven by changes in BA diameter (Restaino et al., 2015). A similar diurnal impairment in microvascular reactivity was observed by Restaino et al. (2015), who observed a 30% blunted forearm reactive hyperemic response following 6 hours of prolonged sitting, indicative of impaired downstream resistance artery vasodilation. Previous work by Tamisier et al. (2011) found that 2 weeks of nocturnal IH increased forearm RH, while 4 weeks of IH led to its impairment. Collectively, it seems that the IH-induced increase in blood pressure precedes micro- and macro-vascular impairment.  Although FMD is considered a surrogate marker of global vascular health (Green et al., 2014), interpretation, particularly in mechanistic studies, is often complex. Had we measured only FMD, we would have concluded that a single, six-hour exposure to IH does not immediately impair vascular function. However, it is possible that other hemodynamic factors known to lead to FMD impairment could provide a more sensitive assessment of the factors affecting the endothelium. As a result, we also assessed the influence of IH on OSI and vascular strain. Alterations in these measurements may precede or directly contribute to reductions in FMD.    Common Carotid and Common Femoral Artery Vascular Strain 5.4For the first time, vascular strain mechanics were assessed during IH. Peak systolic strain is the percent deformation of the vessel during systole. Early systolic strain rate is associated with the arterial blood pressure rise following ventricular ejection and late systolic strain rate is indicative of vascular recoil. Acutely, IH does not alter strain parameters in the CCA, however after 6 hours of IH exposure there was a reduction in CCA peak systolic strain that was not observed during SHAM. Both CCA early and late systolic strain rates were blunted from AM to PM, but were unaffected by IH. Therefore, in the CCA, IH reduced wall deformation from AM to PM, but did not change the rate of deformation. Interestingly, CCA peak systolic strain can distinguish between low and intermediate-to-high levels of cardiovascular risk (Catalano et al., 2011). Peak systolic strain, early systolic strain rate and  85 late systolic strain rate were shown to be superior to conventional measures of vascular stiffness in distinguishing between young and old individuals (Bjallmark et al., 2010; Rosenberg et al., 2014). Impaired visco-elastic properties of the CCA may impair responsiveness and hence reactivity, similar to that observed in the cerebral arteries of older individuals (Fluck et al., 2014).   Indices of CFA circumferential strain and strain rate showed a trend towards reduced peak systolic strain and early systolic strain rate during IH compared to SHAM (P=0.055 and P=0.080). The CFA is a muscular conduit artery whereas the CCA is an elastic-type artery (van Sloten et al., 2014). As a consequence, the CFA is a less compliant vessel, hence the considerably lower strain and strain rate values compared to CCA (Charwat-Resl et al., 2015). The reduced peak systolic strain and early systolic strain rate in the CFA appear after brief exposure to IH (during the AM measurement), similar to the alterations in CFA blood flow. It remains unknown whether the impairment of strain parameters is a cause or consequence of the disturbed blood flow. Increased downstream resistance causes an increase in upstream transmural pressure and encourages the progression of hypertension in young adults (Franklin et al., 1997). Vascular resistance and arterial stiffness contribute to increases in SBP, whereas DBP increases occur independent of arterial stiffness (Franklin et al., 1997). Our findings of an increase in SBP throughout the night while DBP was normalized might suggest that IH may have exerted its hypertensive effects through a persistent rise in arterial stiffness, although this was not measured at night. However, each of the parameters of vascular strain reflect deformation or rate of deformation during phases of systole and thus show how well the arteries absorb the ejection of blood during systole. Vessels become less distensible when exposed to higher pressures (Bergel, 1961), whether arterial stiffness precedes the rise in blood pressure or vice versa remains elusive (Franklin, 2005).   Sympathetic activity is associated with a reduction of arterial distensibility in the CCA and CFA in rats (Mangoni et al., 1997). In humans, resting MSNA levels are related to aortic stiffness in men (Swierblewska et al., 2010; Casey et al., 2011). Sympathetic tone restrains arterial distensibility in the medium and large muscular arteries, and sympathoexcitation stiffens the radial and carotid arteries (Boutouyrie et al., 1994; Failla et al., 1997; Failla et  86 al., 1999). The sympathoexcitation evoked by IH may have had a direct effect on the reduced vascular strain and indirect effect via increases in blood pressure.   Both the CCA and CFA are clinically relevant vessels, enhanced stiffness of either vessel is associated with cardiovascular events and mortality (van Sloten et al., 2014). The present study extends the utility of two-dimensional speckle-tracking vascular strain measurements and proposes that a single exposure of IH is sufficient to impair vessel mechanics in the CCA and maybe the CFA.    Upper and Lower Limb Hemodynamics 5.5The experimental protocol produced a marked reduction in BA flow and shear rate while promoting a pro-atherogenic shear pattern (greater OSI) regardless of the experimental condition. While not a primary purpose of the investigation, we recognize that the protocol closely resembled prolonged sitting and sedentary activity studies. Previous research has identified that 3 hours of sitting reduces BA antegrade shear and increases OSI by approximately 30%, but does not alter FMD (Thosar et al., 2014). Extending the prolonged sitting period to 6 hours, Restaino et al. (2015) found a near 50% reduction in shear rate and blunted RH SRAUC response. Furthermore, their protocol abolished the diurnal increase in FMD, similar to the present study. Therefore, a period of disturbed blood flow (greater OSI) appears to precede a functional reduction in FMD. The effects of disturbed shear patterns on endothelial function have been tested experimentally by inflating a forearm cuff, increasing OSI in the BA. Thirty minutes of disturbed blood flow impairs BA FMD (Thijssen et al., 2009; Totosy de Zepetnek et al., 2015). Administration of vitamin C beforehand prevents the impairment, suggesting that shear-induced oxidative stress contributes to the reduced endothelium-dependent vasodilation (Johnson et al., 2013). Antioxidant treatment has a similar endothelial-protective effect on prolonged sitting (Thosar et al., 2015).  In addition to oxidative stress, sympathoexcitation has been implicated in altered limb hemodynamics. Assuming a seated position augments MSNA (Burke et al., 1977) and causes calf blood pooling, which is compensated for by increased peripheral resistance (Shvartz et al., 1983). Acute increases in MSNA disturb BA shear patterns by increasing retrograde  87 shear and OSI (Padilla et al., 2010; Thijssen et al., 2014) causing reductions in FMD (Ghiadoni et al., 2000; Hijmering et al., 2002; Lind et al., 2002; Spieker et al., 2002), although findings appear to be dependent upon the sympathetic stimulus (Dyson et al., 2006). Albeit incompletely understood, prolonged sitting causes venous pooling and subsequent increases in MSNA to increase peripheral resistance. Consequently, blood flow patterns are altered, inducing a pro-atherogenic milieu and oxidative stress that impairs endothelial function. Administration of IH did not contribute to an additive impairment of upper limb hemodynamics.  The lower limb, conversely, did not display any time of day effect. Human lower limbs are exposed to greater hydrostatic pressures compared to upper limbs and appear to have preserved vascular reactivity and hemodynamics following prolonged periods of increased hydrostatic pressure (Padilla et al., 2009b). Therefore, the evaluation of lower limb hemodynamics may offer greater insight on the specific vascular effects of IH that may have been masked by the influences of prolonged sitting in the upper limb. Common femoral artery blood flow and shear rate were lower and OSI higher during the IH condition. Indeed, such an effect was noticeable acutely (AM measurement occurred only 10 minutes into the IH exposure) and persisted throughout the experimental day. Acute severe hypoxemia causes lower limb small artery vasoconstriction without altering conduit artery hemodynamics (Barthelemy et al., 2001). Since IH produces more potent sympathoexcitation and increases in oxidative stress than continuous hypoxia (Prabhakar & Semenza, 2012), hypoxia-induced increases in downstream resistance may have caused a reduction in flow and consequently shear rate even after brief exposure. Obstructive apneas during sleep cause an increase in lower limb vascular resistance (Imadojemu et al., 2002) and MSNA bursts decrease LVC (Fairfax et al., 2013). Since IH causes sympathoexcitation, it is possible that it alters lower limb hemodynamics by decreasing LVC. Finally, 2 weeks of nightly IH decreases calf blood flow RH, but improves forearm blood flow RH, supporting limb-specific consequences of IH (Gilmartin et al., 2010; Tamisier et al., 2011). The forearms have a reduced vasoconstriction response to phenylephrine (α1-agonist) than the lower legs, suggesting the arms may be less responsive to increased SNA than the legs (Pawelczyk & Levine, 2002). Therefore, IH may have caused sufficient sympathoexcitation to increase downstream vasoconstriction in the  88 lower limbs, but not to overcome the influence of prolonged sitting in the upper limb. Since the lower limb is protected against hydrostatic pressure, 6 hours of sitting did not impair its hemodynamics, suggesting the impairments were in fact a result of IH. Contrary to our investigation, OSA patients experience nocturnal IH, while laying down asleep. Blood pressures in the upper and lower limbs are similar in the supine position, whereas in an upright position, the legs are exposed to higher blood pressures (Malhotra et al., 2002). Investigation of nocturnal IH may eliminate the confounding effect of altered hemodynamics induced by an upright body position and better simulate OSA. Exposure to IH during sleep causes sleep fragmentation (Tamisier et al., 2009), and arousals from sleep cause vasoconstriction and are sympathoexcitatory (Morgan et al., 1996). However, repeated arousals without IH do not produce a sustained rise in blood pressure in animals (Brooks et al., 1997; Bao et al., 1999). Future investigations should aim to elucidate upper and lower limb vascular responses following a single exposure of nocturnal isocapnic IH.   Carotid Baroreflex Control of Mean Arterial Pressure and Heart Rate 5.6Carotid baroreflex control of MAP was shifted upwards, operating at higher arterial blood pressures, following IH exposure. While the MAP sensitivity (G parameter) was increased following IH in a subset of participants, neither the maximal gain (Gmax) nor the gain about the operating point (Gop) were different between conditions, opposing the conclusion of increased BRS. Furthermore, when expressed as change in MAP, thereby correcting for prestimulus MAP, response curves were nearly identical. These findings agree with our hypothesis and are supported by a previous investigation using a human experimental IH model, where BRS is unaltered, but shifted to operate at a higher set-point (Monahan et al., 2006). We did not observe an increase in OP (SHAM=85.9±2.4 mmHg vs IH=88.1±1.3 mmHg, P=0.262), perhaps due to a small sample size (n=10). Instead our finding was present as an effect of condition across each level of neck pressure and suction. The cardiac carotid baroreflex, on the other hand, was not affected by IH, implicating the vascular response as the mediator of the alterations in carotid baroreflex control of MAP. Over a longer period of time, Tamisier et al. (2011) demonstrated increased cardiovagal gain and decreased sympathetic vascular gain. Since the 14 night protocol produced marked increases in SBP and DBP (+8 mmHg and +5 mmHg), the authors proposed that the more robust bradycardiac  89 responses following exposure represent an incomplete compensation to offset the augmented pressure. Since the robust bradycardiac responses observed by Tamisier et al. (2011) were insufficient to prevent the rise in blood pressure, there appears to be a predominance of the vascular sympathetic baroreflex in shifting the arterial baroreflex to operate at higher pressures. Carotid baroreflex-mediated changes in blood pressure are primarily mediated by changes in vascular conductance, which contributes to 76% of the peak MAP response (Ogoh et al., 2002). We observed a shift of the carotid baroreflex control of MAP to operate at higher pressures. The cardiac response was not different between conditions, suggesting changes were caused by impairments in vascular conductance.   Carotid Baroreflex Control of Leg Vascular Conductance  5.7The carotid baroreflex MAP response is primarily mediated by alterations in vascular conductance as opposed to HR or SV (Ogoh et al., 2002). The MAP response was shifted upwards following IH, while the vascular response was characterized by a reduced percent reduction in LVC in response to neck suction, implicating hindered sympathetic withdrawal or lower limb vasodilation. The percent change in LVC is reflective of vascular responsiveness (Buckwalter et al., 2001), and an impairment in the hypertensive range suggests impaired vasodilation. Neck suction stretches the carotid sinus, simulating an increase in blood pressure (Eckberg et al., 1975). The carotid baroreflex is subsequently activated, causing a withdrawal in MSNA and passive peripheral vasodilation (Keller et al., 2004). We displayed a blunted LVC response to hypertensive stimuli. The observed reduction in passive vasodilation following IH may relate to the decrease in CFA strain. A reduced arterial compliance blunts the vasodilation response to baroreflex activation, suggesting an impaired vessel reactivity (Kingwell et al., 1995). An impaired reactivity may be further exacerbated by endothelial dysfunction caused by increased OSI (Schreuder et al., 2014; Totosy de Zepetnek et al., 2014). Alternatively, the blunted vasodilation may have been caused by impaired carotid baroreflex withdrawal of SNA, as observed by Tamisier et al. (2011) after 14 nights of IH and using the modified oxford technique.  Intermittent hypoxia may alter baroreflex function at multiple sites in the baroreflex pathway including the carotid baroreceptors, the brain stem associated with processing of afferent  90 information from baroreceptor afferents and vascular reactivity (Gu et al., 2007; Silva & Schreihofer, 2011; Peng et al., 2012). In rats, 2 weeks of IH attenuates the carotid baroreflex response to increased carotid sinus pressure due to reactive oxygen species-mediated upregulation of ET-1 signalling in the carotid sinus region (Peng et al., 2012). Increased ET-1 at the carotid body sensitizes the hypoxic chemoreflex response to hypoxia (Rey et al., 2006). Thus, ET-1 appears to serve a dual role of supressing the carotid baroreflex and sensitizing the carotid chemoreceptor, promoting sympathetic activation. Additionally, since ET-1 is a vasoconstrictor peptide, Peng et al. (2012) suggest the blunted response to baroreflex activation may be due to impaired compliance at the carotid sinus. Another vasoconstrictor peptide with central effects, Ang II, shifts the baroreflex control of HR to operate at higher levels of blood pressure in rabbits (Brooks et al., 1993), but does not reset the MAP-renal sympathetic nerve activity curve (Barrett et al., 2003). Angiotensin II acts centrally at the level of the nucleus tractus solitarii, where AT1R are expressed (Song et al., 1992). Pharmacological blockade of AT1R at the nucleus tractus solitarii abolishes the Ang II-induced baroreflex resetting in rats, highlighting the potency of its central effects on blood pressure regulation (Matsumura et al., 1998). Furthermore, Ang II is sympathoexcitatory, causes vasoconstriction, increases renin secretion and increases catecholamine release from the adrenal medulla, collectively acting to increase blood pressure (Reid, 1992). Both endothelin-1 and Ang II are vasoconstrictor peptides that also act centrally to disrupt baroreflex function and have been shown to be elevated in OSA patients (Phillips et al., 1999; Moller et al., 2003). Therefore, IH may have elevated circulating ET-1 and Ang II, thus promoting blood pressure elevations and a resetting of the carotid baroreflex control of absolute MAP to higher pressures without effecting cardiac baroreflex.   A reduction in sympathetic withdrawal may be due to the mechanical properties of the carotid sinus. The neck chamber technique exerts its effects via alterations in transmural pressure. It is therefore conceivable that alterations in CCA stiffness may influence the degree of distension of the large elastic arteries. Stiffening of the CCA and aortic arch reduce mechanotransduction and limit the capability of the baroreceptors to detect blood pressure fluctuations (Bonyhay et al., 1996; Kornet et al., 2002; Lipponen et al., 2012). Indeed, a reduced CCA compliance dampens responses to baroreflex activation (increased blood  91 pressure) and is associated with decreased baroreflex sensitivity (Kingwell et al., 1995; Monahan et al., 2001). Transmission of suction to the carotid using a neck chamber is 82%, but it remains unknown whether transmission is altered in the case of increased CCA stiffness (Querry et al., 2001). Intermittent hypoxia produced a decrease in peak systolic strain (see Common Carotid and Common Femoral Artery Vascular Strain, page 84), indicative of a reduced vessel deformation during systole, and a measure of CCA stiffness. A significant inverse correlation between sympathetic baroreflex sensitivity and carotid artery stiffness has been observed in elderly men and women (Okada et al., 2012). Furthermore, the same group found that hypertensive subjects who experience large SBP morning surges (>35 mmHg) have reduced sympathetic baroreflex sensitivity and increased arterial stiffness, suggesting that the increased stiffness impairs the ability to buffer large increases in SBP (Okada et al., 2013). Therefore, the blunted LVC response to hypertensive stimuli may be due to increased CCA stiffness (and hence carotid sinus) and may have contributed to the impairment of the carotid baroreflex response to simulated hypertension.   Methodological Considerations 5.8The present investigation sought to assess the influence of IH independent of confounding factors and to characterize the resulting effects on the global vascular and carotid baroreflex response. Such an inherently integrative study cannot control for all potential contributions and the limitations are discussed below.  5.8.1 Intermittent Hypoxia In an attempt to maintain isocapnic IH, PETCO2 was controlled by operator-dependent titration of CO2 triggered by inspiration. After baseline acquisition of PETCO2, IH was started and the first 10 minutes (5 cycles) was spent ensuring proper administration of CO2. While generally effective, PETCO2 was statistically lower during IH than SHAM (-1.4±0.5 mmHg, P=0.027). Arterial hypoxemia stimulates the peripheral chemoreceptors causing a reflex increase in   E. Consequently, the hyperventilation causes a reduction in PaCO2. Hypocapnia produces vasodilation and a decrease in arterial blood pressure (Burnum et al., 1954). Hypercapnia, in contrast, is sympathoexcitatory and OSA produces hypercapnic hypoxia (Somers et al., 1988). We were concerned solely with the effects of IH, as the hypoxia, but  92 not hypercapnia induce LTF (Xie et al., 2001). The degree of hypocapnia observed was likely insufficient to influence our conclusions, indeed we still produced significant increases in 24-hour arterial blood pressure.  5.8.2 Carotid Baroreflex Assessment The strengths and weaknesses of the neck chamber technique have been thoroughly discussed above (see Baroreflex Assessment Techniques, page 11) and elsewhere (Fadel et al., 2003). Concerning the present investigation, several limitations must be acknowledged.   We were the first to employ the neck chamber technique to assess carotid baroreflex function following IH. Six hours of IH shifted the absolute MAP-carotid baroreflex response to operate at higher pressures, however we were only able to model responses to the four-parameter logistic function in a subset of participants (n=5). Therefore comparison of BRS (i.e., gain, Gmax and Gop), minimum and maximum responses, and saturation and threshold between SHAM and IH were performed with less statistical power. The change in MAP-response curve was successfully modelled in a larger subset of participants (n=7), and eliminated the differences in gain and threshold between conditions that were present when absolute MAP responses were modelled. Because the change in MAP abolished such findings, we concluded that the carotid baroreflex control of MAP sensitivity was unaltered. The carotid baroreflex control of HR was successfully modelled in 6 and 5 participants for absolute HR and change in HR, respectively, and did not reveal any differences between conditions. Collectively, we were able to describe an upwards shift in absolute MAP-response curve and attribute this to a resetting of the baroreflex set-point. Because we investigated and compared both the absolute and change in MAP and HR with and without using four-parameter logistic modelling, we are confident that our interpretation of the effect of IH on carotid baroreflex function are accurate.    Applications of neck suction (simulated hypertension) were well maintained, but neck pressure often dissipated during the 5-second application. To mitigate any leak, external pressure was applied on the collar by an investigator from 10 seconds before, until 10  93 seconds after stimulus. When a baroreflex response was evoked by the externally applied pressure, evidenced by a prestimulus rise in MAP, the trial was excluded and repeated.   Application of neck suction and pressure are usually applied following a 10-15 seconds end-expiratory apnea. The cardiac response depends on the respiratory phase when neck suction/pressure are applied, with delivery during inspiration failing to produce a carotid baroreflex response due to gated baroreceptor activity (Eckberg et al., 1980). Since we were primarily interested in the MAP and LVC responses, which are more delayed than the cardiac response, an apnea would have needed to be held for close to 30 seconds. Pilot testing found the prolonged apnea too burdensome for the participants and led to irreproducible carotid baroreflex responses. Instead, we permitted normal breathing, ensuring that participants avoided gasping or holding their breath from 10 seconds prior until 15 seconds after application of neck suction/pressure. Previous investigations concerned with the carotid baroreflex control of the vasculature have permitted free-breathing (Cooper & Hainsworth, 2001; Cooper et al., 2005). Foster et al. (2010) found that   E was unaltered between SHAM and IH exposures, therefore a change in   E between conditions was unlikely.   Because the investigation was powered according to changes in MAP, the sample size of 10 may have been insufficient for the 2x7 repeated measures ANOVA employed for the carotid baroreflex assessment to detect changes. Thus, it is possible that we observed a Type II error and failed to identify interaction effects that could have been present.   5.8.3 Ultrasound Measurements Impairments in vascular strain were observed. Importantly, however, simultaneous measure of beat-by-beat arterial blood pressure and CCA blood flow were not performed. Therefore, no index of PP or CCA flow and shear rate were possible. Bjallmark et al. (2010) identified that circumferential strain parameters independently distinguish between young and old participants and Catalano et al. (2011) were able to classify those at low-risk compared to those at intermediate or high cardiovascular risk with CCA peak systolic strain even without normalization for PP. Daytime SBP and DBP increased equally with IH (~3 mmHg),  94 producing equivocal PP. Had we observed an increase in SBP absent any change in DBP, an increased PP would have been observed and may alter interpretation of the vascular mechanics. It would have been advantageous to assess both CCA flow and shear rate, and PP, however the current findings support the conclusion of IH-induced impairment of CCA strain.   Given the condition effect of CFA flow and shear, it would have been beneficial to assess lower limb endothelial function via superficial femoral artery (SFA) or popliteal artery FMD. Brachial artery FMD itself is not a systemic index of endothelial function, the lower and upper limb are heterogeneous in their vascular responses (Proctor & Newcomer, 2006; Thijssen et al., 2011b). The lower limbs have blunted endothelium-dependent and independent vasodilation compared to the upper limb (Newcomer et al., 2004). Importantly, limb-specific responses exist in response to increased hydrostatic pressure (Padilla et al., 2009b). Three hours of prolonged sitting has been shown to impair SFA FMD, but not BA FMD (Thosar et al., 2014) and a 6-hour sitting protocol reduces popliteal artery FMD and RH, indicating lower limb micro- and macro-vascular reactivity  impairment (Restaino et al., 2015). Similar to BA, altered shear patterns acutely impair SFA FMD (Schreuder et al., 2014; Totosy de Zepetnek et al., 2014), therefore the disturbed blood flow during IH may have been reflected by an impaired SFA FMD. The lower limbs are more susceptible to atherosclerotic lesion formation (Kroger et al., 1999) due to chronically lower mean shear rate and turbulent flow, making them a more clinically relevant site than the BA (Wu et al., 2004; Wood et al., 2006; Newcomer et al., 2008). Given the divergent vascular responses of the upper and lower limbs, future investigations should study the lower limb FMD response following 6 hours of IH rather than the upper limb.    Perspectives and Significance 5.9The present investigation sought to provide insight on the pathogenesis of IH-induced hypertension by assessing 1) the 24-hour arterial blood pressure responses, 2) upper limb endothelium-dependent vasodilation, 3) CCA and CFA vascular strain and strain rate, 4) upper and lower limb conduit artery hemodynamics, and 5) carotid baroreflex control of MAP, HR and LVC in response to 6 hours of IH. The investigation found 1) an increase in  95 24-hour SBP, DBP and MAP with an increase in nocturnal SBP and PP following IH, 2) no effect of IH on upper limb macro or microvascular reactivity, 3) IH impaired CCA and CFA vascular strain, 4) IH induced a reduction in CFA flow and shear rate, and 5) increased carotid baroreflex control of MAP set-point and a blunted LVC response to hypertensive stimuli following IH. Together, these findings help to characterize the global vascular and carotid baroreflex responses to acute IH, providing insight on the initial adaptations that facilitate an increase in arterial blood pressure.   The lower limb vasculature appears to be more susceptible to acute IH, and leg macro and microvascular reactivity in a similar protocol deserves exploration. Reduced LVC buffering of simulated hypertension may be reflective of impaired sympathetic withdrawal or lower limb vasodilation. The finding of increased CCA and potentially CFA stiffness may contribute to the impaired LVC response at the level of the vessel reactivity or baroreceptor stretch. The hypertensive response to acute IH appears to be at least initially mediated by heterogeneous changes in the vasculature that depend on the vascular bed. The unexpected finding of disturbed shear patterns and impaired vascular reactivity in the upper limb regardless of condition was attributed to the prolonged sitting. The impact of prolonged sitting merits consideration when developing experimental protocols. Exposing participants to IH in a supine position, may offer insight on the effects of IH on different vascular beds independent of the influences of prolonged sitting. Alterations in blood flow patterns in the lower limbs, impaired vascular strain and blunted baroreflex responses may represent the acute adaptations involved in IH-induced hypertension.   Future Directions 5.10The heterogeneous effects of IH on the vasculature was observed in the present investigation. The reduced blood flow and increased OSI in the lower limb during IH suggests disturbed hemodynamics. Future investigations in the vascular effects of IH should include functional measures of lower limb vascular function, for example FMD in the SFA or popliteal artery. We did not measure SNA, but suggest that percent LVC responses to hypertensive stimuli may be blunted following IH due to reduced sympathetic withdrawal. Measuring MSNA is a logical extension in understanding vascular regulation following IH. Finally, a similar  96 investigation should be performed in middle-aged adults. We studied young, healthy men who may be better protected against the cardiovascular consequences of IH. Indeed, studying middle-aged adults would be of greater clinical relevance, as the prevalence of OSA increases with age (Bixler et al., 1998). 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Circulation research 91, 1038-1045.       130 Appendices Appendix A  : Forms  A.1 University of British Columbia Ethics Certificate of Full Board Approval        The University of British Columbia Office of Research (Services) Ethics Clinical Research Ethics Board – Room 210, 828 West 10th Avenue, Vancouver, BC V5Z 1L8       ETHICS CERTIFICATE OF FULL BOARD APPROVAL  PRINCIPAL INVESTIGATOR: INSTITUTION / DEPARTMENT: UBC CREB NUMBER: Glen E. Foster  UBC/UBCO Health & Social Development/UBCO Health and Exercise Sciences  H14-01151 INSTITUTION(S) WHERE RESEARCH WILL BE CARRIED OUT:   Institution Site UBC Okanagan Other locations where the research will be conducted: N/A   CO-INVESTIGATOR(S): Michael Tymko Joshua C. Tremblay   SPONSORING AGENCIES: - Natural Sciences and Engineering Research Council of Canada (NSERC) - "Intermittent Hypoxia and Cardiopulmonary Adaptation " - UBCO Faculty of Health and Social Development - "Start-Up Funds" - UBCO Internal Research Funds - "Angiotensin receptor blockers as a novel treatment for cardiovascular disease associated with obstructive sleep apnea "  PROJECT TITLE: Renin-angiotensin system control of baroreflex following exposure to intermittent hypoxia.  THE CURRENT UBC CREB APPROVAL FOR THIS STUDY EXPIRES:  June 10, 2015 The full UBC Clinical Research Ethics Board has reviewed the above described research project, including associated documentation noted below, and finds the research project acceptable on ethical grounds for research involving human subjects and hereby grants approval.  This approval applies to research ethics issues only. The approval does not obligate an institution or any of its  131 departments to proceed with activation of the study. The Principal Investigator for the study is responsible for identifying and ensuring that resource impacts from this study on any institution are properly negotiated, and that other institutional policies are followed. The REB assumes that investigators and the coordinating office of all trials continuously review new information for findings that indicate a change should be made to the protocol, consent documents or conduct of the trial and that such changes will be brought to the attention of the REB in a timely manner.  REB FULL BOARD MEETING REVIEW DATE:   June 10, 2014     DOCUMENTS INCLUDED IN THIS APPROVAL: DATE DOCUMENTS APPROVED:   Document Name Version Date Protocol: Protocol 2 July 7, 2014 Consent Forms: Informed Consent 2 July 7, 2014 Investigator Brochures: Cozzar N/A December 19, 2007 Advertisements: Advertisement 2 July 7, 2014 Questionnaire, Questionnaire Cover Letter, Tests: Questionnaire 2 July 7, 2014 Inclusion/Exclusion 2 July 7, 2014 Epworth Sleepiness Scale N/A January 1, 1991 Other Documents: External Reviews 1 May 22, 2014    July 16, 2014 CERTIFICATION: In respect of clinical trials:  1. The membership of this Research Ethics Board complies with the membership requirements for Research Ethics Boards defined in Division 5 of the Food and Drug Regulations. 2. The Research Ethics Board carries out its functions in a manner consistent with Good Clinical Practices. 3. This Research Ethics Board has reviewed and approved the clinical trial protocol and informed consent form for the trial which is to be conducted by the qualified investigator named above at the specified clinical trial site. This approval and the views of this Research Ethics Board have been documented in writing.    The documentation included for the above-named project has been reviewed by the UBC CREB, and the research study, as presented in the documentation, was found to be acceptable on ethical grounds for research involving human subjects and was approved by the UBC CREB.    Approval of the Clinical Research Ethics Board by:   Dr. Peter Loewen, Chair      132 A.2 Health Questionnaire Please answer Yes/No for each question (if yes, please explain):  Have you refrained from caffeine, alcohol and vigorous exercise 12 hours prior to the experimental day? YES NO  Do you have a history of fainting or have ever experienced a syncopal episode?     YES NO   Do you have a previous history of or a current respiratory disease or abnormality (e.g., asthma, chronic bronchitis, cystic fibrosis)?   YES  NO  Do you have a previous history of or a current cardiovascular disease or abnormality (e.g., cardiac arrhythmia, hypertension, myocardial infarction)? YES  NO  Do you have a previous history of or a impaired renal function (e.g., kidney failure, chronic kidney disease)? YES  NO  Do you have a previous history of or a current neurological disease or abnormality (e.g., epilepsy, chronic migraines, stroke)? YES  NO  Are you currently on any kind of medication, over the counter or prescribed?   YES  NO         Do you have type I or II diabetes?  YES  NO  Do you smoke?            YES  NO  Do you have any drug allergies?  YES NO  Have you had all of your questions or concerns addressed?    YES NO        133 A.3 Epworth Sleepiness Scale   134 A.4 Diet and Sleep Log Date: Name: Time Food Consumed              Time to bed:_____________ Time awake:_____________ Sleep quality (1-5):__________ Comments (discomforts, etc):   135 A.5 Activity Log Name: Date: Time of Day Activity 16:00  16:15  16:30  16:45  17:00  17:15  17:30  17:45  18:00  18:15  18:30  18:45  19:00  19:15  19:30  19:45  20:00  20:15  20:30  20:45  21:00  21:15  21:30  21:45  22:00  22:15  22:30  22:45  23:00  23:00 and beyond   136 Appendix B  : Individual Raw Data Tables  B.1 Participant Characteristics Table B.1: Baseline common carotid intima-media thickness. Participant ID Posterior CIMT (mm) Anterior CIMT (mm) 01 0.41 0.58 02 0.47 0.57 03 0.63 0.49 04 0.58 0.59 05 0.60 0.69 06 0.42 0.46 07 0.46 0.57 08 0.51 0.56 09 0.45 0.55 10 0.44 0.54 MEAN 0.50 0.56 SEM 0.03 0.02 Definition of abbreviations: CIMT, common carotid artery intima-media thickness; SEM, standard error of the mean.      137 Table B.2: Baseline pulmonary function test individual data. Participant ID FVC (l) (%) FEV1 (l) (%) FEV1/FVC TLC (l) (%) DLCO (ml/min/mmHg) (%) DLCO/VA (ml/min/mmHg/l) (%) 01 6.6 (122) 5.1 (108) 77 7.5 (109) 40.7 (112) 5.4 (102) 02 7.1 (129) 5.9 (125) 83 7.2 (102) 38.8 (107) 5.4 (107) 03 6.0 (116) 4.6 (108) 77 7.7 (115) 34.3 (98) 5.0 (95) 04 5.7 (100) 4.0 (85) 71 6.8 (96) 38.5 (103) 5.5 (103) 05 6.2 (115) 4.6 (99) 74 7.6 (112) 38.5 (106) 5.5 (103) 06 6.1 (109) 4.8 (100) 79 7.4 (105) 36.1 (98) 5.7 (109) 07 5.0 (98) 4.0 (95) 80 6.5 (101) 30.0 (85) 4.9 (90) 08 6.6 (124) 5.3 (124) 79 7.6 (116) 34.9 (95) 5.1 (92) 09 6.0 (118) 4.7 (109) 78 7.0 (101) 49.5 (146) 5.0 (129) 10 5.6 (105) 4.6 (104) 82 6.7 (100) 34.9 (96) 5.2 (96) MEAN 6.1 (113.6) 4.7 (105.7) 78.0 7.2 (105.7) 37.6 (104.6) 5.3 (102.6) SEM 0.2 (3.3) 0.2 (3.9) 1.1 0.1 (2.2) 1.6 (5.2) 0.1 (3.5) Definition of abbreviations: DLCO, diffusion capacity of the lung for carbon monoxide transfer; DLCO/VA, DLCO corrected for alveolar volume; FEV1, forced expiratory volume in one second; FVC, forced vital capacity; SEM, standard error of the mean; TLC, total lung capacity.   138 B.2 Intermittent Hypoxia and SHAM Exposures Table B.3: Mean end-tidal gases throughout SHAM and IH individual data.  PETCO2 (mmHg) PETO2 (mmHg) Participant ID SHAM IH IH - SHAM SHAM IH IH - SHAM 01 36.8 35.0 -1.9 100.7 64.1 -36.6 02 33.4 31.2 -2.2 103.8 70.3 -33.5 03 34.9 34.9 0.0 103.5 65.3 -38.2 04 36.6 33.2 -3.4 98.5 68.1 -30.5 05 31.8 30.4 -1.4 103.4 66.0 -37.3 06 36.7 37.0 0.3 102.0 66.2 -35.8 07 37.6 38.8 1.2 100.5 69.6 -30.9 08 35.3 33.1 -2.2 103.8 67.9 -35.9 09 36.8 33.0 -3.8 101.5 67.1 -34.4 10 33.6 33.3 -0.3 103.2 66.0 -37.2 MEAN 35.4 34.0 -1.4 102.1 67.1 -35.0 SEM 0.6 0.8 0.5 0.6 0.6 0.8 Definition of abbreviations: IH, intermittent hypoxia; PETCO2, end-tidal partial pressure of carbon dioxide; PETO2, end-tidal partial pressure of oxygen; SEM, standard error of the mean.  139 Table B.4: Mean individual hypoxemia parameters throughout SHAM and IH.      SHAM IH Participant ID Mean SpO2 (%) % of Time <90% SpO2 % of Time <85% SpO2 ODI, events/hour Mean SpO2 (%) Mean Minimum SpO2 (%) % of Time <90% SpO2 % of Time <85% SpO2 ODI, events/hour 01 97.3 0.1 0.1 0.0 92.2 80.4 48.3 24.8 27.1 02 98.6 0.0 0.0 0.5 94.6 83.2 31.1 12.3 29.1 03 97.6 0.0 0.0 0.8 92.1 81.8 44.7 21.3 31.9 04 96.8 0.0 0.0 0.3 92.8 80.4 39.1 22.8 30.8 05 97.1 0.0 0.0 1.5 92.5 79.9 42.1 23.4 29.0 06 96.9 0.0 0.0 0.0 91.3 80.7 47.3 24.1 32.2 07 95.7 0.0 0.0 3.0 91.3 80.2 47.5 24.5 29.2 08 96.2 0.0 0.0 2.2 91.2 80.6 46.0 22.7 29.9 09 94.6 0.0 0.0 2.4 91.3 81.6 42.1 14.5 29.2 10 95.9 0.5 0.0 4.9 90.4 78.2 53.1 30.0 30.1 MEAN 96.7 0.1 0.0 1.6 92.0 80.7 44.1 22.0 29.9 SEM 0.4 0.0 0.0 0.5 0.4 0.4 1.9 1.6 0.5 Definition of abbreviations: IH, intermittent hypoxia; ODI, oxygen desaturation index; SEM, standard error of the mean; SpO2, peripheral oxyhemoglobin saturation.    140 B.3 Ambulatory Blood Pressure Monitoring Table B.5: Individual systolic blood pressure data throughout SHAM and IH.  SBP (mmHg)  DAY NIGHT 24-h Participant ID SHAM  IH  IH - SHAM SHAM IH  IH - SHAM SHAM IH  IH - SHAM 01 123.7 127.4 3.7 115.4 120.0 4.7 121.7 125.2 3.5 02 115.1 122.9 7.8 107.1 111.9 4.8 112.5 120.2 7.6 03 117.4 117.4 0.0 107.4 107.1 -0.3 114.5 113.9 -0.5 04 116.5 121.1 4.6 107.9 113.4 5.4 114.3 118.5 4.3 05 124.6 126.8 2.3 102.8 116.1 13.3 118.9 124.3 5.4 06 133.7 132.2 -1.5 117.5 121.3 3.8 129.9 128.9 -1.0 07 118.7 120.2 1.6 106.1 102.9 -3.2 114.8 116.0 1.2 08 124.1 125.8 1.8 119.2 119.1 -0.1 122.6 124.1 1.5 09 120.8 125.4 4.6 103.4 101.8 -1.6 116.6 120.8 4.2 10 112.4 117.5 5.1 97.9 110.2 12.2 108.1 115.5 7.4 MEAN 120.7 123.7 3.0 108.5 112.4 3.9 117.4 120.7 3.4 SEM 1.9 1.5 0.9 2.2 2.2 1.8 1.9 1.5 1.0 Definition of abbreviations: IH, intermittent hypoxia; SBP, systolic blood pressure; SEM, standard error of the mean    141 Table B.6: Individual diastolic blood pressure data throughout SHAM and IH.  DBP (mmHg)  DAY NIGHT 24-h Participant ID SHAM  IH  IH - SHAM SHAM IH  IH - SHAM SHAM IH  IH - SHAM 01 65.2 70.6 5.4 54.4 60.8 6.4 62.6 67.6 5.1 02 59.6 63.0 3.4 56.3 52.9 -3.4 58.5 60.5 2.0 03 64.8 63.6 -1.2 54.7 55.5 0.8 61.8 60.9 -1.0 04 65.8 72.5 6.7 55.4 56.0 0.7 63.1 67.0 3.9 05 65.7 65.6 -0.1 48.7 51.9 3.3 61.3 62.4 1.2 06 62.5 69.3 6.8 53.3 53.0 -0.3 60.4 64.4 4.0 07 71.0 73.6 2.6 60.5 59.0 -1.5 67.8 70.0 2.3 08 69.3 65.7 -3.6 66.0 60.8 -5.2 68.3 64.4 -3.8 09 72.4 77.6 5.2 55.7 53.5 -2.3 68.4 72.9 4.5 10 62.5 64.6 2.1 52.1 60.4 8.3 59.4 63.5 4.1 MEAN 65.9 68.6 2.7 55.7 56.4 0.7 63.1 65.4 2.2 SEM 1.3 1.6 1.1 1.5 1.1 1.3 1.2 1.3 0.9 Definition of abbreviations: DBP, diastolic blood pressure; IH, intermittent hypoxia; SEM, standard error of the mean.    142 Table B.7: Individual mean arterial pressure data throughout SHAM and IH.  MAP (mmHg)  DAY NIGHT 24-h Participant ID SHAM  IH  IH - SHAM SHAM IH  IH - SHAM SHAM IH  IH - SHAM 01 84.7 89.6 4.8 74.8 80.6 5.8 82.3 86.8 4.5 02 78.1 82.9 4.9 73.3 72.6 -0.7 76.5 80.4 3.8 03 82.3 81.5 -0.8 72.3 72.7 0.5 79.4 78.6 -0.8 04 82.7 88.7 6.0 72.9 75.2 2.3 80.1 84.2 4.0 05 85.3 86.0 0.7 66.7 73.3 6.6 80.5 83.1 2.6 06 86.6 90.3 3.7 74.7 75.7 1.1 83.7 85.9 2.1 07 86.8 89.2 2.4 75.7 73.6 -2.1 83.3 85.3 2.0 08 87.5 85.7 -1.8 83.7 80.2 -3.5 86.4 84.3 -2.1 09 88.6 93.5 5.0 71.6 69.6 -2.1 84.5 88.9 4.4 10 79.2 82.2 3.1 67.4 77.0 9.6 75.6 80.8 5.2 MEAN 84.2 87.0 2.8 73.3 75.0 1.8 81.2 83.8 2.6 SEM 1.1 1.2 0.8 1.5 1.1 1.4 1.1 1.0 0.8 Definition of abbreviations: IH, intermittent hypoxia; MAP, mean arterial pressure; SEM, standard error of the mean.    143 Table B.8: Individual heart rate data throughout SHAM and IH.  HR (bpm)  DAY NIGHT 24-h Participant ID SHAM  IH  IH - SHAM SHAM IH  IH - SHAM SHAM IH  IH - SHAM 01 58.9 56.5 -2.4 50.8 46.4 -4.4 56.9 53.4 -3.5 02 47.0 51.4 4.4 49.7 46.5 -3.2 47.9 50.2 2.4 03 61.6 65.8 4.2 57.9 61.3 3.4 60.5 64.3 3.8 04 68.5 69.5 1.0 54.6 56.3 1.7 64.9 65.1 0.2 05 55.8 48.1 -7.7 39.7 37.9 -1.9 51.6 45.7 -5.9 06 57.1 63.0 5.9 42.9 46.4 3.5 53.8 58.0 4.2 07 72.6 69.7 -2.8 64.5 56.6 -7.9 70.1 66.5 -3.6 08 62.3 66.3 3.9 57.1 56.7 -0.4 60.8 63.8 3.0 09 49.1 56.6 7.4 45.3 44.9 -0.4 48.2 54.3 6.1 10 54.7 58.1 3.4 49.5 49.8 0.3 53.2 55.8 2.6 MEAN 58.8 60.5 1.7 51.2 50.3 -0.9 56.8 57.7 0.9 SEM 2.5 2.4 1.5 2.4 2.3 1.1 2.3 2.2 1.2 Definition of abbreviations: HR, heart rate; IH, intermittent hypoxia; SEM, standard error of the mean.    144 Table B.9: Individual pulse pressure data throughout SHAM and IH.  PP (mmHg)  DAY NIGHT 24-h Participant ID SHAM  IH  IH - SHAM SHAM IH  IH - SHAM SHAM IH  IH - SHAM 01 58.5 56.8 -1.7 60.9 59.2 -1.7 59.1 57.5 -1.6 02 55.5 59.9 4.4 50.7 58.9 8.2 54.0 59.7 5.7 03 52.6 53.9 1.2 52.7 51.6 -1.1 52.7 53.1 0.4 04 50.7 48.6 -2.1 52.6 57.3 4.7 51.2 51.5 0.3 05 58.8 61.2 2.4 54.1 64.1 10.0 57.6 61.9 4.3 06 71.2 62.9 -8.2 64.3 68.3 4.1 69.6 64.5 -5.0 07 47.7 46.6 -1.0 45.7 43.9 -1.7 47.0 46.0 -1.1 08 54.8 60.2 5.3 53.2 58.3 5.1 54.3 59.7 5.3 09 48.4 47.8 -0.6 47.7 48.3 0.6 48.2 47.9 -0.3 10 49.9 52.9 3.0 45.8 49.8 3.9 48.7 52.0 3.4 MEAN 54.8 55.1 0.3 52.8 56.0 3.2 54.2 55.4 1.1 SEM 2.2 1.9 1.2 1.9 2.4 1.3 2.1 2.0 1.1 Definition of abbreviations: IH, intermittent hypoxia; PP, pulse pressure; SEM, standard error of the mean.    145 B.4 Flow-Mediated Dilation Table B.10: Individual baseline brachial artery diameter throughout SHAM and IH in the morning (AM) and afternoon (PM).  Baseline Diameter (cm)  AM PM Participant ID SHAM IH SHAM IH 01 0.41 0.41 0.40 0.40 02 0.40 0.43 0.38 0.44 03 0.44 0.47 0.45 0.45 04 0.38 0.41 0.38 0.43 05 0.47 0.50 0.48 0.48 06 0.46 0.46 0.42 0.45 07 0.40 0.39 0.40 0.38 08 0.35 0.37 0.35 0.37 09 0.50 0.51 0.52 0.51 10 0.40 0.40 0.40 0.39 MEAN 0.42 0.43 0.42 0.43 SEM 0.01 0.02 0.02 0.01 Definition of abbreviations: IH, intermittent hypoxia; SEM, standard error of the mean.     146 Table B.11: Flow-mediated dilation individual peak response brachial artery diameter throughout SHAM and IH in the morning (AM) and afternoon (PM).  Peak Response Diameter (cm)  AM PM Participant ID SHAM IH SHAM IH 01 0.46 0.45 0.44 0.43 02 0.44 0.46 0.41 0.48 03 0.47 0.51 0.47 0.47 04 0.42 0.44 0.41 0.47 05 0.50 0.53 0.50 0.50 06 0.48 0.49 0.44 0.47 07 0.43 0.42 0.42 0.41 08 0.38 0.41 0.37 0.40 09 0.53 0.54 0.54 0.54 10 0.43 0.43 0.43 0.43 MEAN 0.45 0.47 0.44 0.46 SEM 0.01 0.02 0.02 0.01 Definition of abbreviations: IH, intermittent hypoxia; SEM, standard error of the mean.    147 Table B.12: Individual absolute change in brachial artery diameter throughout SHAM and IH in the morning (AM) and afternoon (PM).  Peak Response Diameter – Baseline Diameter (cm)  AM PM Participant ID SHAM IH SHAM IH 01 0.048 0.035 0.043 0.027 02 0.033 0.027 0.030 0.043 03 0.030 0.024 0.039 0.023 04 0.044 0.035 0.033 0.036 05 0.029 0.020 0.030 0.018 06 0.029 0.019 0.027 0.026 07 0.029 0.024 0.030 0.026 08 0.025 0.023 0.035 0.027 09 0.032 0.021 0.029 0.028 10 0.030 0.037 0.035 0.036 MEAN 0.033 0.027 0.033 0.029 SEM 0.002 0.002 0.002 0.002 Definition of abbreviations: IH, intermittent hypoxia; SEM, standard error of the mean.    148 Table B.13: Individual percent flow-mediated dilation throughout SHAM and IH in the morning (AM) and afternoon (PM).  FMD (%)  AM PM Participant ID SHAM IH SHAM IH 01 11.74 10.46 8.71 6.72 02 8.19 7.04 7.07 9.89 03 6.85 8.28 5.38 5.12 04 11.70 8.15 9.26 8.35 05 6.14 6.06 4.14 3.76 06 6.37 5.86 4.54 5.80 07 7.32 7.79 6.05 6.86 08 7.12 9.38 6.57 7.32 09 6.47 5.70 4.08 5.50 10 7.46 8.86 9.37 9.23 MEAN 7.94 7.76 6.52 6.85 SE 0.66 0.50 0.65 0.60 Definition of abbreviations: FMD, flow-mediated dilation; IH, intermittent hypoxia; SEM, standard error of the mean.    149 Table B.14: Individual allometrically scaled percent flow-mediated dilation throughout SHAM and IH in the morning (AM) and afternoon (PM).  Allometric FMD (%)  AM PM Participant ID SHAM IH SHAM IH 01 10.64 9.49 7.88 6.08 02 7.41 6.41 6.36 9.02 03 6.25 7.62 4.92 4.69 04 10.51 7.38 8.32 7.61 05 5.66 5.61 3.82 3.47 06 5.84 5.38 4.12 5.31 07 6.61 7.02 5.46 6.17 08 6.35 8.42 5.85 6.56 09 5.98 5.29 3.79 5.11 10 6.75 8.00 8.46 8.32 MEAN 7.20 7.06 5.90 6.23 SEM 0.58 0.44 0.57 0.54 Definition of abbreviations: FMD, flow-mediated dilations; IH, intermittent hypoxia; SEM, standard error of the mean.    150 Table B.15: Individual flow-mediated dilation total shear rate response (SRAUC) until peak diameter throughout SHAM and IH in the morning (AM) and afternoon (PM).  SRAUC (au)  AM PM Participant ID SHAM IH SHAM IH 01 36426.76 37077.24 27437.42 19340.14 02 22605.46 17514.62 12206.61 33022.79 03 24081.31 48204.83 13439.81 14791.82 04 64405.14 41212.53 35684.89 24360.36 05 20595.37 18121.21 17490.45 15975.03 06 18971.85 32211.29 15320.94 13109.9 07 32546.95 24252.84 12711.37 16489.34 08 27390.5 30846.62 24415.36 21115.24 09 24234.91 18614.33 8546.543 10506.43 10 43402.14 26419.62 32404.3 15414.01 MEAN 31466.04 29447.51 19965.77 18412.5 SEM 4383.621 3303.619 2964.515 2053.091 Definition of abbreviations: IH, intermittent hypoxia; SEM, standard error of the mean; SRAUC, shear rate area under the curve.    151 Table B.16: Flow-mediated dilation normalized to shear rate area under the curve throughout SHAM and IH in the morning (AM) and afternoon (PM).  FMD/SRAUC (103[%/au])  AM PM Participant ID SHAM IH SHAM IH 01 0.32 0.28 0.32 0.35 02 0.36 0.40 0.58 0.30 03 0.28 0.17 0.40 0.35 04 0.18 0.20 0.26 0.34 05 0.30 0.33 0.24 0.24 06 0.34 0.18 0.30 0.44 07 0.22 0.32 0.48 0.42 08 0.26 0.30 0.27 0.35 09 0.27 0.31 0.48 0.52 10 0.17 0.34 0.29 0.60 MEAN 0.27 0.28 0.36 0.39 SEM 0.02 0.02 0.04 0.03 Definition of abbreviations: FMD, flow-mediated dilation; IH, intermittent hypoxia; SEM, standard error of the mean; SRAUC, shear rate area under the curve.    152 Table B.17: Time from cuff release to peak vasodilation throughout SHAM and IH in the morning (AM) and afternoon (PM).  Time to Peak Vasodilation (s)  AM PM Participant ID SHAM IH SHAM IH 01 72.4 52.3 62.9 41.6 02 38.2 28.2 37.3 73.8 03 64.2 35.2 93.5 40.3 04 78.2 50.5 72.5 65.8 05 57.3 40.5 54.8 46.9 06 54.7 46.4 46.9 42.2 07 55.2 29.7 45.6 30.2 08 47.6 42.1 46.2 39.6 09 59.9 33.5 32.8 43.8 10 73.0 58.0 42.9 30.7 MEAN 60.1 41.6 53.5 45.5 SEM 3.9 3.2 5.8 4.4 Definition of abbreviations: IH, intermittent hypoxia; SEM, standard error of the mean.     153 Table B.18: Reactive hyperemia velocity envelope throughout SHAM and IH in the morning (AM) and afternoon (PM).  Velocity Envelope (cm/min)  AM PM Participant ID SHAM IH SHAM IH 01 2175 1960 2247 1212 02 2032 912 1426 2149 03 2279 935 3352 1199 04 2795 2533 2735 1773 05 1982 1329 1639 1072 06 1322 1189 3172 1368 07 2233 1455 2058 1624 08 1886 1608 2613 1862 09 1944 1091 2104 896 10 2685 2039 2129 1501 MEAN 2133 1505 2347 1466 SEM 132 168 196 123 Definition of abbreviations: IH, intermittent hypoxia; SEM, standard error of the mean.     154 Table B.19: Reactive hyperemia area under the curve envelope flow throughout SHAM and IH in the morning (AM) and afternoon (PM).  Blood Flow AUC (au)  AM PM Participant ID SHAM IH SHAM IH 01 40103 30839 49318 21637 02 30904 13155 25164 51641 03 36072 18235 77928 20480 04 34689 42073 47093 26297 05 36237 31736 41301 26843 06 26903 16849 45838 19223 07 41448 19532 37844 16002 08 23473 19390 25364 21381 09 46512 18492 53610 21079 10 51572 39437 34663 21837 MEAN 36791 24974 43812 24642 SEM 2709 3224 4852 3159 Definition of abbreviations: AUC, area under the curve; IH, intermittent hypoxia; SEM, standard error of the mean.      155 Table B.20: Reactive hyperemia shear rate area under the curve throughout SHAM and IH in the morning (AM) and afternoon (PM).  SRAUC (au)  AM PM Participant ID SHAM IH SHAM IH 01 48209 38998 53880 26763 02 37003 16791 26050 52822 03 37316 16626 56479 18523 04 45742 59888 54475 30997 05 27022 26405 25113 20192 06 21905 18473 37026 16965 07 51638 24773 50276 23014 08 43265 37272 43067 32407 09 30640 10819 35862 12889 10 60213 48570 43387 29158 MEAN 40295 29861 42562 26373 SEM 3732 5018 3616 3569 Definition of abbreviations: IH, intermittent hypoxia; SEM, standard error of the mean; SRAUC, shear rate area under the curve.     156  Table B.21: Reactive hyperemia peak blood flow response throughout SHAM and IH in the morning (AM) and afternoon (PM).  Peak Blood Flow Response (ml/min)  AM PM Participant ID SHAM IH SHAM IH 01 794.6 602.6 771.7 729.2 02 763.4 919.2 700.6 647.1 03 857.9 657.8 438.5 875.5 04 856.0 1067 735.1 805.4 05 948.3 719.8 596.5 480.3 06 724.9 1020.4 862.3 876.2 07 862.4 1124.6 572.9 543.3 08 477.3 550.3 434.8 542.3 09 1061.0 575.6 434.7 526.1 10 794.6 1128.2 695.9 731.3 MEAN 811.8 849.2 607.4 658.8 SEM 48.4 72.1 45.6 48.0 Definition of abbreviations: IH, intermittent hypoxia; SEM, standard error of the mean.    157 B.5 Vascular Strain Table B.22: Peak systolic strain individual data during morning and afternoon bouts of SHAM and IH. ID Carotid Artery Peak Systolic Strain (%) Femoral Artery Peak Systolic Strain (%) SHAM AM SHAM PM HX AM HX PM NX AM NX PM SHAM AM SHAM PM HX AM HX PM NX AM NX PM 01 7.16 7.86 7.65 6.09 7.67 6.49 1.37 1.62 2.08 1.53 2.06 3.54 02 7.06 7.42 8.30 8.64 9.4 9.20 1.73 1.44 2.26 1.04 3.18 1.19 03 9.44 7.05 9.25 7.91 9.86 7.61 4.11 4.37 2.41 2.54 2.45 2.27 04 9.57 9.45 7.56 7.61 11.43 7.27 2.95 2.46 2.20 2.63 2.9 3.13 05 6.64 7.63 8.71 7.49 8.33 7.48 1.49 2.13 1.61 1.46 1.56 1.81 06 9.03 8.46 8.06 7.26 11.02 8.60 2.07 3.62 2.12 1.41 2.25 1.86 07 8.04 6.57 9.11 5.55 7.44 5.65 1.47 1.22 0.83 0.84 1.29 1.06 08 11.09 10.50 12.25 10.81 13.29 11.79 3.13 3.58 1.58 1.44 1.54 2.05 09 8.62 11.46 7.67 6.13 7.05 7.14 1.58 2.02 0.61 1.08 2.25 1.73 10 10.44 10.34 9.04 8.98 11.72 8.22 2.12 2.77 3.05 2.17 2.63 1.99 MEAN 8.71 8.67 8.76 7.65 9.72 7.95 2.20 2.52 1.88 1.61 2.21 2.06 SEM 0.47 0.53 0.44 0.50 0.67 0.53 0.29 0.33 0.23 0.20 0.19 0.24 Definition of abbreviations: HX, hypoxia; NX, normoxia; SEM, standard error of the mean.    158 Table B.23: Early systolic strain rate individual data during morning and afternoon bouts of SHAM and IH. ID Carotid Artery Early Systolic Strain Rate (1/s) Femoral Artery Early Systolic Strain Rate (1/s) SHAM AM SHAM PM HX AM HX PM NX AM NX PM SHAM AM SHAM PM HX AM HX PM NX AM NX PM 01 0.36 0.40 0.43 0.34 0.44 0.33 0.07 0.13 0.12 0.09 0.12 0.20 02 0.45 0.37 0.50 0.53 0.55 0.58 0.11 0.10 0.15 0.06 0.21 0.07 03 0.58 0.44 0.53 0.47 0.58 0.46 0.26 0.24 0.15 0.19 0.15 0.12 04 0.55 0.55 0.42 0.50 0.67 0.42 0.20 0.17 0.15 0.18 0.19 0.20 05 0.37 0.44 0.46 0.46 0.45 0.42 0.09 0.13 0.09 0.09 0.09 0.10 06 0.51 0.44 0.47 0.39 0.54 0.44 0.11 0.19 0.15 0.09 0.15 0.10 07 0.47 0.40 0.54 0.29 0.44 0.30 0.08 0.07 0.04 0.05 0.08 0.05 08 0.68 0.64 1.09 0.88 1.05 0.99 0.21 0.25 0.12 0.14 0.12 0.20 09 0.63 0.73 0.40 0.35 0.35 0.37 0.12 0.16 0.03 0.08 0.16 0.10 10 0.84 0.81 0.67 0.76 0.81 0.69 0.20 0.20 0.25 0.21 0.21 0.16 MEAN 0.54 0.52 0.55 0.50 0.59 0.50 0.15 0.16 0.13 0.12 0.15 0.13 SEM 0.05 0.05 0.06 0.06 0.07 0.07 0.02 0.02 0.02 0.02 0.01 0.02 Definition of abbreviations: HX, hypoxia; NX, normoxia; SEM, standard error of the mean.   159 Table B.24: Late systolic strain rate individual data during morning and afternoon bouts of SHAM and IH. ID Carotid Artery Early Late Strain Rate (1/s) Femoral Artery Late Systolic Strain Rate (1/s) SHAM AM SHAM PM HX AM HX PM NX AM NX PM SHAM AM SHAM PM HX AM HX PM NX AM NX PM 01 -0.11 -0.13 -0.14 -0.13 -0.12 -0.11 -0.03 -0.02 -0.07 -0.04 -0.08 -0.07 02 -0.12 -0.10 -0.14 -0.16 -0.12 -0.13 -0.02 -0.05 -0.07 -0.03 -0.10 -0.02 03 -0.19 -0.16 -0.17 -0.14 -0.2 -0.17 -0.16 -0.15 -0.11 -0.07 -0.10 -0.07 04 -0.18 -0.13 -0.14 -0.18 -0.26 -0.12 -0.11 -0.09 -0.09 -0.12 -0.10 -0.11 05 -0.11 -0.11 -0.14 -0.14 -0.13 -0.14 -0.04 -0.05 -0.06 -0.06 -0.08 -0.09 06 -0.18 -0.13 -0.18 -0.19 -0.19 -0.18 -0.06 -0.06 -0.08 -0.07 -0.08 -0.04 07 -0.18 -0.15 -0.17 -0.13 -0.16 -0.14 -0.04 -0.03 -0.03 -0.03 -0.05 -0.03 08 -0.22 -0.20 -0.29 -0.24 -0.33 -0.20 -0.08 -0.14 -0.06 -0.07 -0.04 -0.08 09 -0.16 -0.14 -0.15 -0.15 -0.12 -0.11 -0.05 -0.05 -0.05 -0.04 -0.05 -0.03 10 -0.22 -0.18 -0.20 -0.18 -0.27 -0.13 -0.08 -0.06 -0.11 -0.04 -0.08 -0.07 MEAN -0.17 -0.14 -0.17 -0.16 -0.19 -0.14 -0.07 -0.07 -0.07 -0.06 -0.08 -0.06 SEM 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 Definition of abbreviations: HX, hypoxia; NX, normoxia; SEM, standard error of the mean.    160 B.6 Limb Blood Flow and Shear Table B.25: Brachial and common femoral artery diameter individual data during morning and afternoon bouts of SHAM and IH. Participant ID Brachial Artery Diameter (cm) Common Femoral Artery Diameter (cm) SHAM AM SHAM PM HX AM HX PM NX AM NX PM SHAM AM SHAM PM HX AM HX PM NX AM NX PM 01 0.42 0.41 0.43 0.41 0.43 0.41 - - - - - - 02 0.41 0.41 0.44 0.43 0.44 0.42 0.96 0.98 1.00 0.96 1.00 0.96 03 0.48 0.46 0.48 0.47 0.48 0.47 0.94 0.90 0.93 0.91 0.92 0.88 04 0.40 0.39 0.39 0.39 0.39 0.39 0.83 0.82 0.90 0.92 0.90 0.93 05 0.48 0.48 0.53 0.51 0.53 0.51 1.26 1.18 1.27 1.30 1.28 1.31 06 0.44 0.41 0.46 0.46 0.46 0.47 - - - - - - 07 0.39 0.39 0.38 0.37 0.38 0.37 0.95 0.93 0.90 0.87 0.92 0.87 08 0.35 0.36 0.40 0.37 0.40 0.37 0.83 0.78 0.90 0.86 0.89 0.88 09 0.49 0.50 0.47 0.48 0.48 0.48 1.16 1.17 1.19 1.14 1.19 1.12 10 0.41 0.38 0.41 0.41 0.41 0.40 1.05 1.01 1.03 1.02 1.00 1.02 MEAN 0.43 0.42 0.44 0.43 0.44 0.43 1.01 0.97 0.99 0.99 0.99 0.99 SEM 0.01 0.02 0.01 0.02 0.01 0.02 0.04 0.04 0.04 0.05 0.04 0.05 Dashes indicate exclusion from analysis due to inconsistent image quality. Definition of abbreviations: HX, hypoxia; NX, normoxia; SEM, standard error of the mean.    161 Table B.26: Brachial and common femoral artery mean shear rate individual data during morning and afternoon bouts of SHAM and IH. Participant ID Brachial Artery Mean Shear Rate (1/s) Common Femoral Artery Mean Shear Rate (1/s) SHAM AM SHAM PM HX AM HX PM NX AM NX PM SHAM AM SHAM PM HX AM HX PM NX AM NX PM 01 96.5 39.3 64.2 48.3 66.6 44.7 - - - - - - 02 57.4 40.8 71.3 62.1 54.0 76.7 31.2 34.0 33.2 30.1 31.7 29.9 03 137.6 27.3 108.0 33.4 128.4 38.1 38.7 58.7 41.1 39.9 54.5 37.4 04 213.5 165.3 154.4 68.1 170.9 115.1 81.6 71.9 33.5 31.1 42.9 28.4 05 62.2 36.5 56.3 34.5 61.2 32.1 27.5 27.0 17.5 24.3 19.4 21.5 06 36.5 34.7 56.9 42.7 62.0 41.6 - - - - - - 07 101.0 35.4 96.2 36.9 98.0 38.6 45.2 31.0 38.9 32.1 43.8 41.0 08 212.4 43.3 176.0 69.8 203.7 69.2 70.1 73.8 35.1 40.0 31.7 45.5 09 43.5 37.6 88.4 29.8 109.5 36.7 21.5 11.1 15.0 14.0 14.0 17.7 10 145.2 45.2 138.2 47.5 116.8 52.8 49.6 35.9 50.5 38.7 47.6 41.1 MEAN 110.6 50.5 101.0 47.3 107.1 54.6 47.0 39.3 31.2 29.9 33.0 31.0 SEM 20.6 12.9 13.4 4.7 15.9 8.2 5.9 6.8 3.6 3.0 4.4 3.5 Dashes indicate exclusion from analysis due to inconsistent image quality. Definition of abbreviations: HX, hypoxia; NX, normoxia; SEM, standard error of the mean.    162 Table B.27: Brachial and common femoral artery oscillatory shear index individual data during morning and afternoon bouts of SHAM and IH. Participant ID Brachial Artery Oscillatory Shear Index Common Femoral Artery Oscillatory Shear Index SHAM AM SHAM PM HX AM HX PM NX AM NX PM SHAM AM SHAM PM HX AM HX PM NX AM NX PM 01 0.07 0.26 0.11 0.23 0.10 0.28 - - - - - - 02 0.12 0.20 0.06 0.06 0.12 0.05 0.17 0.14 0.16 0.20 0.19 0.23 03 0.02 0.34 0.05 0.27 0.05 0.26 0.31 0.28 0.26 0.30 0.22 0.33 04 0.03 0.05 0.02 0.22 0.03 0.10 0.18 0.21 0.21 0.26 0.19 0.29 05 0.08 0.11 0.07 0.15 0.06 0.17 0.13 0.16 0.17 0.16 0.16 0.19 06 0.32 0.31 0.22 0.32 0.23 0.33 - - - - - - 07 0.06 0.17 0.06 0.22 0.08 0.21 0.15 0.19 0.19 0.20 0.19 0.17 08 0.00 0.26 0.04 0.23 0.02 0.29 0.16 0.13 0.26 0.27 0.29 0.25 09 0.16 0.22 0.03 0.28 0.01 0.24 0.32 0.40 0.37 0.35 0.38 0.33 10 0.08 0.27 0.07 0.22 0.13 0.22 0.21 0.19 0.20 0.19 0.23 0.19 MEAN 0.09 0.22 0.07 0.22 0.08 0.21 0.20 0.24 0.24 0.26 0.25 0.27 SEM 0.03 0.03 0.02 0.02 0.02 0.03 0.02 0.03 0.02 0.02 0.03 0.02 Dashes indicate exclusion from analysis due to inconsistent image quality. Definition of abbreviations: HX, hypoxia; NX, normoxia; SEM, standard error of the mean.     163 Table B.28: Brachial and common femoral artery mean blood flow individual data during morning and afternoon bouts of SHAM and IH. Participant ID Brachial Artery Mean Blood Flow (ml/min) Common Femoral Artery Mean Blood Flow (ml/min) SHAM AM SHAM PM HX AM HX PM NX AM NX PM SHAM AM SHAM PM HX AM HX PM NX AM NX PM 01 31.0 12.1 22.4 13.1 22.4 12.2 - - - - - - 02 16.6 11.9 25.0 20.9 19.6 25.1 123.8 139.3 143.8 121.2 139.2 116.4 03 62.2 12.0 51.3 15.1 60.8 16.8 138.0 199.6 149.6 141.6 184.5 123.3 04 57.0 41.4 39.4 17.6 44.1 30.4 212.6 184.8 112.9 115.5 144.4 106.6 05 31.3 16.5 39.2 23.0 41.2 20.3 242.9 179.5 156.6 234.1 184.7 209.9 06 13.4 11.2 23.7 18.4 25.8 17.4 - - - - - - 07 26.4 8.9 22.2 8.0 23.2 8.3 165.0 106.8 124.8 89.9 144.2 118.3 08 80.9 17.4 95.9 30.5 106.7 31.4 357.9 313.7 229.6 236.3 208.7 279.3 09 23.5 21.7 41.1 14.3 47.6 18.0 150.0 76.7 105.1 93.6 104.2 111.9 10 42.2 11.2 39.4 14.1 34.8 14.9 253.4 168.4 256.1 188.1 189.5 192.3 MEAN 38.5 16.4 40.0 17.5 42.6 19.5 218.8 157.9 142.5 144.2 144.2 147.3 SEM 7.0 3.0 7.0 2.0 8.3 2.4 24.0 22.6 19.1 20.6 15.7 21.6 Dashes indicate exclusion from analysis due to inconsistent image quality. Definition of abbreviations: HX, hypoxia; NX, normoxia; SEM, standard error of the mean.     164 B.7 Carotid Baroreflex Table B.29: The coefficient of determination (r2) of baroreflex modelling following SHAM and IH.  Absolute MAP Change in MAP Absolute HR Change in HR Participant ID SHAM IH SHAM IH SHAM IH SHAM IH 01 - - 0.997 0.970 0.973 0.996 0.885 0.993 02 0.912 0.966 - - - - - - 03 - - 0.998 0.990 0.936 0.996 0.916 0.989 04 - - 0.985 0.983 0.967 1.000 0.975 0.968 05 0.917 0.978 0.966 0.959 - - - - 06 - - - - - - - - 07 - - 0.997 0.978 0.996 0.978 0.993 0.976 08 0.961 0.970 - - - - 0.965 0.908 09 0.986 0.904 0.962 0.975 0.984 0.939 - - 10 0.942 0.943 0.985 0.989 0.995 0.941 - - MEAN 0.944 0.952 0.984 0.978 0.975 0.975 0.947 0.967 SEM 0.014 0.013 0.006 0.004 0.009 0.011 0.020 0.015 Dashes indicate exclusion from analysis due to poor modelling. Definition of abbreviations: HR, heart rate; IH, intermittent hypoxia; MAP, mean arterial pressure; SEM, standard error of the mean.   165 Table B.30: SHAM baroreflex parameters for mean arterial pressure.  Participant ID Operating Point Minimum Maximum Range Gain Centering Point Gmax Gop Threshold Saturation 01 - - - - - - - - - - 02 74.0 77.1 96.2 19.1 -0.09 64.2 -0.43 -0.36 31.9 96.6 03 - - - - - - - - - - 04 - - - - - - - - - - 05 82.4 79.1 89.2 10.1 -0.20 78.6 -0.51 -0.44 64.1 93.2 06 - - - - - - - - - - 07 - - - - - - - - - - 08 92.5 80.6 100.4 19.8 -0.05 104.4 -0.24 -0.22 42.5 166.2 09 86.7 76.3 95.0 18.7 -0.06 91.8 -0.28 -0.28 43.0 140.5 10 76.0 74.1 94.3 20.2 -0.06 75.3 -0.32 -0.32 28.2 122.3 MEAN 82.3 77.5 95.0 17.6 -0.09 82.9 -0.36 -0.32 41.9 123.8 SEM 3.4 1.1 1.8 1.9 0.03 6.9 0.05 0.04 6.3 13.7 Dashes indicate exclusion from analysis due to poor modelling. The units for operating point, minimum, maximum, range, centering point, threshold and saturation are mmHg and for gain, Gmax and Gop are mmHg/mmHg. Definition of abbreviations: Gmax, maximal gain; Gop, gain at operating point; SEM, standard error of the mean.     166 Table B.31: Intermittent hypoxia baroreflex parameters for mean arterial pressure. Participant ID Operating Point Minimum Maximum Range Gain Centering Point Gmax Gop Threshold Saturation 01 - - - - - - - - - - 02 79.7 77.8 85.9 8.0 -0.14 102.2 -0.29 -0.04 81.7 122.6 03 - - - - - - - - - - 04 - - - - - - - - - - 05 87.8 83.9 103.6 19.7 -0.31 83.1 -1.53 -0.93 73.6 92.6 06 - - - - - - - - - - 07 - - - - - - - - - - 08 84.0 79.8 94.1 14.3 -0.18 79.3 -0.65 -0.55 63.2 95.4 09 88.0 87.0 98.0 11.0 -0.26 79.3 -0.72 -0.25 68.0 90.7 10 87.3 83.0 98.2 15.2 -0.11 74.6 -0.42 -0.27 48.2 101.0 MEAN 85.4 82.3 95.9 13.6 -0.20 83.7 -0.72 -0.41 67.0 100.5 SEM 1.6 1.6 2.9 2.0 -0.04 4.8 0.22 0.15 5.6 5.8 Dashes indicate exclusion from analysis due to poor modelling. The units for operating point, minimum, maximum, range, centering point, threshold and saturation are mmHg and for gain, Gmax and Gop are mmHg/mmHg. Definition of abbreviations: Gmax, maximal gain; Gop, gain at operating point; SEM, standard error of the mean.    167 Table B.32: SHAM baroreflex parameters for change in mean arterial pressure. Participant ID Operating Point Minimum Maximum Range Gain Centering Point Gmax Gop Threshold Saturation 01 93.4 -5.4 5.3 10.7 -0.17 93.9 -0.45 -0.45 76.3 111.5 02 - - - - - - - - - - 03 91.0 -5.7 9.3 15.0 -0.09 97.0 -0.32 -0.30 62.9 131.0 04 78.9 -9.7 10.7 20.4 -0.05 80.0 -0.25 -0.25 18.8 141.2 05 82.4 -8.6 7.2 15.7 -0.04 85.6 -0.18 -0.17 19.6 151.6 06 - - - - - - - - - - 07 95.7 -6.5 5.4 11.9 -0.05 98.0 -0.13 -0.13 32.6 163.4 08 - - - - - - - - - - 09 86.7 -11.2 2.0 13.2 -0.20 95.4 -0.66 -0.33 80.7 110.1 10 76.0 -9.3 8.4 17.7 -0.05 82.8 -0.20 -0.20 18.7 147.0 MEAN 86.3 -8.1 6.9 15.0 -0.09 90.4 -0.31 -0.26 44.2 136.6 SEM 2.8 0.8 1.1 1.3 0.02 2.8 0.07 0.04 10.6 7.6 Dashes indicate exclusion from analysis due to poor modelling. The units for operating point, minimum, maximum, range, centering point, threshold and saturation are mmHg and for gain, Gmax and Gop are mmHg/mmHg. Definition of abbreviations: Gmax, maximal gain; Gop, gain at operating point; SEM, standard error of the mean.    168 Table B.33: Intermittent hypoxia baroreflex parameters for change in mean arterial pressure. Participant ID Operating Point Minimum Maximum Range Gain Centering Point Gmax Gop Threshold Saturation 01 89.9 -6.0 6.2 12.2 -0.08 90.7 -0.25 -0.25 54.8 126.6 02 - - - - - - - - - - 03 91.3 -6.9 6.7 13.7 -0.10 93.0 -0.35 -0.35 64.3 121.7 04 86.7 -7.2 12.3 19.4 -0.10 82.9 -0.50 -0.48 54.2 111.6 05 87.8 -6.6 8.6 15.2 -0.13 89.4 -0.48 -0.48 66.3 112.6 06 - - - - - - - - - - 07 94.4 -6.9 2.9 9.8 -0.08 103.0 -0.20 -0.18 67.4 138.6 08 - - - - - - - - - - 09 88.0 -12.6 2.4 15.0 -0.08 109.6 -0.29 -0.16 71.4 147.7 10 87.3 -6.8 8.1 14.9 -0.04 86.9 -0.14 -0.14 8.0 165.9 MEAN 89.3 -7.6 6.7 14.3 -0.09 93.6 -0.32 -0.29 55.2 132.1 SEM 1.0 0.9 1.3 1.1 0.01 3.5 0.05 0.06 8.2 7.5 Dashes indicate exclusion from analysis due to poor modelling. The units for operating point, minimum, maximum, range, centering point, threshold and saturation are mmHg and for gain, Gmax and Gop are mmHg/mmHg. Definition of abbreviations: Gmax, maximal gain; Gop, gain at operating point; SEM, standard error of the mean.     169 Table B.34: SHAM baroreflex parameters for heart rate. Participant ID Operating Point Minimum Maximum Range Gain Centering Point Gmax Gop Threshold Saturation 01 93.4 45.6 56.8 11.2 -0.25 92.0 -0.69 -0.67 80.2 103.8 02 - - - - - - - - - - 03 91.0 61.5 72.9 11.4 -0.11 68.5 -0.31 -0.09 41.1 96.0 04 78.9 56.0 69.8 13.8 -0.14 80.5 -0.48 -0.48 59.5 101.4 05 - - - - - - - - - - 06 - - - - - - - - - - 07 95.7 64.8 75.9 11.2 -0.05 95.8 -0.13 -0.13 32.9 158.7 08           09 86.7 42.0 57.0 15.0 -0.10 70.7 -0.38 -0.21 41.6 99.9 10 76.0 47.8 57.6 9.8 -0.22 74.0 -0.54 -0.51 60.6 87.3 MEAN 86.9 52.9 65.0 12.1 -0.14 80.2 -0.42 -0.35 52.6 107.8 SEM 3.3 3.8 3.6 0.8 0.03 4.6 0.08 0.10 7.1 10.4 Dashes indicate exclusion from analysis due to poor modelling. The units for operating point, centering point, threshold and saturation are mmHg. The units for minimum, maximum and range are beats per minute (bpm) and for gain, Gmax and Gop are bpm/mmHg. Definition of abbreviations: Gmax, maximal gain; Gop, gain at operating point; SEM, standard error of the mean.    170 Table B.35: Intermittent hypoxia baroreflex parameters for heart rate. Participant ID Operating Point Minimum Maximum Range Gain Centering Point Gmax Gop Threshold Saturation 01 89.9 44.1 63.3 19.2 -0.07 74.3 -0.33 -0.25 30.8 117.8 02 - - - - - - - - - - 03 91.3 55.2 65.8 10.6 -0.20 91.1 -0.53 -0.53 76.4 105.7 04 86.7 53.0 74.0 21.0 -0.05 80.6 -0.28 -0.27 24.6 136.7 05 - - - - - - - - - - 06 - - - - - - - - - - 07 94.4 62.9 73.0 10.1 -0.15 101.9 -0.38 -0.28 82.5 121.2 08 - - - - - - - - - - 09 88.0 46.4 59.4 13.0 -0.06 102.7 -0.21 -0.17 56.2 149.2 10 87.3 47.3 50.3 3.1 -0.19 103.9 -0.14 -0.02 88.4 119.4 MEAN 89.6 51.5 64.3 12.8 -0.12 92.4 -0.31 -0.25 59.8 125.0 SEM 1.2 2.9 3.6 2.7 0.03 5.2 0.06 0.07 11.1 6.3 Dashes indicate exclusion from analysis due to poor modelling. The units for operating point, centering point, threshold and saturation are mmHg. The units for minimum, maximum and range are beats per minute (bpm) and for gain, Gmax and Gop are bpm/mmHg. Definition of abbreviations: Gmax, maximal gain; Gop, gain at operating point; SEM, standard error of the mean.    171 Table B.36: SHAM baroreflex parameters for change in heart rate. Participant ID Operating Point Minimum Maximum Range Gain Centering Point Gmax Gop Threshold Saturation 01 93.4 -5.1 4.9 10.0 -0.15 97.7 -0.37 -0.33 77.5 117.9 02 - - - - - - - - - - 03 91.0 -4.4 2.5 6.9 -0.09 96.5 -0.16 -0.15 64.3 128.7 04 78.9 -7.0 6.7 13.7 -0.11 80.8 -0.36 -0.36 53.2 108.5 05 - - - - - - - - - - 06 - - - - - - - - - - 07 95.7 -11.4 4.5 15.9 -0.05 116.0 -0.21 -0.16 59.4 172.6 08 92.5 -11.5 3.2 14.8 -0.13 101.3 -0.48 -0.35 78.7 123.9 09 - - - - - - - - - - 10 - - - - - - - - - - MEAN 90.3 -7.9 4.3 12.2 -0.11 98.5 -0.31 -0.27 66.6 130.3 SEM 2.9 1.5 0.7 1.7 0.02 5.6 0.06 0.05 5.0 11.1 Dashes indicate exclusion from analysis due to poor modelling. The units for operating point, centering point, threshold and saturation are mmHg. The units for minimum, maximum and range are beats per minute (bpm) and for gain, Gmax and Gop are bpm/mmHg. Definition of abbreviations: Gmax, maximal gain; Gop, gain at operating point; SEM, standard error of the mean.    172 Table B.37: Intermittent hypoxia baroreflex parameters for change in heart rate. Participant ID Operating Point Minimum Maximum Range Gain Centering Point Gmax Gop Threshold Saturation 01 89.9 -5.1 4.0 9.0 -0.15 91.8 -0.33 -0.32 71.6 111.9 02 - - - - - - - - - - 03 91.3 -6.3 4.6 10.9 -0.10 95.5 -0.27 -0.26 65.6 125.3 04 86.7 -8.4 6.5 14.9 -0.06 96.2 -0.21 -0.19 43.8 148.7 05 - - - - - - - - - - 06 - - - - - - - - - - 07 94.4 -11.0 6.5 17.5 -0.05 106.2 -0.21 -0.20 45.3 167.0 08 84.0 -11.3 4.7 16.0 -0.07 100.1 -0.27 -0.20 56.0 144.2 09 - - - - - - - - - - 10 - - - - - - - - - - MEAN 89.3 -8.4 5.3 13.7 -0.08 97.9 -0.26 -0.24 56.5 139.4 SEM 1.8 1.2 0.5 1.6 0.02 2.4 0.02 0.03 5.5 9.6 Dashes indicate exclusion from analysis due to poor modelling. The units for operating point, centering point, threshold and saturation are mmHg. The units for minimum, maximum and range are beats per minute (bpm) and for gain, Gmax and Gop are bpm/mmHg. Definition of abbreviations: Gmax, maximal gain; Gop, gain at operating point; SEM, standard error of the mean.    173 Table B.38: Baseline measures prior to carotid baroreflex testing following SHAM and intermittent hypoxia.  MAP (mmHg) HR (bpm) LBF (ml/min) LVC (ml/min/mmHg) Participant ID SHAM IH SHAM IH SHAM IH SHAM IH 01 93.4 89.9 50.1 49.4 102.2 62.2 1.09 0.69 02 74.0 79.7 44.1 44.4 213.3 257.8 2.88 3.24 03 91.0 91.3 62.5 60.4 157.3 94.1 1.74 1.03 04 78.9 86.7 63.3 61.9 103.2 206.4 1.31 2.43 05 82.4 87.8 38.5 41.2 211.2 278.3 2.57 3.17 06 88.5 91.8 56.4 60.6 236.8 235.1 2.68 2.56 07 95.7 94.4 75.5 70.5 104.7 108.0 1.10 1.15 08 92.5 84.0 61.3 57.7 91.7 145.0 0.99 1.73 09 86.7 88.0 44.5 54.4 76.9 265.7 0.89 3.03 10 76.0 87.3 51.7 48.1 220.9 197.2 2.92 2.26 MEAN 85.9 88.1 54.8 54.9 151.8 185.0 1.82 2.13 SEM 2.4 1.3 3.5 2.9 19.9 24.6 0.27 0.29 Definition of abbreviations: HR, heart rate; IH, intermittent hypoxia; LBF, leg blood flow; LVC, leg vascular conductance; MAP, mean arterial pressure; SEM, standard error of the mean.    174 Table B.39: Carotid baroreflex-mediated absolute changes in leg blood flow following SHAM and intermittent hypoxia.  SHAM LBF (ml/min) IH LBF (ml/min) Target Pressure (mmHg) -80 -60 -40 -20 20 40 -80 -60 -40 -20 20 40 01 - 20.7 14.6 3.7 -41.6 -42.3 14.4 - 9.8 3.5 -12.8 -6.7 02 42.0 11.6 8.1 14.7 -12.8 -17.5 34.3 52.1 5.3 18.3 -40.4 -70.2 03 38.4 54.9 36.6 8.7 -27.6 -92.2 27.6 23.5 25.8 33.9 -21.4 -29.4 04 39.9 21.0 11.6 4.5 -47.9 -69.7 7.7 21.5 5.8 9.6 -38.1 -73.4 05 50.7 33.2 25.0 19.6 -58.2 -148.6 54.0 43.7 23.9 23.0 -87.2 -105.5 06 36.9 24.4 23.2 14.1 -47.3 -98.2 45.2 12.6 53.8 28.0 -63.2 -98.2 07 19.1 15.4 41.5 15.2 -24.4 -74.1 39.7 16.1 10.6 19.0 0.0 -9.7 08 24.2 16.2 15.0 14.7 -24.9 -27.0 25.8 34.9 22.2 24.4 -51.9 -128.3 09 - 50.3 18.2 14.9 -51.4 -55.8 115.1 28.5 53.9 34.4 -84.0 -96.2 10 108.3 92.1 46.1 38.0 -57.9 -30.8 60.3 47.9 33.3 29.6 -17.3 -46.4 MEAN 45.0 34.0 24.0 14.8 -39.4 -65.6 42.4 31.2 24.5 22.4 -41.6 -66.4 SEM 9.7 7.9 4.2 3.0 5.0 12.6 9.6 4.7 5.7 3.2 9.4 13.3 Dashes indicate exclusion from analysis due to poor image quality. Definition of abbreviations: IH, intermittent hypoxia; LBF, leg blood flow; SEM, standard error of the mean.     175 Table B.40: Carotid baroreflex-mediated percent changes in leg blood flow following SHAM and intermittent hypoxia.  SHAM LBF (%) IH LBF (%) Target Pressure (mmHg) -80 -60 -40 -20 20 40 -80 -60 -40 -20 20 40 01 - 22.7 9.2 4.0 -25.4 -25.5 26.1 - 14.3 5.9 -17.3 -8.2 02 26.7 7.0 5.9 8.4 -5.4 -9.1 12.8 11.9 2.1 3.0 -8.1 -12.8 03 15.1 29.5 21.3 3.0 -12.3 -20.4 17.0 16.2 13.8 16.4 -7.8 -16.4 04 20.8 22.2 8.7 3.7 -19.7 -29.1 5.2 13 4.6 5.8 -20.5 -30.5 05 17.5 18.1 10.5 6.5 -16.8 -36.1 14.5 11.7 6.2 6.1 -19.4 -23.6 06 20.0 14.4 12.3 6.8 -19.0 -35.4 17.3 4.1 14.6 16.2 -18.7 -35.4 07 19.6 11.4 28.0 10.5 -14.0 -28.8 32.6 12.5 10.4 19.0 0.3 -11.3 08 25.2 20.4 18.7 18.3 -15.9 -15.5 9.5 12.3 16.7 11.7 -15.0 -34.9 09 - 32.4 14.4 10.1 -26.8 -28.8 31.0 8.7 16.4 8.4 -20.3 -25.3 10 45.6 40.3 19.6 19.7 -16.4 -16.4 38.9 26.1 23.1 20.5 -7.6 -22.6 MEAN 23.8 21.8 14.9 9.1 -17.2 -24.5 20.5 12.9 12.2 11.3 -13.5 -22.1 SEM 3.4 3.2 2.2 1.8 2.0 2.8 3.5 2.0 2.0 2.0 2.3 3.1 Dashes indicate exclusion from analysis due to poor image quality. Definition of abbreviations: IH, intermittent hypoxia; LBF, leg blood flow; SEM, standard error of the mean.     176 Table B.41: Carotid baroreflex-mediated absolute changes in leg vascular conductance following SHAM and intermittent hypoxia.  SHAM LVC (ml/min/mmHg) IH LVC (ml/min/mmHg) Target Pressure (mmHg) -80 -60 -40 -20 20 40 -80 -60 -40 -20 20 40 01 - 0.33 0.25 0.09 -0.49 -0.52 0.22 - 0.16 0.08 -0.16 -0.11 02 0.74 0.40 0.29 0.32 -0.24 -0.20 0.80 1.13 0.31 0.52 -0.63 -1.14 03 1.07 0.77 0.57 0.18 -0.43 -1.13 0.46 0.40 0.42 0.52 -0.36 -0.42 04 0.88 0.31 0.28 0.07 -0.67 -0.77 0.30 0.41 0.14 0.21 -0.58 -0.90 05 1.03 0.66 0.49 0.49 -0.77 -1.72 0.95 0.77 0.58 0.34 -0.99 -1.33 06 0.69 0.40 0.39 0.28 -0.54 -1.21 0.68 0.29 0.67 0.40 -0.90 -1.24 07 0.29 0.27 0.56 0.21 -0.38 -0.85 0.54 0.32 0.18 0.27 -0.05 -0.11 08 0.46 0.30 0.26 0.22 -0.26 -0.36 0.70 0.65 0.29 0.26 -0.78 -1.66 09 - 0.79 0.53 0.40 -0.86 -0.52 1.84 0.82 1.08 1.10 -0.92 -0.90 10 1.88 1.35 0.87 0.61 -0.63 -0.45 0.86 0.70 0.45 0.40 -0.10 -0.53 MEAN 0.88 0.56 0.45 0.29 -0.53 -0.77 0.74 0.61 0.43 0.41 -0.55 -0.83 SEM 0.17 0.11 0.06 0.05 0.07 0.15 0.14 0.09 0.09 0.09 0.11 0.17 Dashes indicate exclusion from analysis due to poor image quality. Definition of abbreviations: IH, intermittent hypoxia; LVC, leg vascular conductance; SEM, standard error of the mean.     177 Table B.42: Carotid baroreflex-mediated percent change in leg vascular conductance following SHAM and intermittent hypoxia.  SHAM LVC (%) IH LVC (%) Target Pressure (mmHg) -80 -60 -40 -20 20 40 -80 -60 -40 -20 20 40 01 - 31.4 13.6 8.4 -26.9 -29.9 36.7 - 21.5 12.6 -21.0 -12.6 02 42.5 20.4 16.5 15.6 -8.7 -8.4 25.2 22.2 7.9 7.2 -10.3 -16.8 03 40.3 39.1 27.9 5.6 -17.7 -24.8 26.9 25.0 21.5 23.3 -13.0 -20.8 04 38.1 29.8 17.3 5.2 -24.7 -30.4 18.7 21.4 9.7 10.8 -29.5 -34.2 05 31.1 31.9 17.8 13.5 -19.5 -36.8 22.6 18.0 13.9 7.5 -21.4 -28.5 06 35.5 21.4 18.9 12.7 -20.0 -37.8 26.0 8.9 17.0 20.7 -25.7 -38.3 07 28.3 18.5 34.1 14.8 -22.0 -32.5 40.6 23.3 16.5 25.0 -3.6 -13.0 08 44.8 33.2 30.1 25.4 -16.2 -19.0 21.9 19.4 19.1 11.8 -19.6 -40.0 09 - 43.7 33.5 24.4 -34.4 -25.6 51.5 24.3 28.9 29.0 -21.2 -23.7 10 64.3 48.6 30.1 25.6 -17.6 -20.9 51.9 35.0 27.1 22.9 -4.0 -23.5 MEAN 40.6 31.8 24.0 15.1 -20.8 -26.6 32.2 22.0 18.3 17.1 -16.9 -25.2 SEM 3.9 3.2 2.5 2.5 2.2 2.8 3.9 2.3 2.2 2.5 2.8 3.1 Dashes indicate exclusion from analysis due to poor image quality. Definition of abbreviations: IH, intermittent hypoxia; LVC, leg vascular conductance; SEM, standard error of the mean.     178 Appendix C  : Reliability  C.1 Carotid intima-media thickness Table C.1: Reliability testing for carotid intima-media thickness. Participant ID Posterior CIMT 1 (mm) Posterior CIMT 2 (mm) 01 0.41 0.41 02 0.44 0.49 03 0.66 0.59 04 0.57 0.60 05 0.59 0.62 06 0.43 0.40 07 0.45 0.46 08 0.45 0.45 MEAN 0.50 0.50 SEM 0.03 0.03 RMS COV ± SEM 4.72±2.77% Measures were made in the morning under standardized conditions (identical to the above study) on two occasions separated by one week. Coefficient of variation (COV) was calculated using the root-mean squared (RMS) method. Definition of abbreviations: CIMT, common carotid artery intima-media thickness; SEM, standard error of the mean.     179 C.2 Flow-Mediated Dilation Measurements  Table C.2: Unpublished intraobserver flow-mediated dilation. ID FMD 1 (%) FMD 2 (%) Mean of FMD 1 and 2 (%) SD of FMD 1 and 2 (%) COV of FMD 1 and 2 COV2 1 3.3 3.4 3.3 0.1 0.035 0.001 2 12.4 13.5 12.9 0.7 0.057 0.003 3 9.3 6.7 8.0 1.8 0.225 0.051 4 13.4 14.1 13.8 0.5 0.037 0.001 5 9.5 10.7 10.1 0.8 0.083 0.007 6 3.0 3.2 3.1 0.1 0.035 0.001 Mean 8.5 8.6 8.5 0.7 0.079 0.011 SEM 1.8 2.0 1.9 0.3 0.030 0.008 RMS COV 10.37 RMS COV SEM 8.94 Coefficient of variation (COV) was calculated using the root-mean squared method, where the COV (SD/mean within participant) is squared, and the root mean multiplied by 100 to generate a percent COV. Definition of abbreviations: COV, coefficient of variation; FMD, flow-mediated dilation; RMS, root mean squared; SD, standard deviation; SEM, standard error of the mean.      180 Table C.3: Reliability testing for baseline flow-mediated dilation in the current study.  Participant ID FMD 1 (%) FMD 2 (%) COV of 1 and 2 COV2 01 11.7 10.5 0.081 0.007 02 8.2 7.0 0.107 0.011 03 6.8 8.3 0.134 0.018 04 11.7 8.1 0.253 0.064 05 6.1 6.1 0.010 0.000 06 6.4 5.9 0.060 0.004 07 7.3 7.8 0.044 0.002 08 7.1 9.4 0.194 0.038 09 6.5 5.7 0.089 0.008 10 7.5 8.9 0.121 0.015 MEAN 7.9 7.8 0.109 0.017 SEM 0.7 0.5 0.023 0.006 RMS COV Mean ± SEM 12.9±7.9% Measures were acquired in the morning of each experimental visit, before outfitting with the facemask. Definition of abbreviations: COV, coefficient of variation; FMD, flow-mediated dilation; RMS, root mean squared; SEM, standard error of the mean.    181 Table C.4: Baseline measurements of flow-mediated dilation measurements from the current study.  SHAM IH RMS COV P-value Allometrically Scaled FMD, % 7.25±0.62 6.96±0.45 12.8% 0.776 Baseline Diameter, cm 0.42±0.01 0.43±0.02 3.3% 0.021 Response Diameter, cm 0.45±0.01 0.47±0.01 3.3% 0.029 Delta Diameter, cm 0.033±0.002 0.033±0.002 12.7% 0.924 SRAUC, 1/s 31466±4383 29448±3304 26.4% 0.652 FMD/SRAUC, 103(%/au) 0.27±0.018 0.28±0.024 24.8% 0.675 Time to Peak Response, s 60.1±3.8 53.5±6.0 20.5% 0.233 Measures were acquired in the morning of each experimental visit, before outfitting with the facemask. Definition of Abbreviations: RMS COV, root mean squared coefficient of variation; FMD, flow-mediated dilation; IH, intermittent hypoxia; SRAUC, shear rate area under the curve.     182 C.3 Vascular Strain Measures  Table C.5: Reliability testing for peak systolic strain measurements.   CCA Peak Systolic Strain (%) CFA Peak Systolic Strain (%) Participant ID 1 2 1 2 01 8.30 8.29 1.84 2.08 02 5.03 9.83 2.77 2.41 03 9.60 10.70 3.77 3.29 04 9.94 10.74 3.09 2.48 05 5.80 8.66 3.67 1.98 06 12.59 9.28 2.57 3.15 07 8.84 8.14 1.30 1.49 08 11.59 12.34 3.42 2.06 09 8.56 7.59 2.90 3.58 10 10.39 13.44 2.61 2.89 MEAN 9.06 9.90 2.79 2.54 SEM 0.74 0.60 0.25 0.21 RMS COV Mean ± SEM 19.7±14.3% 20.2±13.8% Measures were made in the morning under standardized conditions (identical to the above study) on two occasions separated by one week. Coefficient of variation was calculated using the root-mean squared method. Definition of abbreviations: CCA, common carotid artery CFA, common femoral artery; COV, coefficient of variation; RMS, root mean squared; SEM, standard error of the mean.    183 Table C.6: Reliability testing for early systolic strain rate measurements.   CCA Early Systolic Strain Rate (1/s) CFA Early Systolic Strain Rate (1/s) Participant ID 1 2 1 2 01 0.51 0.46 0.12 0.13 02 0.29 0.60 0.17 0.16 03 0.61 0.66 0.22 0.21 04 0.64 0.64 0.21 0.18 05 0.28 0.49 0.23 0.12 06 0.78 0.53 0.15 0.17 07 0.52 0.50 0.08 0.08 08 0.77 0.97 0.26 0.20 09 0.62 0.44 0.24 0.19 10 0.91 1.16 0.25 0.23 MEAN 0.59 0.65 0.19 0.17 SEM 0.06 0.08 0.02 0.01 RMS COV Mean ± SEM 24.2±15.9% 17.0±13.8% Measures were made in the morning under standardized conditions (identical to the above study) on two occasions separated by one week. Coefficient of variation was calculated using the root-mean squared method. Definition of abbreviations: CCA, common carotid artery CFA, common femoral artery; COV, coefficient of variation; RMS, root mean squared; SEM, standard error of the mean.      184 Table C.7: Reliability testing for late systolic strain rate measurements.  CCA Late Systolic Strain Rate (1/s) CFA Late Systolic Strain Rate (1/s) Participant ID 1 2 1 2 01 -0.15 -0.14 -0.03 -0.05 02 -0.09 -0.15 -0.08 -0.09 03 -0.22 -0.26 -0.13 -0.14 04 -0.24 -0.15 -0.11 -0.08 05 -0.12 -0.16 -0.10 -0.07 06 -0.29 -0.18 -0.08 -0.11 07 -0.25 -0.19 -0.06 -0.07 08 -0.25 -0.29 -0.07 -0.07 09 -0.15 -0.12 -0.08 -0.06 10 -0.30 -0.32 -0.09 -0.12 MEAN -0.21 -0.20 -0.08 -0.09 SEM 0.02 0.02 0.01 0.01 RMS COV Mean ± SEM 21.8±12.3% 19.8±10.9% Measures were made in the morning under standardized conditions (identical to the above study) on two occasions separated by one week. Coefficient of variation was calculated using the root-mean squared method. Definition of abbreviations: CCA, common carotid artery CFA, common femoral artery; COV, coefficient of variation; RMS, root mean squared; SEM, standard error of the mean.     

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