Health and Social Development, Faculty of (Okanagan)
Health and Exercise Sciences, School of (Okanagan)
DSpace
UBCO
Tymko, Michael Martin
Doctor of Philosophy - PhD
University of British Columbia
Previous work has demonstrated that ascent to high altitude results in endothelial impairment (i.e. reduced); however, the mechanism(s) governing this impairment remain unclear. The overall objective of the current thesis, addressed in three separate studies, was to determine the role of the sympathetic nervous system (SNS) on endothelial function after ascent to high altitude. Study #1 investigated the effects of exercise-induced elevations in sympathetic nervous activity (SNA) on endothelial function [via non-invasive flow-mediated dilation (FMD)] at sea level (344m) and high altitude (3800m). The findings from this study demonstrated that endothelial function was not reduced at high altitude compared to sea level at rest. Furthermore, at sea-level, endothelial function was reduced via SNS related pathways after moderate intensity exercise; however, this was not observed at high altitude. Study #2 examined the influence of transient manipulations of SNA [via lower-body differential pressure] on endothelial function (via FMD) at sea level (344m) and high altitude (5050m). The primary findings indicated that endothelial function was impaired at rest, and at both sea level and high altitude, endothelial function was not altered after manipulating SNA. Based on previous investigations and studies #1 and #2 of the current thesis, we hypothesized that endothelial function was only reduced at altitudes >4000m. Study #3 assessed vascular function (via intra-arterial infusion of acetylcholine and sodium nitroprusside) at rest before and after local forearm autonomic blockade at sea level (344m) and high altitude (4300m) in lowlanders and Andean highlanders with and without excessive erythrocytosis (EE). The primary findings were that endothelial function was reduced in lowlanders at high altitude, and this reduction was abolished after local autonomic blockade. In addition, Andean participants with EE had reduced endothelial function compared to healthy Andean highlanders, which was partially normalized after local autonomic blockade. Collectively, the findings from study #3 demonstrate that high altitude related reductions in endothelial function are governed by elevated SNA. The observed disparity on the impact of high altitude on endothelial function at rest is likely related to the severity, and potentially the length of altitude exposure. In summary, we observed that endothelial function is reduced at rest at altitudes >4000m (e.g. study #2 and #3), and provide data indicating that manipulating SNA at high altitude via exercise and lower-body differential pressure has no impact on endothelial function (e.g. study #1 and #2), but endothelial function is fully restored in lowlanders, and partially restored in Andean highlanders with EE, after administration of full autonomic blockade (e.g. study #3).
The Impact of High Altitude on Endothelial Function in Humans by Michael Martin Tymko B.Sc., Mount Royal University, 2012 MSc. University of British Columbia, 2015 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctorate of Philosophy in THE COLLEGE OF GRADUATE STUDIES (Interdisciplinary Graduate Studies) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) May 2019 © Michael Martin Tymko, 2019 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled: The Impact of High Altitude on Endothelial Function in Humans submitted by Michael Martin Tymko in partial fulfillment of the requirements of the degree of Doctorate of Philosophy . Prof. Philip N. Ainslie Supervisor Dr. Neil Eves Supervisory Committee Member Dr. Glen Foster Supervisory Committee Member Dr. Chris West University Examiner Prof. Peter Raven External Examiner iii Abstract Previous work has demonstrated that ascent to high altitude results in endothelial impairment (i.e. reduced); however, the mechanism(s) governing this impairment remain unclear. The overall objective of the current thesis, addressed in three separate studies, was to determine the role of the sympathetic nervous system (SNS) on endothelial function after ascent to high altitude. Study #1 investigated the effects of exercise-induced elevations in sympathetic nervous activity (SNA) on endothelial function [via non-invasive flow-mediated dilation (FMD)] at sea level (344m) and high altitude (3800m). The findings from this study demonstrated that endothelial function was not reduced at high altitude compared to sea level at rest. Furthermore, at sea-level, endothelial function was reduced via SNS related pathways after moderate intensity exercise; however, this was not observed at high altitude. Study #2 examined the influence of transient manipulations of SNA [via lower-body differential pressure] on endothelial function (via FMD) at sea level (344m) and high altitude (5050m). The primary findings indicated that endothelial function was impaired at rest, and at both sea level and high altitude, endothelial function was not altered after manipulating SNA. Based on previous investigations and studies #1 and #2 of the current thesis, we hypothesized that endothelial function was only reduced at altitudes >4000m. Study #3 assessed vascular function (via intra-arterial infusion of acetylcholine and sodium nitroprusside) at rest before and after local forearm autonomic blockade at sea level (344m) and high altitude (4300m) in lowlanders and Andean highlanders with and without excessive erythrocytosis (EE). The primary findings were that endothelial function was reduced in lowlanders at high altitude, and this reduction was abolished after local autonomic blockade. In addition, Andean participants with EE had reduced endothelial function compared to healthy Andean highlanders, which was partially normalized after local autonomic blockade. Collectively, the findings from study #3 demonstrate that high altitude related reductions in endothelial function are governed by elevated SNA. The observed disparity on the impact of high altitude on endothelial function at rest is likely related to the severity, and potentially the length of altitude exposure. In summary, we observed that endothelial function is reduced at rest at altitudes >4000m (e.g. study #2 and #3), and provide data indicating that manipulating SNA at high altitude via exercise and lower-body differential pressure has no impact on endothelial function (e.g. study #1 and #2), but endothelial function is fully restored in lowlanders, and partially restored in Andean highlanders with EE, after administration of full autonomic blockade (e.g. study #3). iv Lay Summary Exposure to low oxygen conditions (i.e. high altitude) has been shown to impair blood vessel function; however, it is currently unknown why this impairment exists. The current thesis encompasses three separate studies conducted at sea level and high altitude, which examined the neural-related mechanism(s) associated with blood vessel impairment after exposure to high altitude. We demonstrated that blood vessel function was impaired above 4000m in altitude, and the reasoning for this impairment was due to heightened levels of neural activity.. v Preface The University of British Columbia Clinical Research Ethics Board approved all experiment chapters in this thesis (CRED IDs: H15-01513; H16-01028; and H17-02687). Chapter 1. Chapter one was written by Michael Tymko, and edited by Prof. Ainslie Chapter 2. No aspects of this chapter are currently, or under consideration, for publication. Michael Tymko drafted and Prof. Ainslie edited this chapter. Chapter 3. This chapter was published in the Journal of Physiology: Tymko, M.M., Tremblay, J.C., Hansen, A.B., Howe, C.A., Willie, C.K., Stembridge, M., Green, D.J., Hoiland, R.L., Subedi, P., Anholm, J.D., and Ainslie, P.N. (2017). The effect of 1-adrenergic blockade on post-exercise brachial artery flow-mediated dilatation at sea-level and high-altitude. Journal of Physiology. 595(5): 1671-1686. Michael Tymko and Prof. Ainslie designed the experimental protocols in this chapter. Michael Tymko drafted the manuscript, was responsible for all tables & figures, and is the corresponding author. Co-authors edited and approved the final version of the manuscript. CRED ID: H15-01513 Chapter 4. This chapter was published in the Journal of Applied Physiology: Tymko, M.M., Tremblay, J.C., Steinback, C.D., Hansen, A.B., Patrician, A., Howe, C.A., Hoiland, R.L., and Ainslie, P.N. (2017). UBC-Nepal Expedition: acute alterations in sympathetic nervous activity do not influence brachial artery endothelial function at sea-level and high-altitude. Journal of Applied Physiology. 123(5): 1386-1396. Michael Tymko and Prof. Ainslie designed the experimental protocols in this chapter. Michael Tymko drafted the manuscript, was responsible for all tables & vi figures, and is the corresponding author. Co-authors edited and approved the final version of the manuscript. CRED ID: H16-01028. Chapter 5. This chapter is currently being drafted for publication and will be submitted to the Circulation. Michael Tymko and Prof. Ainslie designed the experimental protocols in this chapter. Michael Tymko drafted the manuscript, was responsible for all tables & figures, and is the corresponding author. CRED ID: H17-02687. Chapter 6. This chapter was written by Michael Tymko, and edited by Prof. Ainslie Appendix A. This appendix was published in the Canadian multidisciplinary journal Facets: Tymko, M.M. (2016). How to build a lower-body differential pressure chamber integrated on a tilt-table: a pedagogy tool to demonstrate the cardiovagal baroreflex. Facets. 1: 225-244. Michael Tymko was responsible for the concept and design of the manuscript. Michael Tymko drafted the manuscript, was responsible for all tables & figures, and is the corresponding author. Appendix B. This appendix is currently under review in BMC Medical Research Methodology: Tymko, M.M., Hoiland, R.L., Tremblay, J.C., Stembridge, M., Dawkins, T., Coombs, G., Patrician, A., Howe, C.A., Gibbons, T., Moore, J., Steinback, C.D., Stacey, B.S., Bailey, D.M., MacLeod, D., Gasho, C., Anholm, J., Bain, A., Lawley, J., Hansen, A.B., Villafuerte, F., Vizcardo, G., Ainslie, P.N. (2018). The 2018 Global Research Expedition on Altitude-related Chronic Health (REACH) to Cerro de Pasco, Peru: An Experimental Overview. BMC Medical Research Methodology. UNDER REVIEW. Michael Tymko and Prof. Ainslie designed the experimental protocols in this chapter. Michael Tymko drafted the manuscript, was responsible for all tables & figures, and is the corresponding author. CRED ID: H17-02687. vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ........................................................................................................................ vii List of Tables ................................................................................................................................xv List of Figures ............................................................................................................................. xvi Acknowledgements .................................................................................................................... xix Dedication .....................................................................................................................................xx Glossary ...................................................................................................................................... xxi Chapter 1: Research Objectives, Aims, and Hypotheses ............................................................ 1 1.1 Study 1 (Chapter 3) ....................................................................................................... 1 1.1.1 Brief Background .................................................................................................... 1 1.1.2 Study 1 – Aim ......................................................................................................... 1 1.1.3 Study 1 – Hypotheses.............................................................................................. 2 1.2 Study 2 (Chapter 4) ....................................................................................................... 2 1.2.1 Study 2 – Brief Background ................................................................................... 2 1.2.2 Study 2 – Aim ......................................................................................................... 3 1.2.3 Study 2 – Hypotheses.............................................................................................. 3 1.3 Study 3 – (Chapter 5) .................................................................................................... 3 1.3.1 Study 3 – Brief Background ................................................................................... 3 1.3.2 Study 3 – Aims........................................................................................................ 4 1.3.3 Study 3 - Hypotheses .............................................................................................. 4 Chapter 2: Literature Review ...................................................................................................... 5 viii 2.1 The history of atmospheric oxygen............................................................................... 5 2.2 Sojourn to high altitude ................................................................................................. 7 2.3 Physiological overview of high altitude acclimatization .............................................. 8 2.3.1 Respiratory adaptation to high altitude ................................................................... 9 2.3.2 Cardiovascular and hematological adaptation to high altitude ............................. 11 2.4 The neural response to high altitude ........................................................................... 13 2.5 The Endothelium ......................................................................................................... 15 2.5.1 Methods of assessing vascular function ................................................................ 16 2.5.1.1 Non-invasive flow mediated dilation ............................................................ 16 2.5.1.2 Intra-arterial infusion of vasoactive substances ............................................ 19 2.5.1.3 Acetylcholine and sodium nitroprusside infusion......................................... 20 2.6 The influence of sympathetic nervous activity on endothelial function ..................... 21 2.6.1 Organization of the sympathetic nervous system ................................................. 21 2.6.2 Sympathetic nervous activity during cycling exercise (Study 1).......................... 23 2.6.3 Lower body differential pressure (Study #2) ........................................................ 26 2.6.4 Resting sympathetic hyper-activity at high altitude (Study #3) ............................ 28 2.7 Effect of high altitude on endothelial function ........................................................... 30 2.8 Other potential mechanisms of endothelial dysfunction at high altitude - oxidative stress 34 2.9 Permanent high altitude populations ........................................................................... 35 2.9.1 General overview of high altitude populations ..................................................... 35 2.9.2 Endothelial function in high altitude Andean dwellers ......................................... 37 2.10 Synopsis of literature review ...................................................................................... 38 ix Chapter 3: The effect of α1-adrenergic blockade on post-exercise brachial artery flow-mediated dilatation at sea-level and high-altitude .................................................................... 39 3.1 Background ................................................................................................................. 39 3.2 Aims and Hypotheses ................................................................................................. 41 3.3 Methods and Materials ................................................................................................ 41 3.3.1 Ethical Approval. .................................................................................................. 41 3.3.2 Participants. ........................................................................................................... 42 3.3.3 Experimental Design. ............................................................................................ 42 3.3.3.1 Maximal exercise protocols. ......................................................................... 43 3.3.3.2 Moderate-intensity exercise protocol. ........................................................... 44 3.3.4 Experimental Measurements. ................................................................................ 45 3.3.4.1 Cardiovascular measurements. ..................................................................... 45 3.3.4.2 Brachial artery imaging................................................................................. 45 3.3.5 Data Analysis ........................................................................................................ 46 3.3.5.1 Brachial artery diameter and blood flow analysis......................................... 46 3.3.6 Statistics ................................................................................................................ 46 3.3.6.1 Maximal exercise. ......................................................................................... 47 3.3.6.2 Moderate-intensity exercise. ......................................................................... 47 3.3.6.3 Adjusted flow-mediated dilatation. ............................................................... 48 3.4 Results ......................................................................................................................... 48 3.4.1 Participants and resting FMD data ........................................................................ 48 3.4.2 Maximal exercise data .......................................................................................... 49 3.4.3 Moderate intensity exercise at sea-level ............................................................... 51 x 3.4.4 Moderate intensity exercise at high-altitude ......................................................... 52 3.5 Discussion ................................................................................................................... 64 3.5.1 Endothelial function between sea-level and high-altitude at rest ......................... 64 3.5.2 Endothelial function before and after maximal exercise at sea-level and high-altitude65 3.5.3 Endothelial function before and after moderate-intensity exercise at sea-level and high-altitude ...................................................................................................................... 66 3.5.3.1 Sea-level:....................................................................................................... 66 3.5.3.2 High-altitude: ................................................................................................ 67 3.5.4 Effects of 1-adrenergic blockade on endothelial function at sea-level and high-altitude............................................................................................................................... 68 3.6 Methodological considerations. .................................................................................. 69 3.7 Synopsis ...................................................................................................................... 70 Chapter 4: Transient alterations in sympathetic nervous activity do not influence brachial artery endothelial function at sea-level and high-altitude ......................................................... 71 4.1 Background ................................................................................................................. 71 4.2 Aims and Hypotheses ................................................................................................. 73 4.3 Methods and Materials ................................................................................................ 74 4.3.1 Ethical Approval. .................................................................................................. 74 4.3.2 Participants. ........................................................................................................... 74 4.3.3 Experimental Design. ............................................................................................ 75 4.3.3.1 Experimental protocol. .................................................................................. 76 4.3.4 Experimental Measurements. ................................................................................ 77 xi 4.3.4.1 Cardiovascular measurements. ..................................................................... 77 4.3.4.2 Brachial artery imaging................................................................................. 77 4.3.4.3 Lower-body differential pressure chamber. .................................................. 78 4.3.4.4 Muscle sympathetic nerve activity. ............................................................... 78 4.3.5 Data Analysis ........................................................................................................ 79 4.3.6 Statistics ................................................................................................................ 80 4.3.6.1 Adjusted flow-mediated dilatation. ............................................................... 80 4.4 Results ......................................................................................................................... 82 4.4.1 Participants ............................................................................................................ 82 4.4.2 Endothelial function between sea-level and high-altitude. ................................... 82 4.4.3 Muscle sympathetic nervous activity at sea-level and high-altitude. ................... 83 4.4.4 Cardiovascular variables during LBNP and LBPP. .............................................. 83 4.4.4.1 Sea-level:....................................................................................................... 83 4.4.4.2 High-altitude: ................................................................................................ 84 4.4.5 Brachial artery responses during LBNP and LBPP. ............................................. 84 4.4.5.1 Sea-level:....................................................................................................... 84 4.4.5.2 High-altitude: ................................................................................................ 85 4.4.6 Endothelial function during LBNP and LBPP. ..................................................... 85 4.4.6.1 Sea-level:....................................................................................................... 85 4.4.6.2 High-altitude: ................................................................................................ 85 4.5 Discussion ................................................................................................................... 93 4.5.1 Effect of high-altitude on endothelial function ..................................................... 93 xii 4.5.2 Altering sympathetic nervous activity non-invasively with lower-body negative and lower-body positive pressure. .................................................................................... 94 4.5.3 Effect of sympathetic nervous activity on endothelial function at sea-level. ....... 95 4.5.4 Effect of sympathetic nervous activity on endothelial function at high-altitude. . 97 4.5.5 Methodological considerations ............................................................................. 98 4.5.6 Synopsis .............................................................................................................. 100 Chapter 5: Global REACH 2018: α1-adrenergic signaling impairs endothelial function in lowlanders and Andean highlanders with excessive erythrocytosis at 4300m. ...................... 101 5.1 Introduction ............................................................................................................... 101 5.2 Methods and Materials .............................................................................................. 104 5.2.1 Ethical Approval ................................................................................................. 104 5.2.2 Participants .......................................................................................................... 104 5.2.3 Experimental Design ........................................................................................... 105 5.2.4 General procedures. ............................................................................................ 106 5.2.5 Experimental measurements ............................................................................... 107 5.2.5.1 Brachial artery catheterization .................................................................... 107 5.2.5.2 Brachial artery imaging............................................................................... 108 5.2.6 Statistics .............................................................................................................. 108 5.3 Results ....................................................................................................................... 110 5.3.1 Participants .......................................................................................................... 110 5.3.2 Lowlanders .......................................................................................................... 111 5.3.2.1 Cardiovascular response ............................................................................. 111 5.3.2.2 Forearm vascular response .......................................................................... 111 xiii 5.3.3 Highlanders ......................................................................................................... 116 5.3.3.1 Cardiovascular response ............................................................................. 116 5.3.3.2 Forearm vascular response .......................................................................... 116 5.4 Discussion ................................................................................................................. 123 5.4.1 Effect of high altitude on endothelial function in lowlanders ............................. 123 5.4.2 Endothelial function between EE and non-EE highlanders ................................ 124 5.4.3 Methodological considerations ........................................................................... 126 5.5 Synopsis .................................................................................................................... 127 Chapter 6: Conclusion............................................................................................................. 128 6.1 Overview of research objectives and study findings ................................................ 128 6.2 Overall interpretation ................................................................................................ 130 6.2.1 Endothelial function after exercise induced elevations in sympathetic nervous activity130 6.2.2 Endothelial function during lower body differential pressure ............................ 132 6.2.3 Endothelial function during changes in sympathetic nervous activity at high altitude in lowlanders and Andean highlanders .............................................................. 137 6.2.4 Thesis key points................................................................................................. 139 6.3 The challenge of high altitude research .................................................................... 140 6.4 Future directions ....................................................................................................... 140 6.4.1 Targeting the magnitude of hypoxemia required to reduce endothelial function 141 6.4.2 Exploring the impact of arterial carbon dioxide on endothelial function ........... 141 6.4.3 Endothelial function across different high altitude populations ......................... 142 6.5 Clinical implications of high altitude research ......................................................... 143 xiv 6.6 Final thoughts............................................................................................................ 144 References ...................................................................................................................................145 Appendices ..................................................................................................................................165 Appendix A The 2018 Global Research Expedition on Altitude-related Chronic Health (REACH) to Cerro de Pasco, Peru: An Experimental Overview ............ 165 Appendix B How to build a lower-body differential pressure chamber integrated on a tilt-table: a pedagogy tool to demonstrate the cardiovagal baroreflex ........ 219 xv List of Tables Table 2.1 Summary of studies that investigated the impact of high altitude on brachial artery FMD. ............................................................................................................................................. 32 Table 2.2 Estimates for haematological parameters in high-altitude dwellers and low-landers at ~4000m. ........................................................................................................................................ 37 Table 3.1 Brachial artery diameter, shear, and flow-mediated dilatation data during baseline and post-maximal exercise at sea-level and high-altitude ................................................................... 54 Table 3.2 Brachial artery diameter and flow-mediated dilatation data during baseline and post moderate-intensity exercise at sea-level and high-altitude ........................................................... 55 Table 4.1 Cardiovascular variables at baseline, and during lower-body differential pressure at sea-level and high-altitude. ........................................................................................................... 86 Table 4.2 Brachial artery variables during the control and lower-body differential pressure trials at sea-level and high-altitude. ....................................................................................................... 87 Table 5.1 Lowlander and highlander participant demographics ................................................. 110 Table 5.2 Systemic hemodynamics in lowlanders at sea level and high altitude. ...................... 112 Table 5.3 Systemic hemodynamics in lowlanders at sea level and high altitude. ...................... 113 Table 5.4 Systemic hemodynamics in highlanders at high altitude. ........................................... 118 Table 5.5 Systemic hemodynamics in highlanders ..................................................................... 119 Table 6.1 Summary of studies that investigated the impact of high altitude on brachial artery FMD. ........................................................................................................................................... 135 xvi List of Figures Figure 2.1 An estimation of atmospheric oxygen content over the earth’s lifespan....................... 5 Figure 2.2 The estimated change in oxygen concentration over time. ........................................... 6 Figure 2.3 Central (CCR) and peripheral (PCR) respiratory chemoreceptors traditional negative feedback arcs. ................................................................................................................................ 10 Figure 2.4 The temporal changes to ventilation with high altitude exposure. .............................. 11 Figure 2.5 Relative contributions of PaO2 and [Hb] to the high altitude related increase in CaO2........................................................................................................................................................ 13 Figure 2.6 Recordings of SNA via microneurography at sea level, after 4 weeks at 5260m of altitude, and 3 days post-descent from altitude. ............................................................................ 14 Figure 2.7 Endothelial mediated nitric oxide release resulting in smooth muscle relaxation. Refer to text for a brief overview of these mechanism(s). ...................................................................... 16 Figure 2.8 Shear-stress mediated nitric oxide release resulting in smooth muscle relaxation. Refer to text for an overview of these mechanism(s). ............................................................................ 17 Figure 2.9 A depiction of the brachial shear stress and diameter response during a FMD test. ... 19 Figure 2.10 ACH and SNP mediated NO release resulting in smooth muscle relaxation. Refer to text for an overview of these mechanism(s). ................................................................................ 21 Figure 2.11 Schematic of neural control of the vasculature. ........................................................ 23 Figure 2.12 The SNA response to exercise. Printed with permission from Ichinose et al. (2008)25 Figure 2.13 Picture of the custom built LBNP/LBPP chamber during experimental protocol in Nepal at 5050m. ............................................................................................................................ 28 Figure 2.14 Picture of a typical experimental set-up to assess SNA via microneurography. ....... 30 Figure 2.15 Forest plot of the findings related to brachial artery FMD outlined in the table 2.1. 33 xvii Figure 3.1 FMD data collected during baseline on placebo and prazosin at sea-level and high-altitude........................................................................................................................................... 56 Figure 3.2 Cardiovascular data during baseline, maximal exercise, and post-maximal exercise at sea-level and high-altitude. ........................................................................................................... 57 Figure 3.3 Individual FMD data collected at baseline and post-maximal exercise at sea-level and high-altitude. ................................................................................................................................. 58 Figure 3.4 Cardiovascular data during baseline, moderate-intensity exercise, and post-exercise on placebo and prazosin at sea-level and high-altitude. .................................................................... 60 Figure 3.5 Brachial artery shear rate data during baseline, moderate-intensity exercise, and post-exercise on placebo and prazosin at sea-level and high-altitude. ................................................. 61 Figure 3.6 Brachial artery velocity, diameter, blood flow and conductance data collected during baseline, moderate-intensity exercise, and post-exercise on placebo and prazosin at sea-level and high-altitude. ................................................................................................................................. 62 Figure 3.7 Individual FMD data collected during baseline and post-exercise on placebo and prazosin at sea-level and high-altitude. ......................................................................................... 63 Figure 4.1 A schematic representation of the experimental protocol conducted at sea-level and high-altitude, and raw MSNA neurogram in two participants. ..................................................... 88 Figure 4.2 A comparison of absolute and relative brachial artery flow-mediated dilation response between sea-level and high-altitude. ............................................................................................. 89 Figure 4.3 Muscle sympathetic nervous activity during LBNP, control, and LBPP trials at sea-level. .............................................................................................................................................. 90 Figure 4.4 Muscle sympathetic nervous activity at rest between sea-level and high-altitude. ..... 91 xviii Figure 4.5 Brachial artery shear rate and diameter response to forearm cuff release at sea-level and high-altitude. .......................................................................................................................... 92 Figure 5.1 Schematic of experimental design. ............................................................................ 107 Figure 5.2 Absolute forearm vascular conductance in lowlanders at sea level and high altitude...................................................................................................................................................... 114 Figure 5.3 Relative forearm vascular conductance in lowlanders at sea level and high altitude...................................................................................................................................................... 115 Figure 5.4 Absolute forearm vascular conductance in Andean highlanders at high altitude. .... 120 Figure 5.5 Relative forearm vascular conductance in Andean highlanders at high altitude....... 121 Figure 5.6 Absolute forearm vascular conductance normalized to forearm volume in Andean highlanders at high altitude. ........................................................................................................ 122 Figure 6.1 Updated forest plot of studies that have investigated the impact of high altitude on brachial artery flow mediated dilation. ....................................................................................... 136 xix Acknowledgements Most importantly, I would like to thank Prof. Philip Ainslie - my PhD supervisor - for providing his guidance throughout my undergraduate, MSc, and PhD degrees. You have been one of my most influential mentors from both a personal and academic standpoint. Dr. Christopher Willie, I helped you through your PhD thesis by drinking martini’s with you, and now I wish you could have been here to help me through mine. You were one of my most influential life mentors in my adult years. I will miss you always. Dr. Trevor Day, you are the main reason why I am here today. Thank you for your mentorship and for continuing to challenge me throughout my undergraduate and graduate degrees. Most of all, thank you for being an incredible friend. I would like to give special thanks to my committee members Dr. Neil Eves and Dr. Glen Foster for their support throughout both my MSc and PhD degrees. You two have always made time for me whenever I was in need. To my parents, sisters, and of course, my wife, Courtney. Thank you for your love and support throughout all these years. Lastly, to the boys, rain or shine you guys have been there for me. You will always be an important part of my life. xx Dedication This one is for all the SB’s out there. xxi Glossary ACH, acetylcholine CaO2, arterial oxygen content cGMP, cyclic guanosine 3’,5’-monophosphate CMS, chronic mountain sickness CO, cardiac output EE, excessive erythrocytosis FMD, flow-mediated dilatation FVC, forced vital capacity GTP, guanosine 5’-triphosphate Hb, hemoglobin HR, heart rate LBNP, lower-body negative pressure LBPP, lower-body positive pressure MAP, mean arterial pressure MSNA, muscle sympathetic nervous activity NO, nitric oxide PaCO2, partial pressure of arterial oxygen PaO2, partial pressure of arterial oxygen PKG, protein kinase G ROS, reactive oxygen species SaO2, arterial oxyhemoglobin saturation SNA, sympathetic nervous activity xxii SNP, sodium nitroprusside SNS, sympathetic nervous system SpO2, peripheral oxyhemoglobin saturation SRAUC, shear rate area under the curve SV, stroke volume TPR, total peripheral resistance 1 Chapter 1: Research Objectives, Aims, and Hypotheses 1.1 Study 1 (Chapter 3) 1.1.1 Brief Background Flow-mediated dilation (FMD) is a commonly used, non-invasive measurement of conduit artery diameter in response to an imposed change in shear stress; it is widely accepted to be an index of endothelial function (Thijssen et al., 2011), which is largely nitric oxide (NO) mediated (Green et al., 2014). It has been demonstrated that immediately after cycling exercise, endothelial function, measured by brachial FMD in the non-exercising limb is transiently reduced (Jones et al., 2010, Birk et al., 2013, Dawson et al., 2013, Atkinson et al., 2015), and the reduction in endothelial function is inversely related to exercise intensity (Birk et al., 2013). The observed reduction in post-exercise endothelial function has been attributed to increased sympathetic nervous activity (SNA) since this impairment is abolished after administration of the oral α1-adrenergic blockade (via prazosin). This relationship between endothelial dysfunction and exercise has yet to be determined at high altitude where SNA is chronically elevated. 1.1.2 Study 1 – Aim Our objective was to quantify endothelial function (via brachial artery FMD) at sea-level (344m) and high-altitude (3800m) at rest and following both maximal exercise and 30-minutes of moderate-intensity cycling exercise (50% of maximal achieved wattage) with and without administration of 1-adrenergic blockade. 2 1.1.3 Study 1 – Hypotheses We hypothesized that: 1) at rest, FMD would be impaired at high-altitude compared to sea level, 2) FMD would be reduced to a greater extent at high altitude compared to sea level after moderate and maximal exercise, and 3) reductions in endothelial function following moderate-intensity exercise at both sea-level and high-altitude would be mediated via an α1-adrenergic pathway. 1.2 Study 2 (Chapter 4) 1.2.1 Study 2 – Brief Background The role of SNA on endothelial function has been examined in several investigations in young, healthy humans. These studies have revealed that FMD was impaired under conditions in which SNA was transiently elevated, such as lower-body negative pressure (LBNP) (Victor and Leimbach, 1987, Scherrer et al., 1988), cold pressor test (Dyson et al., 2006), mental stress (Ghiadoni et al., 2000), and immediately after cycling exercise (Tymko et al., 2016, Atkinson et al., 2015, Jones et al., 2010, Birk et al., 2013, Dawson et al., 2013). Additionally, exposure to hypobaric hypoxia (e.g. high altitude), which markedly elevates resting SNA (Hansen and Sander, 2003, Duplain et al., 1999), has been demonstrated to reduce endothelial function in some (Bakker et al., 2015, Lewis, et al., 2014), but not all cases (Tymko et al., 2016, Tremblay et al., 2017, Bruno et al., 2015, Bruno et al., 2016). In our previous study (i.e. Study #1) our research group found that endothelial function was reduced immediately after moderate-intensity exercise at sea level, but not high altitude. Since exercise also elicits significant changes to heart rate (HR), stroke volume (SV), blood pressure, and blood flow patterns, which can all impact endothelial function, we attempted to address this research question using a different methodological strategy. 3 1.2.2 Study 2 – Aim Our objective was to quantify endothelial function (via brachial artery FMD) at sea level (344m) and high altitude (5050m) while manipulating SNA independent of hemodynamics non-invasively using lower-body differential pressure. 1.2.3 Study 2 – Hypotheses We hypothesized that: 1) at rest, SNA would be elevated, and endothelial function would be reduced at high altitude compared to sea level, 2) at sea level and after acclimatization to high altitude, endothelial function would be reduced during a transient increase in SNA (induced by mild lower-body negative pressure), and elevated during a transient decrease in SNA (induced by mild lower-body positive pressure), independent of changes in systemic hemodynamics. 1.3 Study 3 – (Chapter 5) 1.3.1 Study 3 – Brief Background The effects of high altitude on endothelial function in lowlanders has been controversial. For example, several studies report that endothelial function remains unchanged after exposure to hypobaric hypoxia (Tymko et al., 2016, Tremblay et al., 2017, Bruno et al., 2015, Bruno et al., 2016) while others indicate that endothelial function is reduced (Lewis, et al., 2014, Bakker et al., 2015, Tymko et al., 2017, Tremblay et al., 2018). The discrepancy between these studies could be due to the severity of altitude exposure since the studies that have demonstrated that high altitude reduces endothelial function have all been conducted at altitudes >4000m. Additionally, the mechanism(s) associated with the potential endothelial dysfunction at high altitudes >4000m have yet to be fully elucidated. 4 1.3.2 Study 3 – Aims To determine if high altitude (>4000m) induced elevations in SNA contribute to endothelial impairment in lowlanders at high altitude, and in Andean highlanders with and without excessive erythrocytosis (EE). 1.3.3 Study 3 - Hypotheses We hypothesized that 1) high altitude related reductions in endothelial function in lowlanders, and 2) endothelial dysfunction in Andean highlanders with EE compared to non-EE highlanders, would be due to elevated SNA adrenergic signaling. To test these hypotheses we employed a novel, randomized, experimental design involving step-wise intra-arterial infusions of acetylcholine (ACH) and sodium nitroprusside (SNP) to elicit controlled forearm blood flow responses that are mechanistically-linked to endothelial and smooth muscle function, respectively (Tousoulis et al., 2005). These vasoactive drugs (i.e. ACH and SNP) were administered before and after local full adrenergic blockade (via phentolamine and propranolol). 5 Chapter 2: Literature Review 2.1 The history of atmospheric oxygen Our current oxygen enriched environment ceased to exist in the early years of the Earth’s history. Originally, the composition of earth’s atmosphere consisted mainly of hydrogen, carbon dioxide, carbon monoxide, hydrogen sulfide and methane; however, approximately 2.5 billion years ago an explosion of oxygen into the atmosphere occurred - now known as the “Great Oxygenation Event” (see Figure 2.1). This pivotal moment in earth’s antiquity was likely due to the emergence of photosynthesising cyanobacteria, which took advantage of Earth’s anaerobic atmosphere and triggered the forthcoming inception of diverse multi-cellular organisms that we have on earth today. Figure 2.1 An estimation of atmospheric oxygen content over the earth’s lifespan. Adapted from Martin et al. (2017) with permission. 6 Oxygen, once described as the “most important discovery in the history of science” has been continuously monitored over the past three decades by several research stations across the globe. This effort has recently demonstrated that atmospheric oxygen concentration is declining at a parabolic rate much greater than originally suggested (Martin et al., 2017). Assuming that oxygen decline will occur at the same rate that has been observed since 1989, it has been proposed that the concentration of atmospheric oxygen at sea level (currently at ~20.93%), will be reduced by ~50% in ~3600 years (roughly equivalent to the oxygen availability at Mt. Everest basecamp), and be completely absent from earth’s atmosphere within ~5000 years (see Figure 2.2). Reasoning for the observed rapid decline in atmospheric oxygen is due to the imbalance of oxygen consumption (e.g. increase in the number of oxygen consuming animals and burning of fossil fuels) and oxygen destruction (e.g. reduction in the number of photosynthesizing plants). Technological advancements aimed to facilitate human colonization on other planets is well within our species grasp within the next few centuries. However, if humans fail to achieve this interstellar objective within an appropriate time-frame, and/or we populate a low-oxygen environment, understanding the physiological mechanism(s) associated with high altitude acclimatization will be critical for the success of our species. Figure 2.2 The estimated change in oxygen concentration over time. 7 The dotted line represents the estimated time until oxygen availability at sea level declines to the equivalent of Mt. Everest basecamp, which is likely near the limit of long-term human tolerance. Adapted from Martin et al. (2017) with permission. 2.2 Sojourn to high altitude Traveling to the Earth’s highest plateaux used to be accessible only to the worlds adventure seekers and mountaineers; however, with the vast growth of the internet and air travel, these once considered exotic regions of the earth are now well-documented and affordable to reach by many. It has been estimated that >100 million individuals travel to high altitude locations each year (Burtscher et al., 2001, Burtscher, 1999). Popular regions of travel are the mountainous regions of Nepal (e.g. Himalaya), Africa (e.g. Mt. Kilimanjaro), Peru (e.g. Machu Picchu), Switzerland (e.g. The Alps), and North America (e.g. Rocky Mountains). The definitions of low, intermediate, and high altitude vary amongst the literature - below are some generally accepted definitions of altitude. Low altitude is typically defined in-between 0-1000m, and at this altitude, there are no altitude-related physiological adaptations detected in healthy humans. Intermediate altitude is defined as in-between 1000-2500m. Here, some altitude-related physiological adaptations may become noticeable (e.g. changes in ventilation); however, the percentage of hemoglobin bound to oxygen likely remains high and unaltered in healthy individuals at this particular altitude. Altitude illness has been reported at <2500m (Finsterer, 2012), but this is extremely rare. High altitude is typically defined as 2500-3500m. Here, some individuals may experience mild breathlessness, onset of altitude illness, and sleep disturbances (Hackett et al., 1976, Bartsch and Swenson, 2013). Above 3500m of altitude is considered as “very” high altitude where marked hypoxemia exists, and ~50-80% of individuals may experience mild or severe forms of illness (Hackett et al., 1976, Bartsch and Swenson, 2013). Each of the studies conducted in the current thesis were completed at low altitude (344m; Kelowna, BC, Canada), and then were repeated at very high altitude [3800m 8 (White Mountain, California; Study #1), 5050m (Nepal, Ev-K2-CNR pyramid laboratory; Study #2), and 4300m (Cerro de Pasco, Peru; Study #3)]. The process that protects individuals from the detrimental effects of hypoxemia is known as “high altitude acclimatization” and it is a critical adaptation for performance and survival beyond low-to-intermediate altitudes. If the degree of hypobaric hypoxia is substantial enough to reduce arterial oxygen saturation of hemoglobin (SaO2) then the acclimatization process begins immediately, and the majority of adaptations occur within the first days at altitude, but some forms of acclimatization may continue for several months, and potentially years (Ainslie et al., 2013, Porcelli et al., 2017). Despite appropriate acclimatization to altitude performing vigorous tasks can still be challenging. To illustrate this point, and the related physiological challenges of high altitude, the following is a quote by Horace-Benedict de Daussure when he was climbing Mont Blanc (altitude 4810m) in 1787. “I therefore hoped to reach the crest in less than three quarters of an hour; but the rarity of the air gave me more trouble than I could have believed. At last I was obliged to stop for breath every fifteen or sixteen steps….This need of rest was absolutely unconquerable; if I tried to overcome it, my legs refused to move” 2.3 Physiological overview of high altitude acclimatization The physiological processes of high altitude acclimatization are multi-faceted. Upon immediate exposure to high altitude the elevations in respiration that occur are likely the most important, followed by more long-term hematological changes that result in increased hemoglobin (Hb) mass and concentration. These important physiological adaptations (i.e. respiratory and hematological) 9 both contribute to an increase in arterial oxygen content (CaO2), which is crucial for survival at high altitude. Below is a brief overview of these critical physiological mechanism(s). 2.3.1 Respiratory adaptation to high altitude The increase in ventilation that takes place in the first few days at altitude is one of the most important aspects of the acclimatization process. Increases in ventilation resulting in a higher partial pressure of alveolar and arterial oxygen (PaO2) and lower arterial carbon dioxide (PaCO2). Higher PaO2 also facilitates improvements in CaO2, and as such, convective oxygen delivery to tissue. The cause of this respiratory response to high altitude is coordinated in part through central and peripheral respiratory chemoreceptor pathways, each with their own respective sensitivities following a change in chemical stimulus (e.g. PaO2, PaCO2, and pH). The exact location of the central respiratory chemoreceptors has not been identified, but there is good evidence that they are located throughout the brainstem, especially within the retrotrapezoid and the raphe nuclei (Guyenet et al., 2005, Nattie, E., 2001, Nattie, E., 2011, Kumar et al., 2015). The central chemoreceptors respond more slowly to stimuli compared to the peripheral chemoreceptors, which are located in the carotid and aortic bodies (Fitzgerald et al., 2009, Iturriaga and Alcayaga, 2004, Nurse, 2009, Torrance, 1996). The central respiratory chemoreceptors respond to alterations in hydrogen ion concentration within brain extracellular fluid, which is meditated through changes in PaCO2 (Ainslie and Duffin, 2009, Duffin, 2005). The peripheral respiratory chemoreceptors are also responsive to changes in hydrogen ions and respond to changes in both PaO2 and PaCO2 (Duffin, 2005), whereas the central respiratory chemoreceptors are traditionally not thought to be sensitive to PaO2. However, at least in animals, there is good evidence suggesting that central chemoreceptors may respond to hypoxia (Dwinell and Powell, 1999). It is the combination of 10 neural inputs to the central respiratory rhythm/pattern generator from both the central and peripheral chemoreceptors that plays a role in the regulation of breathing in humans when acclimatizing to altitude (summarized in Figure 2.3). Figure 2.3 Central (CCR) and peripheral (PCR) respiratory chemoreceptors traditional negative feedback arcs. Respiratory chemoreceptors are stimulated by changes PaCO2 and PaO2 through minute ventilation (V̇E). The PCR’s detect changes in PaCO2 and PaO2, while the CCR’s detect changes in PaCO2 and [H+] within tissue near the brainstem. Synergistic afferent feedback (solid lines) from these receptors is integrated within the respiratory controller in the medulla oblongata, and efferent feedback (dashed lines) is sent to the respiratory muscles to initiate a change in ventilation (V̇E). + represents excitatory stimulus. The most noticeable adaptation to high altitude is an immediate increase in respiratory rate and depth, thus, an increase in overall ventilation to elevate PaO2 (refer to Figure 2.3 and Figure 2.4). This increase in ventilation at altitude is mediated primarily through peripheral chemoreceptor neural mechanism(s) outlined above. However, the increase in ventilation at high altitude is accompanied by a reduction in PaCO2 (refer to Figure 2.3)(Hultgren et al., 1971), which dampens the central and peripheral chemoreceptor drive to breathe; however, despite this, ventilation remains elevated above sea level values. Acutely, ventilation abruptly increases in response to 11 hypoxia exposure; however, after a period of minutes ventilation is reduced closer to resting values (see Figure 2.4). This reduction is known as hypoxic ventilatory decline and the mechanism(s) responsible for it are currently unknown, but may be due to reduced chemoreceptor drive or elevations in cerebral blood flow (Powell et al., 1998, Sato et al., 1994, Ainslie et al., 2013). Over a period of weeks at altitude ventilation slightly increases, and is then depressed after several months, or even years at altitude (Ainslie et al., 2013). Figure 2.4 The temporal changes to ventilation with high altitude exposure. Immediately upon high altitude exposure ventilation is elevated within minutes, and then is reduced shortly after. Over a period of days and months, ventilation remains relatively constant. Figure is adapted with permission from Ainslie and colleagues (2013). 2.3.2 Cardiovascular and hematological adaptation to high altitude In order to enhance oxygen delivery at high altitude, resting cardiac output, a product of heart rate and stroke volume, increases immediately, which is mediated primarily through an increase in resting heart rate. For example, one study exposed participants to 12.5% oxygen and observed a 22% increase in cardiac output, which was nearly entirely explained (18%) by increased heart rate (Naeije et al., 1982). However, over a period of days at high altitude cardiac output returns to 12 baseline levels, which is governed by a reduction in stroke volume (Klausen, 1966, Vogel and Harris, 1967). Although this acute cardiac output response to high altitude is important to the acclimatization process, more long-term hematological adaptations over a period of days and weeks at altitude are critical for human performance and survival. Immediately upon ascent to altitude, hematocrit is elevated due to reductions in blood volume (Pugh, 1964), and then over a period of weeks hematocrit is further elevated due to increased red blood cell production [i.e. erythropoiesis; (Grover et al., 1998, Pugh, 1964)]. The first study to confirm that red blood cell concentration was elevated after long term exposure to high altitude was conducted by Francois Gilbert Viault. Viault’s landmark report in 1890 reported that his red blood cell concentration increased from 5 to 8 million per cubic/ml after 23 days at 4392m in Peru (Beall, 2006). This physiological adaptation is driven by increases in erythropoietin release, the hormone responsible for signaling for red blood cell production. The mechanism responsible for increased erythropoietin in response to high altitude remained unknown until the 1990’s, when it was discovered that hypoxia inducible factor-1α (HIF-1α) more frequently binds to hypoxia inducible factor-1β to form the HIF complex (Semenza et al., 1994, Wang and Semenza, 1996). This complex binds to the erythropoietin gene located on chromosome 7, which stimulates red blood cell proliferation (Wang and Semenza, 1996). The rapid accumulation of the HIF complex occurs within hours after exposure to hypobaric hypoxia, which results in an immediate rise in erythropoietin, and the degree of erythropoietin release is highly influenced by the severity of altitude reached. For example, erythropoietin has been shown to rise ~30% at 1900m and ~300% at 4500m (Windsor and Rodway, 2007), and there is good evidence that erythropoietin and hemoglobin remain elevated with high altitude exposure at ~3800m for ~90 days (Porcelli et al., 2017); however, this time-frame is likely impacted by the severity of altitude exposure and more 13 research is required to determine the temporal patterns of erythropoietin release at altitude. In some cases, high altitude populations exhibit patterns of maladaptation to high altitude through excessive erythrocytosis (EE) such as the Andean highlanders in Peru. In these extreme cases, [Hb] and red blood cell concentration is elevated to the point where peripheral blood flow is “sluggish” yielding reductions in oxygen delivery (explained in further detail below) (Villafuerte and Corante, 2016). The contributions of the respiratory and hematological responses to altitude exposure on CaO2 is summarized in Figure 2.5. Figure 2.5 Relative contributions of PaO2 and [Hb] to the high altitude related increase in CaO2. Adapted with permission from (Hoiland et al., 2018) 2.4 The neural response to high altitude Although less explored compared to the respiratory and hematological adaptations to high altitude, more attention has been given to the neural responses to hypobaric hypoxia. Normobaric hypoxia has demonstrated to elevate SNA (Saito et al., 1988), which signals for increased blood vessel vasoconstrictor drive and is thought to restrain hypoxic vasodilator mechanism(s) (Marshall, 14 2015). Our research group has previously demonstrated that SNA is elevated in lowlanders at altitude (5050m) (Tymko et al., 2017), which is consistent with previous reports at 4559m (Duplain et al., 1999), and at 5260m (Hansen and Sander, 2003) (see Figure 2.6). Taken together, the available data in the literature [including our own; (Tymko et al., 2017)] from direct microneurography have clearly demonstrated that SNA is elevated at high altitude. However, the health implications of SNA “hyper” activity at altitude has yet to be fully explored. Resting SNA has been directly linked to resting blood pressure (Joyner et al., 2008, Wallin and Charkoudian, 2007), and some studies have demonstrated that blood pressure is elevated at high altitude (Parati et al., 2014, Boos et al., 2017); however, a recent large cohort study (n=672) suggests that high altitude may not increase blood pressure in both normotensive and hypertensive individuals (Keyes et al., 2017). The overriding objective of the current thesis is to determine the impact of high altitude-induced elevations in SNA on endothelial function in humans. Figure 2.6 Recordings of SNA via microneurography at sea level, after 4 weeks at 5260m of altitude, and 3 days post-descent from altitude. Figure is adapted with permission from Hansen and Sander (2003). 15 2.5 The Endothelium Blood vessels are comprised of several important layers (e.g. smooth muscle, elastic membranes etc.). Arguably, the most important tissue layer related to blood vasomotion is the endothelium – a single cell thick lining of the interior surface of the blood vessel. Nitric oxide (NO), a crucial molecule released within the endothelium is responsible for smooth muscle relaxation, and its discovery has led to substantial developments in vascular research [(Furchgott and Zawadzki, 1980); ~15,000 citations]. The landmark manuscript by Furchgott and Zawadzki in 1980 demonstrated that the vascular smooth muscle tissue within the descending thoracic aorta only vasodilated in response to ACH when the endothelium was intact, and contracted when the endothelium was not intact. This impressive finding has since led to decades of vascular research focusing on the endothelium. Below is an overview of techniques, and the related cellular cascades, used within the current thesis to assess endothelial function in humans. There are several mechanism(s) that can result in NO production within the endothelium resulting in smooth muscle relaxation, and each of these mechanism(s) trigger a similar cascade of events. A stimulus (e.g. shear stress, ACH) results in a release of endothelial endogenous calcium, which binds to calmodulin, and converts NO synthase into its activate form eNOS. The production of eNOS converts L-arginine into L-citrulline, and while doing so, releases NO. The newly produced NO is able to diffuse across the endothelial cell membrane and into the nearby smooth muscle cells. Within the smooth muscle cells NO activates guanylate cyclase, and guanylate cyclase converts guanosine 5’-triphosphate (GTP) into cyclic guanosine 3’,5’-monophosphate (cGMP). The cGMP upregulates cGMP dependent protein kinase G (PKG), which acts to inhibit myosin light chain kinase resulting in smooth muscle cell relaxation (Adelstein et al., 1978, Kerrick and 16 Hoar, 1981). Upregulated cGMP also phosphorylates voltage-gated potassium channels resulting in potassium cellular extrusion, contributing to smooth muscle relaxation (Irvine et al., 2003) (see Figure 2.7). In addition, there is some evidence that suggests that NO may independently activate voltage-gated potassium channels (Bolotina et al., 1994). Figure 2.7 Endothelial mediated nitric oxide release resulting in smooth muscle relaxation. Refer to text for a brief overview of these mechanism(s). 2.5.1 Methods of assessing vascular function The following section is dedicated to outlining the methodological background, along with the strengths and weaknesses of the techniques used in the current thesis studies. Below is an outline of FMD and intra-arterial fusion as assessments of endothelial function. 2.5.1.1 Non-invasive flow mediated dilation Flow-mediated dilation is a non-invasive technique commonly used to assess endothelial function within conduit arteries. The technique works based upon increasing luminal blood flow, thus shear stress, which stimulates NO release within the endothelium, which mediates an increase in conduit artery diameter (e.g. brachial and femoral artery) via smooth muscle vasodilation (Thijssen et al., 2011). This technique was first described by Celermajer and colleagues in 1992, where they incorporated the findings by Furchgott and Zawadski (1980; outlined above). Here, they 17 demonstrated that the endothelium is full of mechanosensitive structures that are highly responsive to changes in shear stress (Celermajer et al., 1992) (see Figure 2.8 and Figure 2.9). The FMD technique has been confirmed to be at least in-part NO-dependent (~50%) by a number of studies (Green et al., 2014). For example, it was discovered that FMD in the radial artery was abolished after administration of NO blockade (Joannides et al., 1995, Mullen et al., 2001). It has since been confirmed in multiple different conduit arteries including the brachial artery (Green et al., 2014). This technique was employed to assess endothelial function in Studies #1 and #2 in the current thesis. Figure 2.8 Shear-stress mediated nitric oxide release resulting in smooth muscle relaxation. Refer to text for an overview of these mechanism(s). Due to its non-invasive nature and relatively short experimental protocol (~10-15 minutes in length), the FMD test became popular to assess endothelial function in humans in both clinical and research settings. It has been established as a predictor of cardiovascular disease risk in several studies (Meyer et al., 2010, Gokce et al., 2003, Neunteufl et al., 2000, Fathi et al., 2004, Brevetti et al., 2003, Karatzis et al., 2006, Patti et al., 2005, Kitta et al., 2005). Despite the obvious benefits for using this methodology it has been scrutinized for having several limitations that are worth mentioning. First, the FMD test is operator dependent and relies upon accurate and reliable ultrasound measurements from an experienced technician. High quality ultrasound images are critical for the accurate assessment of FMD, which typically requires several months of training 18 and >100 practice FMD exams in order to perform research quality assessments. Second, slight differences in the FMD protocol make it difficult to compare absolute FMD values between studies. For example, cuff placement to induce blood flow occlusion has been suggested to be done either on the forearm or upper arm (below or above the ultrasound image, respectively). Previous reports indicate that cuff placement may have a significant impact on the degree of NO-mediated FMD and image quality (Thijssen et al., 2011). Third, analysis of FMD has been completed using different types of software. Advanced wall-tracking software has been shown to reduce intra-operator bias when analyzing FMD data (Woodman et al., 2001); however, such software can be expensive to purchase (>15K CAD). Fourth, some research studies have failed to consider the effects of differences in baseline diameter (e.g. children vs adult artery size) and shear stress between participants and whether the intervention changes these parameters since they can directly influence the degree of FMD. Finally, although FMD has been found to be a predictor of future cardiovascular events, the physiological significance of this measurement in healthy humans has been questioned. 19 Figure 2.9 A depiction of the brachial shear stress and diameter response during a FMD test. Immediately after cuff release (time = 0), blood flow surges throughout the lumen of the artery, thus, increasing shear stress onto the vessel walls (i.e. endothelium). The increase in shear stress causes a cascade of events within the endothelium resulting in a release of NO that diffuses into the nearby smooth muscle cells of the blood vessel resulting in smooth muscle relaxation (i.e. increase in diameter). Modified with permission from Thijssen et al. (2011), 2.5.1.2 Intra-arterial infusion of vasoactive substances The forearm intra-arterial infusion experimental model has been utilized to assess vascular function for several decades. This methodology involves the placement of a catheter into an artery (usually the brachial or radial) allowing for the infusion of vasoactive drugs [e.g. ACH and SNP]. Administration of ACH is used to determine endothelial-dependent changes in vasomotor tone, and SNP infusion is used to determine endothelial-independent changes in vasomotor tone. Changes in blood flow can be measured via strain gauge plethysmography (Todd et al., 1995, Dinenno et al., 2002), and/or Duplex ultrasound (Hearon et al., 2018, Hearon et al., 2016, Hearon et al., 2017). Often, interventions that block specific mechanism(s) (e.g. adrenergic, NO, prostaglandins) that may govern endothelial and/or smooth muscle function are incorporated into this technique allowing for the unique opportunity to isolate specific mechanism(s) related to vascular function in a very controlled and reliable manner. One of the primary caveats of the intra-arterial forearm infusion approach is its invasive nature; as such, it requires a wide array of highly trained personnel (e.g. ultrasound technicians, nurses, physicians) to be present in order to perform these experiments in human. Below is an outline of the two commonly used vasoactive substances used for Study 3 that were utilized to assess endothelial-dependent (e.g. ACH), and endothelial-independent vasodilation (e.g. SNP). 20 2.5.1.3 Acetylcholine and sodium nitroprusside infusion The landmark study by Furchgott and Zawadski (1980) was the first to report that ACH resulted in vasodilation in intact endothelial tissue in vivo, but not in isolated blood vessels where the endothelial tissue was damaged. Thus, they came to the novel conclusion that ACH causes endothelium-dependent vascular relaxation in blood vessels. Acetylcholine binds to muscarinic receptors that are located on the outside of endothelial cells (Figure 2.10), which results in a similar cascade of events as outlined above, causing an influx of endogenous calcium leading to NO-mediated smooth muscle cell relaxation. Acetylcholine is typically administered in a range of increasing doses to elicit a step-wise increase in forearm blood flow and has a half life of approximately ~2-3 minutes, thus an approximate elimination time of ~10-15 minutes making it an attractive drug for research use (Richards et al., 2014). In order to isolate endothelium-dependent from endothelial-independent vascular function, SNP, which results in endothelial-independent vasodilation has been a popular drug of choice. Another common drug used to assess endothelial-independent vasodilation is Nitroglycerin. Although nitroglycerin is similar in its mechanism of action it does not evoke as strong of a vasodilation response compared to SNP (Ranadive et al., 2017). Discovered in 1849, SNP is a well known vasodilator commonly used to lower blood pressure (Hottinger et al., 2014). When SNP enters the blood stream it interacts with oxyhemoglobin and dissociates into methemoglobin, and consequentially releases cyanide and NO (Friederich and Butterworth, 1995) (see Figure 2.10). Nitric oxide, then diffuses through the endothelial cells and into the adjacent smooth muscle cells to activate guanylate cyclase, which causes a downstream cascade of events leading to smooth muscle cell relaxation. Similar to ACH, SNP is typically administered in a range of increasing 21 doses to elicit a step-wise increases in forearm blood flow and has a short half life of approximately ~2-3 minutes, thus an approximate elimination time of ~10-15 minutes (Richards et al., 2014). Figure 2.10 ACH and SNP mediated NO release resulting in smooth muscle relaxation. Refer to text for an overview of these mechanism(s). 2.6 The influence of sympathetic nervous activity on endothelial function There are several physiological factors that can impact endothelial function such as elevated oxidative stress (Goel et al., 2007a, Silvestro et al., 2002), changes in hemodynamics such as shear stress and blood pressure (Birk et al., 2013, Dawson et al., 2008, Johnson, Mather et al., 2012, Lamping and Dole, 1987, Tremblay et al., 2018), and elevations in SNA (Tymko et al., 2016, Atkinson et al., 2015, Dyson et al., 2006, Hijmering et al., 2002, Thijssen et al., 2014, Thijssen et al., 2006). The current thesis focuses on the influence of the SNA on endothelial function; therefore, the following sections provide an overview of the methodologies employed in the current thesis that were used to alter SNA. 2.6.1 Organization of the sympathetic nervous system The organization of the peripheral sympathetic nervous system is important for the control of blood flow to various tissues and organs in order to meet metabolic demand on a moment-by-moment basis. Neural information discharged from the brainstem travels down the spinal cord and into myelinated preganglionic (i.e. cholinergic) and postganglionic neural fibers, which innervate 22 effector tissues (e.g. heart, blood vessels, kidneys). Sympathetic neural fibers release norepinephrine, which binds to both α- and β-adrenergic receptors located on the surface of smooth muscle cells (see Figure 2.11). Norepinephrine binding to α-adrenergic receptors results in vasoconstriction, and norepinephrine binding to β-adrenergic receptors results in vasodilation. Blood vessels that contain little smooth muscle (e.g. capillaries) are not innervated by the SNS. 23 Figure 2.11 Schematic of neural control of the vasculature. Sympathetic nervous system outflow is present at rest, and can be elevated or suppressed through different interventions such as drug infusions (e.g. central adrenergic agonists and antagonists), and exercise (e.g. cycling or handgrip exercise). Sympathetic nervous activity can be measured directly or indirectly using various techniques. The technique employed by the current thesis to assess SNA (study #2) used microneurography. Sympathetic nervous outflow results in the release of the release of neurotransmitters (e.g. norepinephrine) that bind to both α- and β-adrenergic receptors on smooth muscle cells on the vasculature. 2.6.2 Sympathetic nervous activity during cycling exercise (Study 1) The autonomic nervous system plays a critical role in the cardiovascular regulation during exercise to ensure adequate blood flow delivery throughout the body. There are several convincing reports that SNA increases linearly with exercise intensity (Lehmann et al., 1981, Ichinose et al., 2008, Galbo et al., 1975, Bloom et al., 1976). However, during “very light” exercise, a study by Ichinose et al. (2008) demonstrated that SNA decreased, but then increased in a linear fashion from light-to-exhaustive exercise. The authors reasoned that the reduction in SNA during very light exercise was due to increased venous return and cardiopulmonary baroreceptor loading (Ichinose et al., 24 2008), however, these findings have yet to be confirmed. Several reports that quantified SNA via venous norepinephrine concentration demonstrated that SNA increases during moderate-intensity treadmill exercise, and during incremental cycling exercise (Davy et al., 1995). The study by Davy and colleagues (1995) demonstrated that venous norepinephrine at ~65% of VO2 max continued to increase with duration, providing strong evidence that both exercise intensity and duration contribute to the SNA response to exercise (Davy et al., 1995). Similar findings were observed when measuring SNA via microneurography (described below in detail) during 30-minutes of cycling at 40% VO2 max (Saito et al., 1997). There is evidence that SNA outflow between active and inactive tissues may exist (Savard et al., 1987). For example, Savard et al. (1987) demonstrated that norepinephrine spillover was higher in the active limb compared to the inactive during knee extensions. Reasoning for higher that signal for blood vessel vasodilation (i.e. functional sympatholysis). However, this still remains to be fully clarified, since other reports have demonstrated that SNA increases to a similar extent be due to offset locally released metabolites from the active tissues After a bout of sustained exercise it has been well documented that blood pressure is reduced in individuals with hypertension (Floras et al., 1989, Somers et al., 1985), and some studies have also demonstrated this in normotensive individuals (MacDonald, 2002, MacDonald et al., 1999). This physiological phenomenon known as “post exercise hypotension” is thought to be partly driven from the consequence of reduced SNA and elevated levels of histamine. However, the study by Ichinose et al. (2008) clearly shows that MSNA remains elevated for at least 10-minutes after cycling exercise (see Figure 2.12) in both active and inactive limbs during exercise (Hansen et al., 1994).SNA to active tissues could This acute elevation in MSNA is thought to be the root cause for post-exercise endothelial impairment immediately after cycling exercise (Atkinson et al 2015), but this has yet to be tested in an environment where SNA is 25 chronically elevated (i.e. high altitude). The first study of this thesis aimed to investigate the effects of elevated SNA (induced via cycling exercise) on endothelial function at both sea level and high altitude. Figure 2.12 The SNA response to exercise. Printed with permission from Ichinose et al. (2008) 26 2.6.3 Lower body differential pressure (Study #2) Lower body negative pressure and lower-body positive pressure (LBNP and LBPP, respectively) are commonly used to alter central blood volume and evoke changes in arterial baroreflex activity. The arterial baroreflex is a bi-directional, stretch-sensitive, negative feedback response to changes in mean arterial pressure (MAP) mediated by the autonomic nervous system (Mancia et al., 1984, Fadel and Raven, 2012). This reflex is critical for moment-to-moment regulation of HR and vascular resistance in humans. Sustained or progressive LBNP (resulting in reduced central blood volume) or LBPP (resulting in elevated central blood volume) is a reliable method to elicit an arterial baroreflex response since it is non-invasive, reproducible (Howden et al., 2001), and the pressure stimulus can be easily adjusted and controlled on an individual basis. Lower-body differential pressure protocols requires the participant to lie in a vacuum sealed chamber, and by using a vacuum source (e.g. shopvac), the pressure within chamber can be increased or decreased relative to atmospheric pressure. Exposure to lower-body differential pressure results in blood translocating between the upper body and the lower extremities, resulting in altered venous return and potentially MAP, which changes the afferent nervous outflow from arterial and cardiopulmonary baroreceptors. The baroreflex involves a cardiac and vascular arm, with responses mediated by the sympathetic and parasympathetic nervous systems. The cardiac response can be easily monitored using recordings of HR (via ECG) and estimations of SV, while the vascular response can be monitored with measures of muscle sympathetic nervous activity (MSNA) or vascular ultrasound (to estimate vascular resistance and/or transduction), or both. Both of these responses (HR and vascular response) simultaneously contribute in the maintenance of MAP. 27 Although LBNP has been used extensively for the past several decades, there has recently been an emergence of the utility of LBPP, particularly with rehabilitation (Takacs et al., 2013). Conversely to LBNP chambers, LBPP chambers have been employed to investigate the effects of increased venous return and MAP, which has profound effects on the cardiovascular and cerebrovascular systems (Nishiyasu et al., 2007, Perry et al., 2013). For example, LBPP chambers have been employed to investigate the regulatory responses of the brain to transient increases in blood pressure (Perry et al., 2013). Lower-body differential pressure chambers can be purchased commercially; however, these are usually expensive ($10,000 to $20,000 CAD) and have a similar performance compared to homemade chambers, which are relatively easy to construct. Helpful documentation on how to construct a basic lower-body differential chamber exists (Esch et al., 2007); however, other literature has lacked an appropriate amount of detail (Hisdal et al., 2003, Lategola and Trent, 1979, Verghese and Prasad, 1993, Russomano et al., 2005). For Study #2, a custom made, portable LBNP/LBPP chamber was built to elicit changes in SNA via mild LBNP and mild LBPP (see Figure 2.13). 28 Figure 2.13 Picture of the custom built LBNP/LBPP chamber during experimental protocol in Nepal at 5050m. Mild levels of LBNP and LBPP have demonstrated to alter SNA via cardiopulmonary and arterial baroreflex pathways largely independent of hemodynamics (e.g. blood pressure, HR, shear patterns) (Millar et al., 2013). For example, in the supine position at rest, mild LBNP (-5 to -10 mmHg) significantly increases SNA by ~30-60% (Cui et al., 2004, Scherrer et al., 1988, Rea and Wallin, 1989), while LBPP (+10 to +20 mmHg) decreases SNA by ~30% in healthy individuals (Fu et al., 1998). Interestingly, the elevations in SNA observed during mild LBNP (~30-60%) are comparable to those achieved with acute hypoxia (FIO2 = 0.11) (DeBeck et al., 2010), which reduces brachial FMD via an α1-adrenergic pathway (Lewis et al., 2014). Study #2 in this thesis used a novel approach to transiently and non-invasively alter SNA via mild levels of LBNP and LBPP to assess the impact of SNA on endothelial function at sea level and high altitude. 2.6.4 Resting sympathetic hyper-activity at high altitude (Study #3) Normobaric hypoxia has demonstrated to elevate SNA (Saito et al., 1988), which signals for increased vasoconstrictor drive, and is thought to restrain hypoxic vasodilator mechanism(s) 29 (Marshall, 2015). There are several different methods that are commonly used to assess SNA such as analysis of catecholamine concentrations (Rostrup, 1998), heart rate variability (Kanai et al., 2001, Duplain et al., 1999), and MSNA via microneurography (Vallbo et al., 2004). The latter method, microneurography (see Figure 2.14), is considered the gold standard to assess SNA in humans and uses a tungsten needle inserted into a peripheral nerve to record SNA in humans. A primary advantage of direct MSNA assessment is that microneurography allows for detailed information on the ‘bursting’ pattern of SNA directed at the vasculature, such as burst occurrence and amplitude. With this approach our research group has previously demonstrated that SNA is elevated in lowlanders at altitude (5050m) (Tymko et al., 2017). These findings are consistent with those reported by Duplain et al. (1999) after 18–24 h at 4559m (Duplain et al., 1999), Hansen & Sander (2003) after 4 weeks at 5260m (Hansen and Sander, 2003), and Lundby et al. (2018) after 10 and 50 days at 4100m (Lundby et al., 2018). Taken together, the available data in the literature (including our own) from direct microneurography clearly demonstrate sympathetic hyperactivity at altitude. Elevations in SNA have been associated with reduced endothelial function (Hijmering et al., 2002). For example, a study by Hijmering et al. (2012) investigated the effects of LBNP (increases SNA) on endothelial function and found that endothelial function was reduced during LBNP via an α-adrenergic mediated pathway. However, the mechanistic link between SNA and endothelial function at high altitude has yet to be fully elucidated. 30 Figure 2.14 Picture of a typical experimental set-up to assess SNA via microneurography. A small tungsten electrode (refer to red arrow) is placed into a peripheral nerve (e.g. radial nerve) and the obtained voltage signal is amplified, filtered, rectified, and then integrated into a mean voltage neurogram This technique was employed for study #2 in the current thesis. Modified with permission from Hart et al. (2017) (Hart et al., 2017). 2.7 Effect of high altitude on endothelial function Exposure to hypoxia is associated with arterial stiffening, but the effects of hypoxia on endothelial function remain unclear (Lewis et al., 2014, Boos et al., 2012, Rhodes et al., 2011). For example, different studies have reported that exposure to simulated high-altitude (i.e. acute normobaric hypoxia), results in no change in FMD [~4000m, FIO2 = 0.13; (Iglesias et al., 2015)], and decreased FMD [~5000m, FIO2 = 0.11 (Lewis et al., 2014)]. Upon ascent to high-altitude between 3800m-5050m (i.e. hypobaric hypoxia), a decrease in FMD has been observed in some (Bakker et al., 2015, Lewis et al., 2014), but not all studies (Bruno et al., 2015, Tremblay et al., 2017). The disparity within this literature might be due to different methodological approaches between 31 investigations. For example, the three studies that reported reductions in FMD involved ascent to high-altitude over 7-10 days of trekking for several hours each day at altitudes >4000m (Lewis et al., 2014, Bakker et al., 2015, Tremblay et al., 2018). In contrast, the two studies that reported no change in FMD involved participants without acute mountain sickness (i.e. AMS) ascending rapidly to high-altitude via cable car (Bruno et al., 2015, Tremblay et al., 2017). The methodological difference between these studies raises the possibility of a moderating impact of trekking exercise at altitude, and the severity of altitude exposure (e.g. <4000m = no observed reductions in FMD). Table 2.1 and Figure 2.15 summarize these studies below. 32 Table 2.1 Summary of studies that investigated the impact of high altitude on brachial artery FMD. Study Location and altitude Ascent Technique Participants Main Findings (Lewis et al., 2014) Nepal (5050m) Trekking Brachial artery FMD 12 lowlanders FMD was reduced at altitude (Bakker et al., 2015) Nepal (4200m) Trekking Brachial artery FMD 11 lowlanders FMD was reduced at altitude (Bruno et al., 2015) France (3842m) Cable car Brachial artery FMD 22 AMS- lowlanders; 12 AMS+ lowlanders FMD was reduced in participants with AMS, but not in AMS- participants (Tremblay et al., 2017) White Mountain, CA (3800m) Automobile Brachial artery FMD 12 lowlanders FMD was not reduced at altitude (Tremblay et al., 2018) Nepal (3400m, 4200m, and 5050m) Trekking Brachial and Femoral FMD 22 lowlanders; 12 Sherpa FMD was reduced at 4200m and 5050m, but not at 3400m 33 -6 -4 -2 0 2 4Overall EffectLewis et al. 2014Bakker et al. 2015Bruno et al. 2015 (AMS-)Bruno et al. 2015 (AMS+)Tremblay et al. 2017Tremblay et al. 2018Study Figure 2.15 Forest plot of the findings related to brachial artery FMD outlined in the table 2.1. Each point represents mean reduction in FMD with 95% confidence intervals. The blue line represents the mean reduction across all studies. The arrows highlight the studies that found endothelial function is reduced at high altitude and NC represents the studies that found that high altitude had no impact (i.e. No Change) on endothelial function. NCNC NCNC 34 2.8 Other potential mechanisms of endothelial dysfunction at high altitude - oxidative stress The current thesis investigates the impact of high altitude related heighted SNA on endothelial function; however, there are several other key mechanism(s) that may alter endothelial function at high altitude – these include oxidative and inflammatory stress, which is elevated at high altitude. A primary modulator of endothelial function is the balance between endothelium derived vasodilators (e.g. NO) and reactive oxygen species (ROS). An imbalance to this system (e.g. NO:ROS) is referred to as oxidative stress and may lead to a reduction in NO bioavailability through the interaction of NO and superoxide to form peryoxynitrite (Meli et al., 2002). Elevations in oxidative stress has been shown to increase structural damage to the vasculature and exists in several different disease states including hypertension, diabetes mellitus, atherosclerosis, myocardial infarction, and heart failure (Neunteufl et al., 2000, Schachinger et al., 2000, Gokce et al., 2002, Heitzer et al., 2001). Markers of oxidative stress have been shown to be elevated after acute hypoxia at sea level (Cosby et al., 2003, Maher et al., 2008), rapid ascent to altitude (>3500m) (Irarrazaval et al., 2017), and after more chronic exposure to hypoxia (5050m) (Lewis et al., 2014). Interestingly, vascular bioavailability of NO has been reported to increase at high altitude (Lewis et al., 2014); therefore, high altitude related vascular dysfunction may be more related to the toxic and vasoconstrictor effects of lipid hydroperoxides (Lewis et al., 2014), or by through other mechanistic pathways (e.g. SNA related). 35 2.9 Permanent high altitude populations 2.9.1 General overview of high altitude populations Humans are the only mammals to have colonized all of Earth’s most extreme environments, and the mechanism(s) of human adaptation in the different domains have naturally attracted the attention of physiologists for centuries. Permanent settlements and sojourners at high altitude are subject to potent environment stressors, specifically low levels of oxygen, as well as colder temperatures and increased ultraviolet radiation, and undergo various physiological adaptations and acclimatization. Hence, populations that have thrived at high altitude for millennia have evolved distinct physiological phenotypes. The indigenous populations of the Tibetan, Andean, and Ethiopian plateaux have developed three separate physiological adaptive strategies to thrive in their respective harsh hypoxic environments. Although there is considerable debate regarding specific durations, the general consensus is that the Old World plateaux (Ethiopian and Tibetan) have been settled for longer than the Altiplano in the New World (Andeans) (Beall, 2006, Beall, 2007, Beall et al., 2002, Alkorta-Aranburu et al., 2012, Zhang et al., 2018). These plateaux have spawned magnificent empires including the Ethiopian, Tibetan, Tiwanaku, and Inka. The different steps of oxygen transport and utilization yield impressive function at high-altitude in these populations compared to low-landers. When native low-landers are transplanted to altitudes above 2,500m, hyperventilation and increased red blood cell formation in the bone marrow (erythropoiesis) compensate for the reduced oxygen levels in the blood. Andeans (Quechua and Aymara populations) present what can be considered an exaggerated form of typical low-lander high-altitude adaptation with increased haemoglobin levels and haematocrit and high oxygen saturation, together increasing the oxygen content in 36 arterial blood, so that it actually exceeds that of low-landers at sea level. In some instances (~15-34%) (Monge et al., 1989, León-Velarde et al., 1993), this adaptive response is perturbed with an excessive production of red blood cells, and thus elevated haematocrit, and reduced oxygen saturation (Villafuerte and Corante, 2016, Corante et al., 2018). The resultant thick and viscous blood combined with hypoxaemia leads to so-called chronic mountain sickness (CMS), which involves cardiovascular and neurogenic sequelae. Andeans appear to be especially susceptible to this maladaptation, and large increases in haemoglobin and blood viscosity may not represent the ideal adaptive strategy. Indeed, Old World high altitude natives, such as Tibetans and Ethiopians, exhibiting alternative adaptive strategies, and perhaps as a result, display a negligible incidence of CMS. Hence, Tibetans (and Sherpa) present lower haemoglobin concentration and SaO2 at similar altitudes compared to Andeans, resembling an unacclimatized lowlander at high altitude. Tibetans, whose genetic composition may have been influenced by Denisovan ancestors (Sankararaman et al., 2016), seem to rely on enhanced blood flow to sustain oxygen delivery and cellular metabolic adaptations to more efficiently utilize oxygen (Gilbert-Kawai et al., 2014, Tremblay et al., 2018). Less is known about Ethiopian highlanders (Amhara) of the Simien Plateau, but they do appear to have low haemoglobin concentration and higher than expected SaO2 due to an increased affinity of haemoglobin for oxygen (Beall, 2006, Beall et al., 2002). Conceivably, these three phenotypes thus represent distinct evolutionarily-driven strategies which permit survival and performance at high-altitude (Beall et al., 2002, Beall, 2006, Beall, 2007) (see Table 2.2 for a summary of the distinct differences between the Tibetan, Peruvian and Ethiopian high altitude natives). In the context of this thesis, differences in SaO2 and CaO2 may exist between populations; it is unknown if differences in SNA also exist. 37 Table 2.2 Estimates for haematological parameters in high-altitude dwellers and low-landers at ~4000m. Population O2 saturation (%) Haemoglobin (g/dl) Haematocrit (%) Arterial O2 content (ml/dl) Low-landers at sea level (Wolfel et al., 1991) 98 14 45 22 Low-landers at altitude (Wolfel et al., 1991) 90 16 50 20 Ethiopians (Beall et al., 2002, Claydon et al., 2008, Hoit et al., 2011, Cheong et al., 2017) 92-96 15-16 48 20-21 Tibetans (Beall, 2006, Beall, 2007) 88-92 14-16 48 16-20 Andeans (Winslow et al., 1989, Jansen and Basnyat, 2011) 92-94 18-20 54 20-26 Andeans with CMS (Maignan et al., 2009, Villafuerte et al., 2014) 84 23 68 26 2.9.2 Endothelial function in high altitude Andean dwellers The final PhD research project in the current thesis (i.e. Study #3) was conducted in both lowlanders and in Peruvian highlanders in Cerro de Pasco (4300m). The Peruvian residents of the Andes mountains have been living at altitude for ~11,000 years (Beall, 2006, Beall, 2007), and a high percentage of long-term high altitude residents in the Cerro de Pasco region (~15-34%; (Monge et al., 1989, León-Velarde et al., 1993)) are prone to suffering from CMS, which is characterized by an excessive production of red blood cells [i.e. excessive erythrocytosis (EE)], which can lead to severe hypoxemia and cardiovascular health consequences (Villafuerte and Corante, 2016, Corante et al., 2018). Surprisingly, there have only been few investigations that have investigated vascular function in Andean highlanders with and without EE (Rimoldi et al., 2012, Bailey et al., 2013, Rexhaj et al., 2016). Each of these studies demonstrated that endothelial function, assessed via FMD, was impaired in Andeans diagnosed with EE. The authors, however, 38 failed to account for differences in blood viscosity between their two cohorts, which has a major impact on FMD assessment. In addition, elevations in SNA have yet to be investigated as a potential mechanism for the observed reductions in endothelial function in Andean high landers. 2.10 Synopsis of literature review This literature review encompassed a brief overview of the physiological adaptations to high altitude exposure, the importance of the endothelium on smooth muscle function, different methodologies to assess endothelial function, and an overview of different mechanism(s) that can alter endothelial function. Further, the literature review focused on the impact of high altitude and on SNA, which could be a potential mechanism responsible for impaired endothelial function in hypoxic environments. The experimental chapters herein contain the three separate experimental protocols designed to test the overriding hypothesis that SNA is a key mechanism involved in endothelial-mediated blood flow regulation. 39 Chapter 3: The effect of α1-adrenergic blockade on post-exercise brachial artery flow-mediated dilatation at sea-level and high-altitude 3.1 Background Flow-mediated dilatation is a commonly used, non-invasive measurement of conduit artery diameter in response to an imposed change in shear rate; it is widely accepted to be an index of endothelial function (Thijssen et al., 2011), which is largely NO mediated (Green et al., 2014). It has been demonstrated that immediately after cycling exercise, endothelial function measured by brachial (i.e. non-exercising limb) FMD is transiently reduced (Jones et al., 2010, Birk et al., 2013, Atkinson et al., 2015, Dawson et al., 2013), and the reduction in FMD is inversely related to exercise intensity (Birk et al., 2013). Although the precise mechanisms responsible for this acute effect of exercise on FMD remain unclear, several possibilities exist, including: 1) elevated oxidative stress (Goel et al., 2007, Silvestro et al., 2002); 2) altered hemodynamics (i.e. shear rate, shear pattern, and blood pressure) (Johnson, Mather et al., 2012, Dawson et al., 2008, Birk et al., 2013, Lamping and Dole, 1987, Millgard and Lind, 1998); 3) changes in baseline artery diameter post-exercise (Padilla et al., 2007, Atkinson, G. et al., 2013), and; 4) elevations in SNA (Hijmering et al., 2002, Dyson et al., 2006, Atkinson et al., 2015). The role of the SNS was recently examined in a study which observed that the exercise-mediated reduction in FMD following exercise was abolished after administration of an 1-adrenergic receptor blockade (Atkinson et al., 2015). Interestingly, the relationship between increased SNS activity and post-exercise FMD has not previously been explored under conditions where resting SNS activity is chronically elevated, such as in aging, pathology (e.g. obstructive sleep apnea, heart failure), or in hypoxia (i.e. normobaric and hypobaric) (Saito et al., 1988, Duplain et al., 1999, Xie et al., 2001, Hansen and Sander, 2003). 40 Exposure to hypoxia is associated with arterial stiffening, but the effects of hypoxia on endothelial function remain unclear (Lewis et al., 2014, Boos et al., 2012, Rhodes et al., 2011). For example, different studies have reported that exposure to simulated high-altitude (i.e. acute normobaric hypoxia), results in no change in FMD [~4000m, FIO2 = 0.13; (Iglesias et al., 2015)], and decreased FMD [~5000m, FIO2 = 0.11 (Lewis et al., 2014)]. Upon ascent to high-altitude between 3700m-5050m (i.e. hypobaric hypoxia), a decrease in FMD has been observed in most (Bakker et al., 2015, Lewis et al., 2014), but not all studies (Bruno et al., 2016, Bruno et al., 2015). The disparity within this literature might be due to different methodological approaches between investigations. For example, the two studies that reported reductions in FMD involved ascent to high-altitude over 7-10 days of trekking for several hours each day (Lewis et al., 2014, Bakker et al., 2015). In contrast, the study that reported no change in FMD involved participants ascending rapidly to high-altitude via cable car (Bruno et al., 2015). The methodological difference between these studies raises the possibility of a moderating impact of trekking exercise at altitude, and total acclimatization time [e.g. 3-10 days of exposure (FMD reduced) vs. 1 day of exposure (no change in FMD)] on vascular responses at high-altitude. In keeping with exercise mediated reductions in FMD, the mechanism responsible for high-altitude induced vascular impairment may involve increased SNS activity (Saito et al., 1988, Duplain et al., 1999, Xie et al., 2001, Hansen and Sander, 2003, Lewis et al., 2014). Augmentation in SNS activity may yield vascular dysfunction either directly (Hijmering et al., 2002), or indirectly by increasing retrograde shear rate (Thijssen et al., 2014). Nevertheless, there has only been one investigation examining the role of the SNS on endothelial function in normobaric hypoxia (Lewis 41 et al., 2014), and no studies have addressed the impacts of exposure to hypobaric hypoxia (high-altitude) at rest or following exercise. 3.2 Aims and Hypotheses By employing a double-blinded, counter-balanced, randomized and placebo-controlled design, the primary purposes of the current study were to investigate: 1) the effects of non-trekking ascent to high-altitude on endothelial function (via brachial FMD) and shear patterns, and 2) the effects of post-exercise related increases in SNS activity on FMD and shear patterns at both sea-level and at high-altitude (3800m). We hypothesized that: 1) at rest, FMD would be impaired at high-altitude compared to sea-level, 2) FMD would be reduced to a greater extent at sea-level compared to high-altitude after maximal exercise, and 3) reductions in endothelial function following moderate-intensity exercise at both sea-level and high-altitude are mediated via an 1-adrenergic pathway. 3.3 Methods and Materials 3.3.1 Ethical Approval. All experimental procedures and protocols were approved by the clinical research ethics board at the University of British Columbia and conformed to the Declaration of Helsinki. All participants provided written informed consent prior to participation in this study. This study was part of a larger research expedition conducted in October 2015. As such, participants took part in a number of studies conducted at the University of British Columbia (Kelowna, BC; 344m) and during two weeks at the Barcroft high-altitude research station (White Mountain, California, USA; 3800m). However, the a priori, primary research questions addressed in the current paper are novel and are 42 exclusively dealt within this study alone – there is no overlap between this investigation and others completed on the research expedition. 3.3.2 Participants. Recruited participants (n=11; 3F) were normotensive (systolic blood pressure <140 and diastolic pressure <90 mmHg) at rest, and completed a medical history questionnaire. Two of the recruited participants were excluded from all mean data analysis; one participant due to illness (i.e. syncope, light-headedness, nausea) caused by our drug intervention (prazosin), and one participant due to illness at high-altitude. The participants (n=9; 2F) included in the data analysis were non-smokers, had no previous history of cardiovascular, cerebrovascular, or respiratory diseases, were not taking any medications during testing besides oral contraceptives (n=1). All participants arrived at the Barcroft high-altitude research facility (elevation = 3800m) on the same day, and approximately at the same time (i.e. within one hour). Nine of these participants drove to the Barcroft high-altitude research facility after staying overnight in Palm Springs, CA, USA (elevation = 146m), while one participant stayed overnight in Bishop, CA, USA (elevation = 1265m). Maximal exercise testing for this study occurred on day three, while moderate-intensity exercise experimentation occurred between days four to seven at high-altitude. 3.3.3 Experimental Design. This study was conducted in two parts: sea-level and high-altitude investigations. Prior to each experiment, all participants abstained from exercise, alcohol, and caffeine for at least 12 hours. Additionally, participants were asked to consume a light meal at least two-hours prior to experimentation, and to keep their diet consistent between experimentation days. In order to 43 determine whether our participants had normal healthy lung function, at sea-level we conducted a forced vital capacity (FVC) test to measure lung function, a vital capacity and inspiratory capacity maneuver to measure lung volumes, and a single breath carbon monoxide test to quantify diffusing capacity on each individual. All testing procedures were conducted in accordance with the American Thoracic Society and European Respiratory Society’s joint guidelines (Macintyre et al., 2005, Miller et al., 2005). For each of these tests, participants sat within a 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 while wearing a nose-clip. All pulmonary function measurements were compared against population-based predictions. Exercise testing was then conducted on three separate lab visits at both sea-level and high-altitude while participants lay in the semi recumbent position. The time of day of the testing sessions were kept the same for each participant, with a minimum of 24-hours between testing sessions. 3.3.3.1 Maximal exercise protocols. Prior to the prolonged moderate-intensity exercise testing at sea-level and high-altitude, participants were required to conduct a maximal exercise protocol in order to obtain peak Watt. In the semi-recumbent position, participants rested during a quiet baseline period on the cycle ergometer (Lode Ergometer, Lode, Groningen, Netherlands) for 15-minutes. Immediately after the baseline period a brachial artery FMD was performed (Thijssen et al., 2011). Exercise began with a two-minute warm-up period (40 watts for females, 60 watts for males), followed by staged exercise with workload increased by 20 watts every minute. This maximal exercise protocol was 44 terminated when participants either 1) reached volitional exhaustion, or 2) cycling cadence could no longer be maintained. Immediately after the maximal exercise protocol was completed, another brachial artery FMD was performed. 3.3.3.2 Moderate-intensity exercise protocol. Following at least 24 hours after the maximal exercise test, participants performed two moderate-intensity exercise protocols on two independent days separated by at least 24 hours. Upon arrival on each of these testing sessions, participants ingested a capsule containing either oral prazosin (1-adrenergic receptor blocker; 0.05 mg kg-1), or Placebo (i.e. sugar pill). The order of condition (placebo and prazosin) was counter-balanced and randomized at both sea-level and high-altitude. The dosage of prazosin used has been demonstrated to provide ~80% 1-adrenergic blockade, and has been used in other studies by our research group (Lewis et al., 2014, Atkinson et al., 2015). Seventy-five-minutes after ingestion, participants were instrumented on the semi-recumbent cycle ergometer (see details below in section Experimental Measurements). Participants were asked to sit in the semi-recumbent position quietly for 15-minutes, and afterwards, a baseline FMD on the left brachial artery was conducted. Immediately after the baseline FMD, participants completed 30-minutes of cycle exercise at 50% of their peak exercise workload. At sea-level, this workload was 137.0  6.9 Watts, while at high-altitude the average workload was 114.3  5.9. Every five-minutes during exercise a one-minute measurement of brachial artery shear rate and blood flow was recorded (total of six recordings during the 30 minutes of exercise). After completion of the 30-minutes of moderate-intensity exercise, a brachial FMD was conducted immediately, and then following 60-minutes of post-exercise rest. 45 3.3.4 Experimental Measurements. 3.3.4.1 Cardiovascular measurements. All continuously recorded cardiovascular measurements were acquired at 200 Hz using an analog-to-digital converter (Powerlab/16SP ML 880; ADInstruments, Colorado Springs, CO, USA) interfaced with a personal computer. Commercially available software was used to analyze cardiovascular variables (LabChart V7.1, ADInstruments, Colorado Springs, CO, USA). Electrocardiogram electrodes were placed in lead II configuration (Bioamp, ML132, ADInstruments, Colorado Springs, CO, USA) to measure heart rate. Beat-by-beat arterial pressure, cardiac output, stroke volume, and total peripheral resistance was measured by finger photoplethysmography (Finometer Pro, Finapres medical systems, Amsterdam, Netherlands). Prior to baseline data collection, the Finometer was calibrated using the return-to-flow function, and blood pressure from the Finometer was confirmed with automated brachial blood pressure readings (HEM-775CAN, Omron Healthcare, Bannockburn, IL, USA). Mean, systolic, and diastolic arterial pressure were quantified from the raw Finometer recordings. 3.3.4.2 Brachial artery imaging. With the participants left arm extended perpendicular (i.e. 80 degrees) from their body while seated on the semi-recumbent cycle ergometer, an inflation/deflation cuff was placed on the participants left forearm, and their arm was fixed into position on a table. Brachial artery image acquisition was obtained using a 10 MHz multifrequency linear array probe attached to a high-resolution ultrasound machine (15L4, Terason t3200, Burlington, MA, USA). All brachial artery images were performed by the same experienced ultrasonographer (JT), whom has a between day coefficient of variation in FMD of 8.3 ± 2.1% (n=10, unpublished data). Following optimal image acquisition, 46 and one-minute of baseline recordings, the forearm was occluded by inflating the cuff to 220-250 mmHg for five-minutes. Recordings of diameter and velocity continued 30-seconds prior to cuff deflation and continuously for three-minutes thereafter (Thijssen et al., 2011). 3.3.5 Data Analysis 3.3.5.1 Brachial artery diameter and blood flow analysis. Ultrasound recordings were continuously screen captured and saved for offline analysis. Blood flow analysis of the brachial artery was performed using automated edge-detection and wall tracking software, which allows for the integration of synchronous diameter and velocity measurements to continuously determine flow, shear, diameter and velocity at 30-Hz, independent of investigator bias (Woodman et al., 2001). Antegrade, retrograde, and mean shear rates were calculated as four times the mean blood velocity, divided by vessel diameter. The FMD was calculated as the percent increase in vessel diameter from resting baseline diameter, where baseline and peak diameters were automatically detected from the continuous data described above. 3.3.6 Statistics All statistical analyses were performed using SigmaStat V11 (Systat, Chicago, IL, USA), and were reported as mean  SEM. Statistical significance was assumed at P<0.05. When significant F-ratios were detected, post-hoc comparisons were made using Tukey’s post hoc test for pair-wise comparisons. Our study is a follow-up from a previous study in our laboratory (Atkinson et al., 2013), which found a ~2% decrease in FMD. To detect a 2% decrease in post-exercise FMD with a standard deviation of 2%, we calculated that a sample size of 10 participants was required in order to achieve a statistical power >0.80 with a statistical difference of P<0.05. Based on this 47 power calculation, we recruited 11 participants for the sea-level and high-altitude arms of the study. 3.3.6.1 Maximal exercise. For the cardiovascular data obtained at sea-level and high-altitude, baseline measurements were averaged over one-minute immediately prior to exercise, peak maximal-exercise data were averaged over the last 30-seconds of the maximal exercise protocol, and post-maximal exercise data were averaged over one-minute, immediately prior to FMD cuff release after maximal exercise was terminated (i.e. approximately four-minutes post-exercise). Differences between peak Watt and maximal exercise time at sea-level and high-altitude were determined using a paired Student’s t-test. For the cardiovascular data, a two-way repeated measures analysis of variance (2RM-ANOVA) was used to detect differences across time (baseline, maximal exercise, post-maximal exercise), and conditions (sea-level and high-altitude). For the brachial artery data measured during FMD, a 2RM-ANOVA was used to detect differences across time (baseline and post-maximal exercise), and conditions (sea-level and high-altitude). 3.3.6.2 Moderate-intensity exercise. For the cardiovascular data obtained at sea-level and high-altitude, baseline measurements were averaged over one-minute immediately prior to exercise. During moderate-intensity exercise, cardiovascular data were averaged over 30-seconds at every five-minute time-point (i.e. time = 5-, 10-, 15-, 20-, 25-, and 30-minutes). Post-exercise cardiovascular data were averaged during the one-minute prior to FMD cuff release, immediately after, and 60-minutes after, moderate-intensity exercise. For the cardiovascular data, a 2RM-ANOVA was used to detect differences across time 48 (baseline, exercise [six time-points], and post-exercise [two time-points]), and conditions (placebo and prazosin), at sea-level and high-altitude, separately. For the brachial artery FMD data, a 2RM-ANOVA was used to detect differences across time (baseline, post-exercise, 60-minutes post exercise), and conditions (placebo and prazosin), at sea-level and high-altitude, separately. 3.3.6.3 Adjusted flow-mediated dilatation. The effects of time and condition were analyzed within and between sea-level and high-altitude for FMD. To determine if our FMD results were different due to changes in baseline arterial diameter and/or shear rate area under the curve (SRAUC), we included these variables as covariates in a logarithmic-linked generalized linear model, where FMD was the dependent variable. This approach has been used to account for any changes in FMD that may be related to differences in baseline diameter or shear rate between conditions (i.e. time and condition) (Atkinson, G. et al., 2013). 3.4 Results 3.4.1 Participants and resting FMD data The participants (n=9; one female) included in the sea-level and high-altitude protocol data analysis had a mean  SEM age of 26.9  1.8 years, height of 176.3  1.5 cm, and weight of 71.1  2.5 kg. Participants had normal pulmonary health with an FVC of 5.5  0.2 L (109.0  3.2% of predicted), forced expiratory volume in one-second (FEV1) of 4.3  0.2 L (100.6  2.8% of predicted), FEV1/FVC of 78.7  1.0 (no individuals reported under an FEV1/FVC <75), total lung capacity of 6.7  0.3 L (101.9  0.3% of predicted), and had a diffusing capacity of the lung for carbon monoxide of 32.1  5.7 ml/min/mmHg (94.1  4.7% of predicted). Recruited participants 49 did not demonstrate any signs of small nor large airway obstruction characterized by an irregular expiratory flow tracing during the FVC maneuver. Additionally, participants were normotensive (systolic blood pressure = 130.6  2.5 mmHg, diastolic blood pressure 68.7  1.9 mmHg). No differences were observed in FMD between sea-level and high-altitude during the placebo (P=0.287) and prazosin (P=0.110) trials at baseline (Figure 3.1). 3.4.2 Maximal exercise data Figure 3.2 illustrates the cardiorespiratory data collected during the maximal exercise protocol at sea-level and high-altitude. At sea-level, peak Watt and maximal exercise protocol time (min) were greater than at high-altitude (275.4  12.4 watts vs. 228.8  11.2 watts, P<0.001; 13.0  0.5 min vs. 10.6  0.5 min, P<0.001). Cardiac output and stroke volume were higher at sea-level compared to high-altitude (P=0.047 and P=0.032, respectively). In contrast, heart rate at high-altitude was elevated compared to sea-level (P=0.038). No differences were observed between sea-level and high-altitude for mean arterial pressure or total peripheral resistance at baseline, peak exercise, and post-exercise (P=0.130 and P=0.055, respectively). As expected, a main effect was observed for SpO2, as it was elevated at sea-level compared to high-altitude (P<0.001). Table 3.1 illustrates brachial diameter, shear, and FMD data during baseline and post-maximal exercise at sea-level and high-altitude. Brachial artery diameter was greater at sea-level compared to high-altitude across all time-points (P=0.030). At both sea-level and high-altitude, brachial artery diameter was reduced post-maximal exercise compared to baseline by 4.8  2.5% and 8.7  1.2%, respectively (P=0.004). Mean shear rate increased post-maximal exercise compared to 50 baseline at sea-level by 211.2  26.5 1 s-1, and at high-altitude by 101.7  20.9 1 s-1 (P<0.001). Although a main effect was not present for mean shear rate between sea-level and high-altitude (P=0.244), an interaction effect was observed post-maximal exercise as mean shear was higher at sea-level by 36.1  19.0% compared to high-altitude (P=0.003). Antegrade shear rate increased after post-maximal exercise compared to baseline at sea-level by 236.2  20.2 1 s-1, and at high-altitude by 163.8  20.3 1 s-1, respectively (both P<0.001); however, similar to mean shear rate, altitude had no effect (P=0.833). Retrograde shear rate increased (i.e. became more negative) between baseline and post-maximal exercise at sea-level by -25.3  7.8 1 s-1 and at high-altitude by 67.4  11.7 1 s-1 (P<0.001). Retrograde shear was also greater (i.e. more negative) at high-altitude compared to sea-level (P=0.031). No differences were found in absolute (mm) or relative changes (FMD) in brachial artery diameter between baseline and post-maximal exercise at sea-level or high-altitude (P=0.453 and P=0.282, respectively), or between sea-level and high-altitude (P=0.380 and P=0.244, respectively). Flow-mediated dilatation SRAUC was greater post-maximal exercise compared to baseline (P=0.025), but there was no difference between sea-level and high-altitude (P=0.312). When taking into account baseline diameter and SRAUC as covariates, the FMD results remained the same as there was no effect of time (P=0.614), altitude (P=0.291), nor was there an interaction between these effects (P=0.717) (refer to Figure 3.3 for individual FMD data at baseline and post-maximal exercise). 51 3.4.3 Moderate intensity exercise at sea-level Figure 3.4 (A-F) illustrates cardiovascular data collected during moderate intensity exercise on placebo and prazosin at sea-level. No differences were detected for cardiac output between placebo and prazosin during baseline, exercise, and post-exercise (P=0.444). In contrast, prazosin increased heart rate (P<0.001), and decreased stroke volume (P=0.026), across all time-points compared to placebo. prazosin had no effect on mean arterial pressure (P=0.701), nor total peripheral resistance (P=0.492) during baseline, exercise, and post-exercise time-points. Additionally, there was no difference in SpO2 between placebo and prazosin trials (P=0.237). Figure 3.5 (A-C) outlines brachial antegrade, retrograde, and mean shear rate data collected during baseline, exercise, and post-exercise on placebo and prazosin at sea-level. Mean and antegrade shear rate were not different between conditions (P=0.567 and P=0.156, respectively. Retrograde shear rate was lower (i.e. more negative) during the prazosin trial compared to placebo (P=0.037). Figure 3.6 (A-D) displays brachial artery velocity, diameter, blood flow and conductance data collected during baseline, exercise, and post-exercise on placebo and prazosin at sea-level. No differences were found in mean blood flow (P=0.285), forearm vascular conductance (P=0.294), artery diameter (P=0.623), nor blood velocity (P=0.400) between the placebo and prazosin trials. Figure 3.7 (A-B) illustrates FMD data collected at baseline, post-exercise, and 60-minutes post exercise on placebo and prazosin at sea-level. No differences in FMD were detected between placebo and prazosin (P=0.916), however, there was a time effect during the placebo trial, where FMD was reduced immediately post exercise compared to baseline (P=0.043), and 60-minutes 52 post-exercise (P<0.001). Additionally, an interaction effect was present between placebo and prazosin immediately post-exercise, where FMD was greater during the prazosin trial by 2.9  1.3% compared to the placebo trial (P=0.039). No differences were found in FMD between placebo and prazosin trials during baseline (P=0.762), nor 60-minutes post-exercise (P=0.107). When taking into account baseline diameter and SRAUC as covariates, the FMD results remained the same as there was still a main effect for time (P=0.016), no effect between conditions (i.e. placebo vs prazosin) (P=0.450), and an interaction effect (P=0.026). 3.4.4 Moderate intensity exercise at high-altitude Figure 3.4 (G-L) illustrates cardiovascular data collected during baseline, moderate intensity exercise, and post-exercise on placebo and prazosin at high-altitude. No differences were detected for cardiac output between placebo and prazosin during baseline, exercise, and post-exercise (P=0.825). In contrast, heart rate was elevated (P<0.001), and stroke volume was decreased (P=0.006), while on prazosin compared to placebo. prazosin resulted in a lower mean arterial pressure (P<0.001), and total peripheral resistance (P<0.001) compared to the placebo trial. Interestingly, SpO2 was elevated by 2.5  0.7% (P=0.005) during the prazosin trial compared to placebo during the moderate-intensity exercise. Figure 3.5 (D-F) illustrates brachial shear rate data collected during baseline, moderate-intensity exercise, and post-exercise on placebo and prazosin at high-altitude. Mean and antegrade shear rates were elevated during the prazosin trial compared to the placebo trial across all time-points (P=0.002 and P<0.001, respectively). However, retrograde shear rate was not different between placebo and prazosin (P=0.983). 53 Figure 3.6 (E-H) illustrates brachial artery velocity, diameter, blood flow and conductance data collected during baseline, exercise, and post-exercise on placebo and prazosin at high-altitude. During the prazosin trial, mean blood flow, conductance, and blood velocity were elevated compared to the placebo trial (P=0.004, P=0.008, and P=0.002, respectively). No differences were found to brachial artery diameter between placebo and prazosin trials (P=0.516). Figure 3.7 (C-D) illustrates FMD data collected at baseline, post-exercise, and 60-minutes post exercise on placebo and prazosin at high-altitude. No differences in FMD were detected between time-points (i.e. baseline, post-exercise, and post-60 exercise) (P=0.474), between placebo and prazosin (P=0.099). When taking into account baseline diameter and SRAUC as covariates, no differences were detected between time-points (P=0.681), nor were any interactions present (P= 0.474), which was consistent with our original results. However, we found that there was a main effect for condition (i.e. prazosin vs placebo), where FMD was higher with prazosin compared to placebo (P=0.032). 54 Table 3.1 Brachial artery diameter, shear, and flow-mediated dilatation data during baseline and post-maximal exercise at sea-level and high-altitude Definition of abbreviations: FMD, flow mediated dilatation; SRAUC, shear rate area under the curve. *P<0.05, pre-max data vs post-max data. P<0.05, interaction effect baseline vs post-max Sea-Level High-Altitude Baseline Post-max Baseline Post-max Diameter (mm) 4.4  0.2 4.2  0.2* 4.3  0.2 3.9  0.2* Time: P=0.004, Altitude: P=0.030, Interaction: P=0.328 Mean Shear (1 s-1) 86.4  13.5 297.6  26.2* 111.8  20.1 213.5  27.7* Time: P<0.001, Altitude: P=0.244, Interaction: P=0.003 Antegrade Shear (1 s-1) 101.7  13.7 337.9  20.5* 144.5  17.3 308.3  24.3* Time: P<0.001, Altitude: P=0.833, Interaction: P=0.012 Retrograde Shear (1 s-1) -15.2  6.2 -40.5  8.6* -22.1  7.1 -89.6  14.9* Time: P=0.001, Altitude: P=0.031, Interaction: P=0.009 Change in diameter (mm) 0.23  0.05 0.25  0.05 0.26  0.04 0.32  0.05 Time: P=0.453, Altitude: P=0.380, Interaction: P=0.581 FMD (%) 5.0  1.1 6.2  1.2 6.3  1.3 8.5  1.6 Time: P=0.282, Altitude: P=0.244, Interaction: P=0.532 FMD SRAUC (103 s-1) 23.3  1.9 30.3  2.8* 23.2  2.7 22.6  1.9* Time: P=0.025, Altitude: P=0.312, Interaction: P=0.185 55 Table 3.2 Brachial artery diameter and flow-mediated dilatation data during baseline and post moderate-intensity exercise at sea-level and high-altitude Definition of abbreviations: FMD, flow mediated dilatation; SRAUC, shear rate area under the curve. *P<0.05, vs pre-exercise data. †P<0.05, Post-60 vs Post. P<0.05, Interaction within Post FMD. Bolded Condition: P=0.099, when accounted for baseline diameter and shear rate, P=0.032 Sea-Level High-Altitude Pre Post Post-60 Pre Post Post-60 Diameter Placebo 4.5  0.2 4.8  0.2* 4.4  0.2* 4.2  0.1 4.2  0.1 4.2  0.2 (mm) Prazosin 4.5  0.2 4.6  0.2 4.6  0.2 4.2  0.2 4.3  0.1 4.2  0.2 Time: P=0.015, Condition: P=0.783, Interaction: P=0.008 Time: P=0.496, Condition: P=0.398, Interaction: P=0.501 Change in diameter Placebo 0.21  0.03 0.12  0.02 0.30  0.05† 0.26  0.03 0.22  0.04 0.25  0.03 (mm) Prazosin 0.20  0.04 0.22  0.05 0.22  0.03† 0.29  0.03 0.31  0.05 0.35  0.05 Time: P=0.022, Condition: P=0.954, Interaction: P=0.074 Time: P=0.502, Condition: P=0.093, Interaction: P=0.501 FMD Placebo 4.9  0.7 2.1  0.8* 7.0  1.2† 6.3  0.8 5.3  1.0 5.8  0.6 (%) Prazosin 4.5  1.0 4.9  1.3 4.0  0.9 6.8  0.8 7.3  1.2 8.5  1.2 Time: P=0.013, Condition: P=0.916, Interaction: P=0.033 Time: P=0.474, Condition: P=0.099, Interaction: P=0.455 FMD SRAUC Placebo 19.8  2.4 37.9  4.5*† 18.3  2.7 26.9  2.8 40.2  5.3*† 25.5  1.7 (103 s-1) Prazosin 27.4  2.1 39.8  4.7*† 23.7  2.5 30.5  2.5 50.2  3.0*† 37.6  3.2 Time: P<0.001, Condition: P=0.060, Interaction: P=0.448 Time: P<0.001, Condition: P=0.005, Interaction: P=0.287 56 FMD (%)02468Placebo Prazosinsea-levelhigh-altitude Figure 3.1 FMD data collected during baseline on placebo and prazosin at sea-level and high-altitude. White bars represent sea-level data  SEM, and black bars represent high-altitude data  SEM in 9 participants. Definitions of abbreviations: FMD, flow-mediated dilatation. These findings illustrate that there were no differences in resting FMD between sea-level and high-altitude after passive ascent to 3800m. 57 A.B.C.D.E.F.SpO2 (%)707580859095100SV (ml)6080100120140160HR (bpm)406080100120140160180200CO (l min-1)481216202428* ******MAP (mmHg)80100120140160TPR (mmHg (l min-1)-1)400800120016002000Baseline Max-EX Post-MaxTime: P<0.001Altitude: P<0.001Interaction: P<0.001Time: P=0.168Altitude: P=0.031Interaction: P=0.068Time: P<0.001Altitude: P=0.038Interaction: P<0.001Time: P<0.001Altitude: P=0.047Interaction: P=0.005Time: P<0.001Altitude: P=0.130Interaction: P=0.136Time: P<0.001Altitude: P=0.055Interaction: P=0.806Baseline Max-EX Post-Maxsea-levelhigh-altitude Figure 3.2 Cardiovascular data during baseline, maximal exercise, and post-maximal exercise at sea-level and high-altitude. Open circles () represent sea-level data  SEM, and closed circles () high-altitude data  SEM in 9 participants. *P<0.05, for interaction effects. Statistics for main effects and interactions are displayed on the top right of each figure panel. Definitions of abbreviations: BL, baseline; Max-Ex, maximal exercise; Post-Max, post-maximal exercise; SpO2, percent oxygen saturation of hemoglobin; SV, stroke volume, HR, heart rate; CO, cardiac output; MAP, mean arterial pressure; TPR, total peripheral resistance. Collectively, these findings reveal that SV, HR, CO, and SpO2 were all elevated at sea-level compared to high-altitude during a maximal exercise test. 58 FMD (%)024681012141618FMD (%)024681012141618Baseline Post-Max Baseline Post-MaxA. B.Sea-Level High-Altitude Figure 3.3 Individual FMD data collected at baseline and post-maximal exercise at sea-level and high-altitude. Mean data (n=9) is represented by the gray line plot. Definitions of abbreviations: BL, baseline; Post-Max, post-maximal exercise; FMD, flow-mediated dilatation. These data reveal that we found no change in FMD post maximal exercise at sea-level and high-altitude. 59 PlaceboPrazosinSV (ml)6080100120140160SV (ml)6080100120140160SpO2 (%) 80859095100Sea-Level High-AltitudeHR (bpm)406080100120140160180CO (l min-1)4812162024MAP (mmHg)708090100110120130TPR (mmHg (l min-1)-1)4008001200160020002400SpO2 (%)80859095100HR (bpm)406080100120140160180CO (l min-1)4812162024MAP (mmHg)708090100110120130TPR (mmHg (l min-1)-1)4008001200160020002400BL 5 10 15 20 25 30 Post Post60 BL 5 10 15 20 25 30 Post Post60Time: P<0.001Condition: P=0.237Interaction: P=0.190Time: P=0.001Condition: P=0.026Interaction: P=0.860Time: P<0.001Condition: P<0.001Interaction: P<0.001Time: P<0.001Condition: P=0.444Interaction: P=0.986Time: P<0.001Condition: P=0.701Interaction: P=0.199Time: P<0.001Condition: P=0.492Interaction: P=0.976Time: P<0.001Condition: P=0.005Interaction: P<0.001Time: P<0.001Condition: P=0.006Interaction: P=0.014Time: P<0.001Condition: P<0.001Interaction: P<0.001Time: P<0.001Condition: P=0.825Interaction: P=0.828Time: P<0.001Condition: P<0.001Interaction: P=0.064Time: P<0.001Condition: P<0.001Interaction: P=0.004*** * * ***** * * ************ ** * * *****A.B.C.D.E.F.G.H.I.J.K.L. 60 Figure 3.4 Cardiovascular data during baseline, moderate-intensity exercise, and post-exercise on placebo and prazosin at sea-level and high-altitude. Open circles () represent placebo data  SEM, and closed circles () represent prazosin data  SEM in 9 participants. *P<0.05, for interaction effects. Statistics for main effects and interactions are displayed on the top right of each figure panel. Definitions of abbreviations: BL, baseline; Post, immediately post-exercise; Post 60, 60-minutes post-exercise; SpO2, percent oxygen saturation of hemoglobin; SV, stroke volume, HR, heart rate; CO, cardiac output; MAP, mean arterial pressure; TPR, total peripheral resistance. These findings demonstrate that at sea-level, SV was elevated, while HR was reduced on placebo compared to prazosin during 30-minutes of moderate-intensity exercise. At high-altitude, SPO2 and HR were lower, while MAP and HR were higher on placebo compared to prazosin during 30-minutes of moderate-intensity exercise. 61 PlaceboPrazosinAntegrade Shear (1 s-1)0100200300400500Antegrade Shear (1 s-1)0100200300400500Mean Shear (1 s-1)0100200300400500Sea-Level High-Altitude Re trogr ade Shear (1 s-1)-180-160-140-120-100-80-60-40-200Mean Shear (1 s-1)0100200300400500Retrograde Shear (1 s-1)-180-160-140-120-100-80-60-40-200BL 5 10 15 20 25 30 Post Post60 BL 5 10 15 20 25 30 Post Post60Time: P<0.001Condition: P=0.567Interaction: P=0.043Time: P=0.001Condition: P=0.156Interaction: P=0.208Time: P<0.001Condition: P=0.037Interaction: P=0.026Time: P<0.001Condition: P=0.002Interaction: P=0.474Time: P<0.001Condition: P<0.001Interaction: P=0.316Time: P<0.001Condition: P=0.983Interaction: P=0.203* ****A.B.C.D.E.F. Figure 3.5 Brachial artery shear rate data during baseline, moderate-intensity exercise, and post-exercise on placebo and prazosin at sea-level and high-altitude. Open circles () represent placebo data  SEM, and closed circles () represent prazosin data  SEM in 9 participants. *P<0.05, for interaction effects. Statistics for main effects and interactions are displayed on the top right of each figure panel. Definitions of abbreviations: BL, baseline; Post, immediately post-exercise; Post 60, 60-minutes post-exercise. These data reveal that retrograde shear rate was greater after prazosin administration at sea-level, while at high-altitude, mean and antegrade shear rate were greater after prazosin administration compared to placebo during 30-minutes of moderate-intensity exercise. 62 PlaceboPrazosinConductance(ml min-1 mmHg-1)0123456Conductance(ml min-1 mmHg-1)0123456Mean Blood Flow (ml min-1)0100200300400500600Sea-Level High-AltitudeDiameter (mm)3.84.04.24.44.64.85.05.2Blood Velocity (cm s-1)0102030405060Mean Blood Flow (ml min-1)0100200300400500600Diameter (mm)3.84.04.24.44.64.85.05.2Blood Velocity (cm s-1)0102030405060BL 5 10 15 20 25 30 Post Post60 BL 5 10 15 20 25 30 Post Post60Time: P<0.001Condition: P=0.285Interaction: P=0.052Time: P=0.001Condition: P=0.294Interaction: P=0.131Time: P<0.001Condition: P=0.623Interaction: P=0.095Time: P<0.001Condition: P=0.400Interaction: P=0.033Time: P<0.001Condition: P=0.004Interaction: P=0.049Time: P<0.001Condition: P=0.008Interaction: P=0.986Time: P<0.001Condition: P=0.516Interaction: P=0.270Time: P<0.001Condition: P=0.002Interaction: P=0.276* ********A.B.C.D.E.F.G.H. Figure 3.6 Brachial artery velocity, diameter, blood flow and conductance data collected during baseline, moderate-intensity exercise, and post-exercise on placebo and prazosin at sea-level and high-altitude. Open circles () represent placebo data  SEM, and closed circles () represent prazosin data  SEM in 9 participants. *P<0.05, for interaction effects. Statistics for main effects and interactions are displayed on the top right of each figure panel. Definitions of abbreviations: BL, baseline; Post, immediately post-exercise; Post 60, 60-minutes post-exercise. These findings illustrate that there were no differences found in brachial artery velocity, diameter, blood flow and conductance at sea-level between placebo and prazosin trials, however, at high-altitude, we found brachial artery velocity, blood flow and conductance were reduced while participants were on placebo compared to prazosin during the 30-minute moderate-intensity exercise test. 63 Baseline Post Post60A.B.C.D.FMD (%)0246810121416FMD (%)0246810121416FMD (%)0246810121416FMD (%)0246810121416Sea-level Placebo High-altitude PlaceboSea-level Prazosin High-altitude Prazosin*Baseline Post Post60 Figure 3.7 Individual FMD data collected during baseline and post-exercise on placebo and prazosin at sea-level and high-altitude. Mean data (n=9) is represented by the gray line plot. *P<0.05, represents time effect, where FMD Post-EX was lower compared to baseline, and Post-60 (for more details, see results section). Definitions of abbreviations: Post, immediately post-exercise; Post 60, 60-minutes post-exercise; FMD, flow-mediated dilatation. Collectively, these findings reveal that at sea-level, FMD was reduced immediately after 30-minutes of moderate-intensity exercise while on placebo, and the reduction in FMD was abolished after administration of prazosin. At high-altitude, there was no observed reduction in FMD immediately after 30-minutes of moderate-intensity exercise after administration of placebo or prazosin. Additionally, after taking into account for changes in brachial baseline diameter and shear rate between placebo and prazosin trials, FMD was elevated after administration of prazosin compared to placebo at high-altitude. 64 3.5 Discussion Using a double-blinded, counter-balanced, randomized and placebo-controlled design, this is the first study to examine the role of the SNS system on post-exercise peripheral vascular endothelial function at both sea-level (344m) and high-altitude (3800m). Our main findings were 1) at rest, brachial artery FMD remained unchanged between sea-level and high-altitude on both placebo and prazosin conditions, 2) flow-mediated dilatation remained unchanged after maximal exercise at both sea-level and high-altitude, and 3) flow-mediated dilatation decreased immediately after moderate-intensity exercise at sea-level, but not high-altitude. Prazosin abolished the observed post-exercise FMD decrease at sea-level, and resulted in an overall increase in FMD at high-altitude compared to placebo when SRAUC and baseline diameter were considered as covariates. These data demonstrate that hypobaric hypoxia counteracts the effect of moderate intensity exercise on FMD. 3.5.1 Endothelial function between sea-level and high-altitude at rest Acclimatization to high-altitude results in several physiological changes, but the effect of high-altitude on endothelial function remains somewhat unclear. At sea-level, recent work has established that increases in SNS activity (Hijmering et al., 2002, Dyson et al., 2006, Atkinson et al., 2015), and altered hemodynamics (e.g. increased retrograde shear) (Johnson, Mather et al., 2012, Dawson et al., 2008, Birk et al., 2013, Lamping and Dole, 1987, Millgard and Lind, 1998) can negatively affect vascular endothelial function as assessed by FMD. Since both of these physiological changes occur during high-altitude exposure, it is logical to hypothesize that endothelial function would be reduced at high-altitude, yet we found that there was no change. Comparisons of endothelial function between sea-level and high-altitude have been made 65 previously, but studies have reported contradictory results such as reduced FMD (Lewis et al., 2014, Bakker et al., 2015), or no change in FMD upon acclimatization to high-altitude (Bruno et al., 2016, Bruno et al., 2015). The major difference between these studies is the mode of transport to altitude – exercise versus cable car ascent. We sought to further investigate the effects of non-trekking arrival to high-altitude on endothelial function, and consistent with the results of Bruno et al. (2015), we found no change in endothelial function between sea-level and high-altitude at rest. This finding opposes our hypotheses that endothelial function would be impaired at high-altitude due to elevated resting SNS activity. Rather, our data indicate that exercise at high-altitude (i.e. trekking) may directly affect endothelial function, and we speculate the mechanism(s) for this may be due to increased vascular inflammation (Bruno et al., 2016), oxidative stress (Quindry et al., 2015), and reductions in nitric oxide bioavailability (Lewis et al., 2014). 3.5.2 Endothelial function before and after maximal exercise at sea-level and high-altitude The degree of observed post-exercise reduction in FMD has been thought to be exercise intensity-dependent (Birk et al., 2013); however, few studies have measured FMD after maximal exercise in healthy individuals in an inactive limb (free from exercise) (Thijssen et al., 2006, Hwang et al., 2012, McClean et al., 2015). Moreover, no studies to date have reported FMD after maximal exercise at high-altitude. Based on previous reports, FMD following maximal exercise remains unchanged in the majority of studies (Thijssen et al., 2006, Hwang et al., 2012, McClean et al., 2015) but this is not a universal finding (Hwang et al., 2012). In support with studies methodologically similar to ours (Thijssen et al., 2006, Hwang et al., 2012, McClean et al., 2015), 66 we demonstrated that at sea-level, and for the first time at high-altitude, FMD remained unchanged post-maximal exercise at both sea-level and high-altitude. This finding opposes the idea that post-exercise related decreases in FMD are inversely related to exercise intensity (Birk et al., 2013). An explanation for this finding is that the exercise duration of the maximal exercise test was too short in duration (i.e. exercise volume) to induce a reduction in FMD (Johnson, Padilla et al., 2012, Dawson et al., 2013). Interestingly, at high-altitude, FMD was trending in the positive direction post-maximal exercise (6.3% to 8.5%), but this observation did not come out statistically significant. 3.5.3 Endothelial function before and after moderate-intensity exercise at sea-level and high-altitude 3.5.3.1 Sea-level: In a different group of subjects, Atkinson et al. (2016) performed a similar investigation to the sea-level component of this study and demonstrated that FMD was transiently impaired immediately after 30-minutes of moderate-intensity exercise; these changes improved back to pre-exercise values 60-minutes post moderate-intensity exercise. Atkinson et al. (2016) attributed this reduction in FMD to exercise related increases in SNS activity. Congruent with Atkinson et al. (2016) and other studies with similar methodology to assess FMD (Goel et al., 2007, Dawson et al., 2008, Jones et al., 2010, Johnson, Mather et al., 2012, Birk et al., 2013), we found similar results. We acknowledge, however, that such transient reductions in FMD following moderate-intensity exercise (at least at sea level) should not necessary be interpreted that endothelial dysfunction; rather, that FMD that it is influenced by a myriad of factors that may well reflect physiological adaptation in other vascular beds, including resistance and conduct arteries. 67 3.5.3.2 High-altitude: Although there has been a recent investigation of post-exercise endothelial function after moderate-intensity exercise in acute normobaric hypoxia (Katayama et al., 2016), our experiment was the first to investigate endothelial function after moderate-intensity exercise at high-altitude (i.e. 3800m). In the study by Katayama et al. (2016), endothelial function was not different post 30-minutes of moderate-intensity (i.e. 60% VO2 max) exercise between normoxia and hypoxia (FIO2 = 0.12-0.13) trials. However, longer exposure to hypoxia (i.e. 4-7 days at high-altitude) may well yield differential results due to cardiovascular adaptation (Lewis et al., 2014, Boos et al., 2012, Rhodes et al., 2011), and perhaps differential tonic SNS activity (Hansen and Sander, 2003). There is evidence suggesting increased SNS activity may be responsible for vascular dysfunction directly (Hijmering et al., 2002), or indirectly by increasing retrograde shear rate (Thijssen et al., 2014). Due to high-altitude related increases in SNS activity, and based on previous reports that endothelial function (via brachial FMD) is reduced upon arrival at high-altitude in some (Bakker et al., 2015, Lewis et al., 2014), but not all studies (Bruno et al., 2016), we anticipated a reduction in FMD immediately post moderate-intensity exercise at high-altitude. Our original hypothesis was not supported as no reduction in FMD was observed post moderate-intensity exercise, indicating that SNS related blood vessel regulation is different between sea-level and high-altitude. This latter notion suggests that post-exercise associated elevations in SNS activity may have a differential transduction to the peripheral vasculature compared to sea-level. 68 3.5.4 Effects of 1-adrenergic blockade on endothelial function at sea-level and high-altitude. 1-adrenergic blockade, results in vasodilatation of the peripheral vasculature by reducing SNS transduction directly to smooth muscle cells. At sea-level, prazosin resulted in an increased heart rate and decreased stroke volume during exercise. However, the differential heart rate response between placebo and prazosin trials did not result in a different blood flow or mean and antegrade shear rate response to exercise. Similar to Atkinson et al. (2016), we reported that prazosin administration abolished the reduction in endothelial function immediately post 30-minutes of moderate-intensity exercise. At rest, SNS blockade has no effect on SpO2 at sea-level (Liu et al., 2007), nor high-altitude (Ainslie et al., 2012). During exercise, SpO2 remained the same at sea-level between conditions, however, at high-altitude, an unexpected observation was that participants on prazosin had a higher SpO2 compared to the placebo trial. This was likely due to increased ventilation during exercise on the prazosin trial, perhaps due to baroreflex-chemoreflex interaction, supported by the increased heart rate, and reduced stroke volume and mean arterial pressure responses to exercise between placebo and prazosin trials. The ventilatory response to changes in arterial blood pressure is related to converging baroreceptor and chemoreceptor afferents at the nucleus tractus solitarius (Richter and Seller, 1975, Eckberg and Orshan, 1977). A reduction in arterial blood pressure potentiates the chemoreflex and results in an increase in ventilation, while an increase in arterial blood pressure dampens ventilation (Heistad et al., 1974). 69 In contrast to our sea-level data, prazosin resulted in an increased mean and antegrade shear rate at high-altitude. These changes were likely facilitated by blockade of SNS vasoconstriction, which was likely enhanced at altitude. Increases in antegrade shear rate has been demonstrated to have a positive effect on endothelial function (Thijssen et al., 2014). This enhanced antegrade shear response at altitude versus sea-level may explain the lack of post exercise FMD impairment, and could be in part responsible for the increase in endothelial function observed during the prazosin trial at altitude. The increase in FMD observed at high-altitude during the prazosin trial compared to the placebo trial suggests that there is indeed some SNS related vascular constraint. These data collected at high-altitude indicate that SNS activity is, in part, responsible for the FMD response. 3.6 Methodological considerations. The intervention to reduce SNS activity was an 1 specific adrenergic receptor blockade, and the dose of administration has been used to establish ~80% blockade in SNS activity, but this was not confirmed on an individual basis. Second, we can not exclude the possibility that the vascular response to changes in vasodilatory substances is also altered (Calbet et al., 2014), and could hence influence our findings. A final consideration is that we did not directly measure SNS activity directly via microneurography. Obviously, obtaining muscle SNS measurements in the leg (via peroneal nerve) would not be possible during exercise; however, past studies have reported in humans in the arm (via radial nerve) (Rea and Wallin, 1989). This approach would not have been feasible given our small sample size and the amount of measurements needed in each participant (i.e. twice at both sea-level and high-altitude). Nevertheless, both at sea level and high altitude we have for 70 the first time employed a powerful double-blinded, counter-balanced, randomized and placebo-controlled study to examine the role of SNS blockade both at rest and during exercise. 3.7 Synopsis Our findings illustrate that at sea-level, exercise related increases in SNS activity reduce endothelial function immediately following moderate-intensity exercise, but not at high-altitude. These findings indicate that differential governing mechanisms exist for endothelial function between sea-level and high-altitude. Together, our findings have implications for better understanding the chronic impacts of hypoxemia and exercise, and the interactions on sympathetic activity and vascular function. 71 Chapter 4: Transient alterations in sympathetic nervous activity do not influence brachial artery endothelial function at sea-level and high-altitude 4.1 Background Brachial artery FMD is a non-invasive measurement of artery diameter changes in response to a transient increase in shear stress, and provides a clinical index of endothelial function [reviewed in: (Thijssen et al., 2011)]. Brachial FMD can be altered by several physiological factors such as: a) oxidative stress (Goel et al., 2007, Silvestro et al., 2002); b) shear stress, and hemodynamics (e.g. cardiac output and blood pressure) (Johnson et al., 2012, Dawson et al., 2008, Birk et al., 2013, Lamping and Dole, 1987); c) inflammation (Hartmann et al., 2000, Hingorani et al., 2000), and; d) SNA (Hijmering et al., 2002, Dyson et al., 2006, Thijssen et al., 2014, Thijssen et al., 2006, Tymko et al., 2016, Atkinson et al., 2015). Given that, increased SNA has been linked to cardiovascular disease and aging (Cohn et al., 1984, Lahiri et al., 2008, Matsukawa et al., 1998), from a clinical perspective it is important to clearly understand the effects of SNA on vascular health in humans. In this context, the role of SNA on endothelial function has been examined by several investigations in young, healthy humans. These studies have revealed that FMD is impaired under conditions in which SNA is transiently elevated, such as LBNP (Thijssen et al., 2014, Hijmering et al., 2002), cold pressor test (Dyson et al., 2006), mental stress (Ghiadoni et al., 2000), and immediately after cycling exercise (Tymko et al., 2016, Atkinson et al., 2015, Jones et al., 2010, Birk et al., 2013, Dawson et al., 2013). Additionally, exposure to hypobaric hypoxia (e.g. high-altitude) - which markedly elevates resting SNA (Hansen and Sander, 2003, Duplain et al., 1999), has been demonstrated to reduce endothelial function in some (Bakker et al., 2015, Lewis et al., 2014), but not all cases (Tymko et al., 2016, Tremblay et al., 2017, Bruno et al., 2015, Bruno et al., 2016). Differences in the degree and duration of altitude exposure, shear stress stimulus, and 72 altitude ascent profile (passive vs. active) likely explain these variable findings on the influence of altitude on endothelial function. It is clear that SNA is elevated during moderate or severe LBNP (Victor and Leimbach, 1987, Scherrer et al., 1988), cold pressor test (Seals, 1990, Dyson et al., 2006), acute and chronic hypoxic exposure (Hansen and Sander, 2003, DeBeck et al., 2010, Duplain et al., 1999), and during lower-body cycling exercise (Katayama et al., 2011). However, in addition to increasing SNA, each of these interventions have consequential changes in heart rate, stroke volume, blood pressure, and retrograde shear (i.e. altered hemodynamics) – these physiological factors can directly affect endothelial function (Birk et al., 2013, Lamping and Dole, 1987, Newcomer et al., 2011, Thijssen et al., 2009, Dawson et al., 2013, Laughlin et al., 2008, Padilla et al., 2010). Currently, it remains unclear whether the current observed reductions in brachial artery endothelial function are directly due to SNA related vascular constraint, or by physiological consequences of SNA (e.g., increases in retrograde shear rate), which directly impairs endothelial function (Thijssen et al., 2014). We attempted to address this gap in the literature by investigating the role of SNA on endothelial function independent of altered hemodynamics using an experimental design similar to previous work (Millar et al., 2013), involving mild LBNP (-10 mmHg) and mild LBPP (+10 mmHg), which alters both cardiopulmonary and arterial baroreflex activity (Millar et al., 2013). The distinct advantage of employing a mild LBNP/LBPP model is that both modalities alter SNA that are independent of changes in heart rate (Fu et al., 1998, Rea and Wallin, 1989), stroke volume (Fu et al., 1998), blood pressure (Fu et al., 1998, Cui et al., 2004, Rea and Wallin, 1989), and brachial artery vessel diameter (Padilla et al., 2010). In supine position at rest, mild LBNP (-5 to -10 73 mmHg) has demonstrated to significantly increase SNA by ~30-60% (Cui et al., 2004, Scherrer et al., 1988, Rea and Wallin, 1989), while LBPP (+10 to +20 mmHg) decreases SNA by ~30% (Fu et al., 1998), in healthy individuals. Interestingly, the elevations in SNA observed during mild LBNP (~30-60%) are comparable to those achieved with acute hypoxia (FIO2 = 0.11) (DeBeck et al., 2010), which reduces brachial FMD via an α1-adrenergic pathway (Lewis et al., 2014). In addition, due to SNA withdrawal, the novel approach of using LBPP may serve as a non-pharmacological tool to elevate endothelial function, especially in the presence of hypobaric hypoxia when resting SNA is markedly elevated (Hansen and Sander, 2003, Duplain et al., 1999). 4.2 Aims and Hypotheses By employing a counter-balanced, randomized design, the primary purposes of the current study were to investigate the role of SNA on endothelial function at sea-level (344m), and during chronic exposure to hypobaric hypoxia (5050m) where resting SNA is chronically elevated (Hansen and Sander, 2003, Duplain et al., 1999). By using a novel, purpose built, light-weight, portable lower-body differential pressure chamber used to alter SNA largely independent of hemodynamics, we hypothesized that: 1) at rest, SNA would be elevated, and endothelial function would be reduced at high-altitude compared to sea-level, 2) at sea-level and after acclimatization to high-altitude, endothelial function would be reduced during a transient increase in SNA (induced by mild LBNP), and elevated during a transient decrease in SNA (induced by mild LBPP), independent of changes in systemic hemodynamics 74 4.3 Methods and Materials 4.3.1 Ethical Approval. All experimental procedures and protocols were approved by the clinical research ethics board at the University of British Columbia and conformed to the Declaration of Helsinki. All participants provided written informed consent prior to participation in this study. This study was part of a larger research expedition conducted between September and November 2016. As such, participants took part in a number of studies conducted at the University of British Columbia (Kelowna, British Columbia; 344m) and during three weeks at the Ev-K2 CNR pyramid laboratory (Khumbu Valley, Nepal, 5050m). However, the a priori, primary research questions addressed in the current paper are novel and are exclusively dealt within this study alone. 4.3.2 Participants. Recruited participants (n=15; 1F) were normotensive (systolic blood pressure <140 and diastolic pressure <90 mmHg) at rest, and completed a medical history questionnaire. The participants were non-smokers, had no previous history of cardiovascular, cerebrovascular, or respiratory diseases. During the time of testing, one participant was taking oral contraceptives (i.e. birth control), and another was taking Mesalazine. At sea-level, two participants were excluded from data analyses for the following reasons: 1) testing was terminated on one participant due to being uncomfortable in the lower-body differential chamber, thus, testing was also not continued at high-altitude in this participant, and 2) a participant was omitted from data analysis at sea-level due to inadequate brachial artery imaging. However, this participant was included in our high-altitude data analysis (n=14). In summary, out of the 15 participants recruited for the current study, 13 and 14 participants were included in our data analysis at sea-level and high-altitude, respectively. In 75 addition, cardiac output data was missing in one participant at high-altitude due to equipment malfunction. All participants arrived at the Ev-K2 CNR research facility within two days of each other, after following a similar ascent profile (7-8 day trek) as described in detail elsewhere (Foster et al., 2014, Lewis et al., 2014, Willie et al., 2014). Upon ascent, all participants avoided taking oral acetazolamide (i.e. Diamox), a carbonic anhydrase inhibitor commonly used to prevent/treat high-altitude illness. Experimentation occurred between days 11 and 14 at high-altitude, and no participants had any symptoms of altitude illness during the time of testing, nor were any using aspirin, non-steroidal anti-inflammatory drugs, and phosphodiesterase-5 inhibitors. 4.3.3 Experimental Design. This study was conducted in two parts: sea-level and high-altitude investigations. Prior to each experiment, all participants abstained from exercise, alcohol, and caffeine for at least 12 hours. Additionally, participants were asked to consume a light meal at least four-hours prior to experimentation, and to keep their diet consistent between experimentation days. In order to determine whether our participants had normal healthy lung function, at sea-level we conducted a forced vital capacity (FVC) test to measure lung function, a vital capacity and inspiratory capacity maneuver to measure lung volumes, and a single breath carbon monoxide test to quantify diffusing capacity on each individual. All testing procedures were conducted in accordance with the American Thoracic Society and European Respiratory Society’s joint guidelines (Miller et al., 2005, Macintyre et al., 2005). For each of these tests, participants sat within a 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 76 filter while wearing a nose-clip. All pulmonary function measurements were compared against population-based predictions. 4.3.3.1 Experimental protocol. After becoming comfortable within our custom lower-body differential pressure chamber (described below), participants were instructed to lie motionless in the supine position and breathe normally for 20-minutes to ensure that blood volume was comparably distributed prior to experimentation (Goswami et al., 2008). At sea-level, muscle sympathetic nervous activity (MSNA) in the radial nerve was collected in a subset of participants (attempted: n=10; obtained: n=5) during the LBNP/LBPP protocol. Muscle SNA signals were obtained once the participant was instrumented while laying supine in our custom lower-body differential pressure chamber (described below). At sea-level and high-altitude, the protocol began with a five-minute eupneic breathing baseline period, after which, the pressure within the chamber was altered to one of the following: 1) -10 mmHg (LBNP trial), 2) remained unchanged at zero mmHg (control trial), or 3) +10 mmHg (LBPP trial). Once adequate pressure was achieved in the lower-body differential chamber, the participant was asked to remain quiet and relaxed, and after five-minutes a brachial artery FMD was performed on the participants left arm. Once the brachial artery FMD measurement was collected, the pressure of the lower-body differential pressure chamber was alleviated and the participant was given a five-minute recovery period. The protocol was then repeated for the remaining two randomized conditions (i.e. LBNP, control, or LBPP). Before each condition, a five-minute quiet resting baseline was endured (Figure 4.1). 77 Additionally, out of the five participants that we were able to obtain radial MSNA data at sea-level, MSNA signals were obtained in the peroneal nerve at rest at high-altitude in four of these participants in order to demonstrate the effects of altitude on resting MSNA. Previous work has shown that there are no regional differences in MSNA between the radial and peroneal nerve (Rea and Wallin, 1989). 4.3.4 Experimental Measurements. 4.3.4.1 Cardiovascular measurements. All continuously recorded cardiovascular measurements were acquired at 1000 Hz using an analog-to-digital converter (Powerlab/16SP ML 880; ADInstruments, Colorado Springs, CO, USA) interfaced with a personal computer. Commercially available software was used to analyze cardiovascular variables (LabChart V7.1, ADInstruments, Colorado Springs, CO, USA). Electrocardiogram electrodes were placed in lead II configuration (Bioamp, ML132, ADInstruments, Colorado Springs, CO, USA) to measure heart rate. Beat-by-beat arterial pressure, cardiac output, stroke volume, and total peripheral resistance was measured by finger photoplethysmography (Finometer Pro, Finapres medical systems, Amsterdam, Netherlands). Prior to baseline data collection, the Finometer was calibrated using the return-to-flow function. Mean, systolic, and diastolic arterial pressure were quantified from the raw Finometer recordings. 4.3.4.2 Brachial artery imaging. With the participants left arm extended perpendicular (i.e. 90 degrees) from their body, an inflation/deflation cuff was placed on the participants left forearm, and their arm was fixed into position on a table at the level of the heart. Brachial artery image acquisition was obtained using a 78 10 MHz multifrequency linear array probe attached to a high-resolution ultrasound machine (15L4, Terason t3200, Burlington, MA, USA). All brachial artery images were performed by the same experienced ultrasonographer [J.C.T; performed brachial artery FMD in the following published investigations (Tremblay et al., 2015, Tremblay et al., 2017, Tymko et al., 2016)], whom has a between day coefficient of variation in FMD of 8.3 ± 2.1% (n=10, unpublished data). Following optimal image acquisition, and one-minute of baseline recordings, the forearm was occluded by inflating the cuff to 220 mmHg for five-minutes. Recordings of diameter and velocity resumed 30-seconds prior to cuff deflation and continuously for three-minutes thereafter (Thijssen et al., 2011). 4.3.4.3 Lower-body differential pressure chamber. Mild LBNP and LBPP was elicited using a custom‐built, light-weight, portable, lower-body differential pressure chamber (designed and built by M.M.T). The LBNP chamber was sealed at the level of the iliac crest of each participant using stretchable waist belts. Pressure within the chamber was generated using a 120V house-hold vacuum pump, and measured using a digital manometer (DigiMano 1000, 200–200IN, Netech Corporation, Farmingdale, NY, USA). The magnitude of negative pressure was manipulated using a 120‐volt input/140‐volt output variable transformer (Variac, Cleveland, OH, USA). Stable pressure of -10 mmHg LBNP or +10 mmHg LBPP were achieved within 10-15 seconds after turning on the vacuum pump. 4.3.4.4 Muscle sympathetic nerve activity. Recordings of MSNA were obtained by an experienced microneurographer (C.D.S and J.P.M). A tungsten microelectrode (50 mm long, 200 μm in diameter) was inserted percutaneously into the right radial nerve (at sea-level), and the right peroneal nerve (at high-altitude), using ultrasound 79 guidance (12mHz linear array probe, GE Health Care, Canada) (Curry and Charkoudian, 2011). A reference electrode was positioned subcutaneously 1–3 cm from the recording site. A suitable sympathetic nerve site was searched through manual manipulation of the tungsten microelectrode until a characteristic pulse-synchronous burst pattern was observed. Confirmation that the recorded signal represented MSNA was determined by the absence of skin paresthesia and a signal that increased in response to voluntary apnea but not during arousal to a loud noise. Muscle sympathetic nervous activity was amplified 1,000× through a preamplifier and 100× by a variable-gain, isolated amplifier. The amplified, raw MSNA signal was band-pass filtered at a bandwidth of 700–2,000 Hz, sampled at 10,000 Hz and stored for offline analysis (LabChart V7.1, ADInstruments, Colorado Springs, CO, USA). 4.3.5 Data Analysis Ultrasound recordings were continuously screen captured and saved for offline analysis. Blood flow analysis of the brachial artery was performed using automated edge-detection and wall tracking software, which allows for the integration of synchronous diameter and velocity measurements to continuously determine flow, shear, diameter and velocity at 30-Hz, while minimizing investigator bias (Woodman et al., 2001). Antegrade, retrograde, and mean shear rates were calculated as four times the mean blood velocity, divided by vessel diameter and the oscillatory shear index as |retrograde shear rate| / (|antegrade shear rate| + |retrograde shear rate|). The FMD was calculated as the percent increase in vessel diameter from resting baseline diameter to peak diameter following cuff release, where baseline and peak diameters were automatically detected from the continuous data described above. 80 Muscle sympathetic nervous activity was analyzed using peak analysis software (LabChart V7.1, ADInstruments, Colorado Springs, CO, USA). Two minutes of MSNA data was averaged immediately prior to the end of each LBNP, control, and LBPP trial, and was expressed as the frequency of MSNA bursts per minute, and incidence per 100 heart beats. 4.3.6 Statistics All statistical analyses were performed using SigmaStat V13 (Systat, Chicago, IL, USA), and were reported as mean  SEM. Statistical significance was set at P<0.05. Paired t-tests were used to detect changes in cardiovascular variables between baseline and during the brachial artery FMD during LBNP, control, and LBPP at both sea-level and high-altitude (Table 4.1). One-way and two-repeated measures analysis of variance were used to detect any differences in brachial artery variables (Table 4.2, Figure 4.2, and Figure 4.5). One-way repeated measures analysis of variance was used to detect any differences in MSNA between LBNP, control, and LBPP trials at sea-level (Figure 4.3), and paired t-tests were used to assess any differences in MSNA between sea-level and high-altitude at rest (Figure 4.4). When significant F-ratios were detected, post-hoc comparisons were made using Bonferonni post hoc test for pair-wise comparisons. 4.3.6.1 Adjusted flow-mediated dilatation. The effects condition (i.e. LBNP, control, and LBPP) were analyzed within and between sea-level and high-altitude for FMD. To determine if our FMD results were different due to changes in baseline arterial diameter and/or shear rate area under the curve (SRAUC), we included these variables as covariates in a logarithmic-linked generalized linear model, where FMD was the dependent variable. This approach has been used to account for any changes in FMD that may be 81 related to differences in baseline diameter or shear rate between conditions (i.e. time and condition) (Atkinson, G. et al., 2013). 82 4.4 Results 4.4.1 Participants The participants included in the sea-level (n=13) and high-altitude (n=14) protocol data analysis had a mean  SEM age of 27.2  1.7 years, height of 179.5  1.7 cm, and weight of 74.4  2.5 kg. Participants had normal pulmonary health with an FVC of 5.5  0.1 L (104.3  2.4% of predicted), forced expiratory volume in one-second (FEV1) of 4.3  0.1 L (95.5  3.3% of predicted), FEV1/FVC of 78.3  1.1, total lung capacity of 6.8  0.2 L (98.5  2.3% of predicted), and had a diffusing capacity of the lung for carbon monoxide of 33.1  1.6 ml/min/mmHg (93.1  3.9% of predicted). Recruited participants did not demonstrate any signs of small nor large airway obstruction characterized by an irregular expiratory flow tracing during the FVC maneuver. 4.4.2 Endothelial function between sea-level and high-altitude. At high-altitude, absolute brachial artery FMD was reduced compared to sea-level by 0.10 ± 0.05 mm during the LBNP trial; 0.08 ± 0.05 mm during the control trial, and; 0.07 ± 0.04 mm during the LBPP trial (main effect: P=0.024; Figure 4.2). Additionally, there was no condition effect of LBNP, control, LBPP (P=0.243), nor interaction effect (P=0.835). Similarly, although relative brachial artery FMD was reduced at high-altitude compared to sea-level, this effect marginally missed our statistical significant criteria of P<0.05 (P=0.061). No differences were detected in relative brachial artery FMD for condition (P=0.343), nor interaction (P=0.856; Figure 4.2). In addition, when taking into account SRAUC and changes in baseline brachial artery diameter between sea-level and high-altitude, our results for brachial FMD were the same with a main effect between sea-level and high-altitude (P=0.008), and no differences found for condition (P=0.250), nor interaction (P=0.693). 83 4.4.3 Muscle sympathetic nervous activity at sea-level and high-altitude. Muscle sympathetic nervous activity was collected in a subset of participants (n=5) at sea-level (Figure 4.3). During the -10 mmHg LBNP trial, MSNA bursts per minute was elevated by 59.1 ± 25.2% compared to control (P=0.007), and by 140.1 ± 10.6% compared to the +10 mmHg LBPP trial (P=0.047). No differences were found between LBPP and the control trial with our one-way repeated measures analysis of variance; however, when comparing MSNA bursts per minute using a paired t-test, MSNA was significantly reduced by 39.2 ± 12.3% (P=0.03; Figure 4.3) during the LBPP trial. Similarly, MSNA burst incidence (per 100 heart beats) was elevated by 61.5 ± 25.9% compared to control (P=0.005), and by 131.9 ± 11.7% compared to the +10 mmHg LBPP trial (P=0.03). When comparing MSNA burst incidence using a paired t-test between LBPP and control trials, MSNA was significantly reduced by 35.1 ± 13.2% (P=0.04; Figure 4.3). Out of the five participants that MSNA recordings were obtained at sea-level, we were able to obtain peroneal MSNA signals at rest in four of these participants at high-altitude (Figure 4.4). At high-altitude, MSNA bursts per minute was elevated compared to sea-level by 98.2 ± 39.5% (P=0.03), and although MSNA burst incidence was also higher at high-altitude compared to sea-level by 72.0 ± 35.2%, this elevation did not reach statistical significance (P=0.05; Figure 4.4). 4.4.4 Cardiovascular variables during LBNP and LBPP. 4.4.4.1 Sea-level: As expected, no change was present in HR, SV, CO, and TPR between baseline and FMD during LBNP (P=0.367, P=0.847, P=0.320, and P=0.614, respectively), control (P=0.854, P=0.155, 84 P=0.472, and P=0.892, respectively), and LBPP (P=0.534, P=0.218, P=0.238, and P=0.785, respectively). Mean arterial pressure also remained unchanged from baseline during the LBNP and control trials (P=0.243 and P=0.257, respectively); however, it was elevated by 4.2 ± 1.2 mmHg during the LBPP trial (P=0.003; Table 4.1). 4.4.4.2 High-altitude: At high-altitude, HR, SV, CO, and TPR were the same between baseline and FMD during LBNP (P=0.703, P=0.677, P=0.992, and P=0.063, respectively), control (P=0.054, P=0.233, P=0.313, and P=0.453, respectively), and LBPP (P=0.201, P=0.355, P=867, and P=0.845, respectively). Mean arterial pressure was unchanged during the LBNP and control trial before and after brachial FMD (P=0.099 and P=0.171, respectively). In contrast, it was slightly elevated by 4.5 ± 0.9 mmHg during LBPP (P<0.001; Table 4.1). 4.4.5 Brachial artery responses during LBNP and LBPP. 4.4.5.1 Sea-level: Brachial artery diameter, blood velocity, blood flow, and vascular resistance were the same between LBNP, control, and LBPP trials (main effects: P=0.422, P=0.384, P=0.985, and P=0.867 respectively). Additionally, brachial mean, antegrade, and retrograde shear rates, and the oscillatory shear index were not different between LBNP, control, and LBPP (main effects: P=0.928, P=0.928, P=0.891, and P=0.919, respectively; Table 4.2). 85 4.4.5.2 High-altitude: No differences were detected in brachial artery diameter, blood velocity, blood flow, and vascular resistance was the same between LBNP, control, and LBPP trials (main effects: P=0.993, P=0.224, P=0.405, and P=0.235, respectively). Additionally, brachial mean, antegrade, and retrograde shear rates, and the oscillatory shear index were not different between LBNP, control, and LBPP (main effects: P=0.304, P=0.563, P=0.119, and P=0.186, respectively; Table 4.2) 4.4.6 Endothelial function during LBNP and LBPP. 4.4.6.1 Sea-level: No difference was detected in SRAUC to peak diameter between LBNP, control, and LBPP trials (main effect: P=0.995). At sea-level, one participant was excluded from mean data analysis due to low-quality video files (Figure 4.5). Brachial artery FMD (n=13) did not change between LBNP, control, and LBPP (main effect: P=0.448). 4.4.6.2 High-altitude: There were no differences detected for SRAUC between LBNP, control, and LBPP trials (main effect: P=0.825) during the hypobaric hypoxia trial. Brachial artery FMD (n=14) did not change between LBNP, control, and LBPP trials (main effect: 0.537; Figure 4.5). 86 Table 4.1 Cardiovascular variables at baseline, and during lower-body differential pressure at sea-level and high-altitude. Sea-level High-altitude LBNP Control LBPP LBNP Control LBPP HR (bpm) BL 55.7 ± 3.0 54.9 ± 3.3 55.5 ± 3.2 61.4 ± 3.3 64.0 ± 4.1 61.9 ± 2.6 FMD 54.9 ± 3.1 54.7 ± 2.8 55.1 ± 3.1 62.0 ± 3.4 59.6 ± 2.9 61.3 ± 3.2 SV (ml) BL 99.8 ± 4.7 101.1 ± 3.8 99.9 ± 5.3 78.7 ± 3.4 79.1 ± 5.2 74.8 ± 4.3 FMD 99.4 ± 4.7 104 ± 4.2 103.3 ± 5.5 77.8 ±4.4 82.4 ± 4.6 78.1 ± 3.9 CO (l min-1) BL 5.6 ± 0.5 5.6 ± 0.5 5.5 ± 0.6 4.7 ± 0.3 4.8 ± 0.4 4.5 ± 0.3 FMD 5.5 ± 0.4 5.7 ± 0.5 5.7 ± 0.6 4.7 ± 0.3 4.8 ± 0.3 4.6 ± 0.3 MAP (mmHg) BL 92.0 ± 1.9 91.1 ± 1.7 92.6 ± 2.2 99.3 ± 1.6 103.3 ± 2.2 102.5 ± 1.8 FMD 93.8 ± 3.0 92.2 ± 1.8 96.8 ± 2.3* 101.7 ± 1.6 104.4 ± 1.6 107.0 ± 2.2* TPR [dyn s/cm5] BL 1350.1 ± 106.7 1305.6 ± 94.5 1365.9 ± 96.3 1747.2 ± 125.5 1767.9 ± 135.6 1880.4 ± 132.6 FMD 1370.0 ± 87.5 1309.6 ± 90.5 1352.0 ± 107.2 1822.3 ± 147.2 1752.0 ± 108.3 1863.1 ± 105.9 SpO2 (%) BL 83.3 ± 0.7 83.7 ± 0.9* 83.4 ± 0.9 FMD 82.7 ± 0.7 81.6 ± 0.6 82.2 ± 0.8 Definition of Abbreviations: HR, heart rate; SV, stroke volume; CO, cardiac output; MAP, mean arterial pressure; TPR, total peripheral resistance; SpO2, peripheral capillary oxygen saturation. *P<0.05, BL vs FMD. 87 Table 4.2 Brachial artery variables during the control and lower-body differential pressure trials at sea-level and high-altitude. Definition of Abbreviations: BA, brachial artery Sea-level High-altitude LBNP Control LBPP LBNP Control LBPP BA diameter (mm) 4.6 ± 0.1 4.6 ± 0.1 4.7 ± 0.1 4.2 ± 0.1 4.2 ± 0.1 4.2 ± 0.1 BA velocity (cm s-1) 14.0 ± 3.0 13.7 ± 2.7 14.4 ± 3.7 5.4 ± 1.0 6.3 ± 1.2 6.5 ± 1.2 BA flow (ml min-1) 147.0 ± 33.3 142.7 ± 30.6 146.0 ± 35.5 46.5 ± 9.5 53.1 ± 11.1 54.5 ± 11.4 BA resistance [mm Hg (ml min-1)-1] 1.1 ± 0.3 1.0 ± 0.2 1.1 ± 0.2 3.0 ± 0.5 2.9 ± 0.5 2.7 ± 0.4 BA mean shear (s-1) 127.5 ± 27.4 136.1 ± 37.2 125.6 ± 33.4 50.0 ± 9.1 59.6 ± 10.9 62.4 ± 11.1 BA antegrade shear (s-1) 136.5 ± 26.0 145.6 ± 36.0 135.7 ± 32.0 70.7 ± 8.2 79.0 ± 9.6 77.1 ± 10.8 BA retrograde shear (s-1) 9.0 ± 2.2 9.5 ± 2.5 10.2 ± 3.6 20.6 ± 4.3 19.5 ± 4.0 14.8 ± 2.1 BA oscillatory shear (s-1) 0.09 ± 0.02 0.10 ±0.03 0.10 ± 0.03 0.23 ± 0.03 0.21 ± 0.03 0.18 ± 0.02 88 Figure 4.1 A schematic representation of the experimental protocol conducted at sea-level and high-altitude, and raw MSNA neurogram in two participants. After 20-minutes of supine rest, the protocol began with a five-minute eupneic breathing baseline period, after which, the pressure within the chamber was altered to one of the following: 1) -10 mmHg (LBNP trial), 2) remained unchanged at zero mmHg (control trial), or 3) +10 mmHg (LBPP trial). Once pressure was achieved, and maintained for five-minutes, a brachial artery FMD was performed on the participants left arm. Once the brachial artery FMD measurement was collected, the pressure of the lower-body differential pressure chamber was alleviated and the participant was given a five-minute recovery period. The protocol was then repeated for the remaining two randomized conditions (i.e. LBNP, control, or LBPP). Before each condition, a five-minute quiet resting baseline was endured. 89 FMD (mm)0.00.10.20.30.40.50.60.00.10.20.30.40.50.60.00.10.20.30.40.50.6FMD (%)024681012140246810121402468101214Sea-level High-altitude Sea-level High-altitude Sea-level High-altitudeCondition: P=0.243Altitude: P=0.024Condition*Altitude: P=0.835Condition: P=0.343Altitude: P=0.061Condition*Altitude: P=0.856A. B. C.E. F. G.Lower-body negative pressure Control Lower-body positive pressure Figure 4.2 A comparison of absolute and relative brachial artery flow-mediated dilation response between sea-level and high-altitude. These data highlight that absolute and relative brachial artery flow mediated dilation remained unchanged between sea-level and high-altitude within LBNP, control, and LBPP trials. Taking into account changes in baseline brachial artery diameter and SRAUC between sea-level and high-altitude, we found that relative endothelial function is still reduced at high-altitude compared to sea-level (P=0.008; see results section) 90 MSNA burst s/minute010203040LBNP Control LBPPMSNA burst incidence010203040506070LBNP Control LBPPA. B.P=0.007 P=0.005 Figure 4.3 Muscle sympathetic nervous activity during LBNP, control, and LBPP trials at sea-level. Individual data of MSNA burst frequency (Panel A; bursts/minute), and burst incidence (Panel B; bursts per 100 heart beats) during LBNP (n=5), control (n=5), and LBPP (n=4) at sea-level. These findings illustrate a significant difference in MSNA between LBNP and LBPP. The gray line on the figure depicts the average between individuals during each trial. 91 MSNA burst s/mi nute1015202530354045MSNA burst incidence20304050607080Sea-level High-altitude Sea-level High-altitudeA. B.P=0.03 P=0.05 Figure 4.4 Muscle sympathetic nervous activity at rest between sea-level and high-altitude. Individual data of MSNA burst frequency (Panel A; bursts/minute), and burst incidence (Panel B; bursts per 100 heart beats) between sea-level and high-altitude (n=4). These findings illustrate MSNA was significantly elevated in each individual at high-altitude, compared to sea-level. The gray line on the figure depicts the average between individuals during each trial. 92 Figure 4.5 Brachial artery shear rate and diameter response to forearm cuff release at sea-level and high-altitude. Panels A and B represent mean data ±SEM for shear rate response during brachial artery FMD during LBNP, control, and LBPP trials at sea-level (n=13) and high-altitude (n=14). Panels C and D represent mean data ±SEM for relative FMD during LBNP, control, and LBPP trials at sea-level (n=13) and high-altitude (n=14). These findings demonstrate that LBNP nor LBPP had no effect on brachial artery FMD, despite altering MSNA. The gray line on the figure depicts the average between individuals during each trial. 024681012141618Sea-levelSRAUC (10-3)0510152025303540High-altitude0510152025303540FMD (%)024681012141618LBNP Control LBPP LBNP Control LBPPA. B.C. D. 93 4.5 Discussion Using a novel, and randomized experimental design, we examined the effect of acute alterations of SNA using mild LBNP and LBPP on brachial artery endothelial function at both sea-level (344m) and high-altitude (5050m). Our main findings were the following: 1) in support of previous studies, MSNA was elevated, and brachial artery endothelial function was reduced at high-altitude compared to sea-level after active ascent to 5050m, and 2) despite acutely increasing SNA with LBNP, and decreasing SNA with LBPP, we demonstrated that brachial artery endothelial function remained unchanged at sea-level and high-altitude. Our data indicates that mild and acute changes in SNA, at least in the absence of alterations in systemic hemodynamics, does not influence endothelial function. 4.5.1 Effect of high-altitude on endothelial function The effects of high-altitude acclimatization on endothelial function, as assessed via brachial FMD, has been studied previously. These studies have reported contradictory results such as reduced FMD (Lewis et al., 2014, Bakker et al., 2015), or no change in FMD upon acclimatization to high-altitude (Bruno et al., 2015, Bruno et al., 2016, Tymko et al., 2016, Tremblay et al., 2017). Despite the disparities between these studies, elevations in SNA is proposed to have a profound effect on brachial FMD (Hijmering et al., 2002, Tymko et al., 2016). After four-weeks of acclimatization to high-altitude (5260m), MSNA has been shown to be elevated by ~200% (Hansen and Sander, 2003). The current study confirms these previous findings as we have demonstrated in four participants that MSNA was substantially elevated at rest after acclimatization to 5050m (Figure 94 4.4). This increase in SNA and total peripheral resistance is likely responsible for the substantial decrease in brachial artery blood flow observed at high-altitude An alternative explanation for the reported differences between these high-altitude FMD studies may lie within the mode of travel to high-altitude, and the severity of altitude exposure. For example, the studies that have reported a decrease in brachial FMD took place after 5-10 days of trekking at high-altitude [4200m, (Bakker et al., 2015); and 5050m, (Lewis et al., 2014)]. In contrast, the studies that have reported no change in endothelial function arrived at a more moderate altitude passively by automobile [at 3800m (Tymko et al., 2016, Tremblay et al., 2017)], or cable car [at 3842m (Bruno et al., 2015, Bruno et al., 2016)]. The high-altitude arm of the current study involved trekking ascent to 5050m over 7-10 days, and in support of our hypothesis, and previous reports (Lewis et al., 2014, Bakker et al., 2015), we found that brachial artery endothelial function was reduced at high-altitude compared to sea-level. This reduction may be due to long-term elevations in sympathetic nervous activity or marked elevations in oxidative stress, or both. 4.5.2 Altering sympathetic nervous activity non-invasively with lower-body negative and lower-body positive pressure. There have been several investigations on the role of the SNA on endothelial function assessed by brachial FMD at sea-level (Tymko et al., 2016, Atkinson et al., 2015, Thijssen et al., 2014, Hijmering et al., 2002, Dyson et al., 2006); however, none of these studies have concurrently measured SNA using microneurography. Existing literature indicates that our mode of altering SNA (i.e. mild LBNP and LBPP) could provide an excellent model to evaluate the role of SNA on 95 endothelial function, assuming that this methodology significantly alters SNA independent of hemodynamics (Fu et al., 1998, Rea and Wallin, 1989, Cui et al., 2004, Scherrer et al., 1988, Padilla et al., 2010, Millar et al., 2013). For the current project we developed a novel, light-weight, purpose built lower-body differential pressure chamber and measured its effectiveness of altering radial MSNA, which is representative of global MSNA (Rea and Wallin, 1989), during our sea level trial (n=5). Our radial MSNA data indicates that SNA was elevated during LBNP and reduced during LBPP (Figure 4.3). Here, we established an effective methodological approach, to non-invasively increase and decrease SNA largely independent of systemic hemodynamics, however, the observed alterations in SNA failed to evoke a change in brachial artery resistance (Table 4.2) – a clear indicator of vascular constraint. Thus, since vascular constraint was not significantly altered during acute and mild LBNP/LBPP, it is unclear if the experimental design in its current form is effective when investigating the effects of SNA on endothelial function. Future studies using a similar LBNP/LBPP experimental model should consider a longer duration of stimulus (i.e. LBNP or LBPP), which may be more effective in altering peripheral vascular resistance. 4.5.3 Effect of sympathetic nervous activity on endothelial function at sea-level. Although there have been several reports of SNA influencing endothelial function (Hijmering et al., 2002, Thijssen et al., 2011, Dyson et al., 2006, Tymko et al., 2016, Atkinson et al., 2015), it has been suggested that the method of altering SNA may yield different results (Dyson et al., 2006). For example, Dyson et al. (Dyson et al., 2006) investigated the role of SNA (via epinephrine and norepinephrine spillover) on brachial artery endothelial function, and discovered that the cold pressor test was the only modality that reduced brachial artery FMD. Interestingly, Dyson et al. (Dyson et al., 2006) found that LBNP increased SNA, but it had no effect on brachial artery 96 endothelial function, which contrasts other studies that have found that LBNP reduces brachial artery endothelial function (Hijmering et al., 2002, Thijssen et al., 2014). The first report of LBNP significantly reducing brachial artery endothelial function was by Hijmering et al. (Hijmering et al., 2002). Here, they discovered that the reduction in brachial artery endothelial function was mediated through a α1-adrenergic pathway as endothelial function was restored during LBNP after administration phentolamine. This finding was supported by two recent studies that used exercise as a method of increasing SNA (Tymko et al., 2016, Atkinson et al., 2015). Hijmering et al. (Hijmering et al., 2002) also suggested that the observed reduction in brachial artery endothelial function could be directly due to SNA, or indirectly via other mechanisms during LBNP such as altered hemodynamics (e.g. increases in retrograde shear stress). Thijssen et al. (Thijssen et al., 2014) attempted to address this question by using a local heating stimulus (to one arm) during LBNP in order to abolish the increase in retrograde shear stress typically observed during moderate-to-severe magnitudes of LBNP (Thijssen et al., 2014, Padilla et al., 2010). Their findings revealed that brachial artery endothelial function was restored after the heat stimulus was applied and retrograde shear rate was reduced (Thijssen et al., 2014). However, altered hemodynamics (e.g. increased heart rate and reduced stroke volume) during LBNP were still present (Thijssen et al., 2014), and these physiological changes can directly affect endothelial function [reviewed in (Green et al., 2017)]. The current study attempts to address this research question by manipulating SNA largely independent of changes in hemodynamics. This is the first study to investigate the role of SNA on endothelial function by increasing and decreasing SNA using LBNP and LBPP, respectively, findings confirmed (at sea-level) via microneurography. In contrast to our hypothesis, we did not 97 observe any change in brachial artery endothelial function during LBNP - a finding that is consistent with at least one previous study (Dyson et al., 2006), but opposes other reports (Thijssen et al., 2014, Hijmering et al., 2002). It is possible that we did not increase SNA activity enough in order to influence endothelial function; however, the experimental design may be more important than the magnitude of SNA increase. For example, Dyson et al. (Dyson et al., 2006) demonstrated that the only intervention that altered endothelial function during elevated SNA was not the intervention that evoked the largest SNA response. Interestingly, acute hypoxia (FIO2 = 0.11) has shown to reduce brachial artery endothelial function after 60-minutes (Lewis et al., 2014), and this severity of hypoxia has been shown to increase SNA to approximately the same extent as our -10 mmHg LBNP stimulus (DeBeck et al., 2010). Additionally, it is possible that the current experimental design was too short in duration to evoke a change in vascular resistance and endothelial function. For example, a recent study demonstrated that 30-minutes of sustained moderate exercise reduced endothelial function via an α1-adrenergic pathway; however, a ~10-minute maximal exercise test did not evoke the same results (Tymko et al., 2016). Nevertheless, our data indicates that acute and mild SNA activation and deactivation via LBNP and LBPP does not alter brachial artery endothelial function. 4.5.4 Effect of sympathetic nervous activity on endothelial function at high-altitude. Lower-body negative pressure has been previously used to measure orthostatic tolerance in high-altitude Andean natives at high-altitude [4338m; (Claydon et al., 2004)]; however, this is the first investigation to use LBNP above 5000m where MSNA is markedly elevated (Figure 4.4), in addition, this is the first study to use LBPP at high-altitude. Our research group has published the only other report investigating the role of SNA on endothelial function at high-altitude (Tymko et 98 al., 2016). Using moderate-intensity exercise to increase SNA, we found that brachial artery endothelial function is not reduced at high-altitude, indicating that after acclimatization to high-altitude neurovascular control may be altered (Tymko et al., 2016). It is also unknown whether our previously reported findings were unique to exercise, and hence potentially, a different strategy to alter SNA may yield different results (Dyson et al., 2006). Additionally, SNA stimulus (e.g. exercise) has been shown to be augmented with cycling exercise during hypoxia (Katayama et al., 2011) – meaning that the alteration in SNA via LBNP and LBPP could be exacerbated at high-altitude, leading to a more pronounced effect on endothelial function. Therefore, we hypothesized that altering SNA using LBNP and LBPP would result in a decrease and increase in brachial artery endothelial function, respectively. To our surprise, similar to our sea-level data, we found that LBNP and LBPP did not change endothelial function at high-altitude. However, the lack of effect of LBNP and LBPP on endothelial function at high-altitude could be also be due to similar methodological reasoning outlined above: (a) our mode of altering SNA does not alter brachial endothelial function, and/or (b) the duration of SNA activation/deactivation was not long enough to elicit a change in endothelial function. 4.5.5 Methodological considerations The degree of LBPP chosen for the current research project (i.e. +10 mmHg) was determined based on previous literature, which reported no changes of MAP (Fu et al., 1998, Millar et al., 2013). However, during our LBPP trials at sea-level and high-altitude, LBPP elevated MAP elevated by ~4-5 mmHg, potentially due to LBPP associated transient fluid shifts. This result was likely not due to measurement drift from our continuous blood pressure monitor (i.e. finometer), since LBPP selectively increased MAP in both sea-level and high-altitude protocols, and the finometer was 99 carefully calibrated before each trail. Changes in blood pressure could have a direct effect on brachial FMD (Green et al., 2017); however, since mild LBNP and LBPP did not alter our other physiological variables (especially shear patterns), we feel that the small change in blood pressure is likely trivial. Another consideration is that due to methodological constraints at high-altitude, we were unable to measure SNA via microneurography, therefore, the absolute effect of LBNP and LBPP on SNA at high-altitude is unknown. Additionally, it is important to consider that neurovascular transduction may be different at high-altitude compared to sea-level, but this is still under debate as there is evidence that neurovascular transduction is reduced (Leuenberger et al., 1991), or increased (Tan et al., 2013), with exposure to hypoxia. We did, however, obtain MSNA recordings in the peroneal nerve at rest in a subset of participants (n=4) at both sea-level and high-altitude. We acknowledge that our MSNA data collected at sea-level and high-altitude were in the radial and peroneal nerves, respectively, but it has been previously demonstrated that MSNA does not differ between these two nerves during mild lower-body negative pressure and are both a reflection of global MSNA (Rea and Wallin, 1989). Although our MSNA sample size was small, we still detected statistical significance between LBNP and LBPP trials, which were recorded using a within subject design at sea-level. Our experimental design warrants further comment. Our LBNP/LBPP methodological approach to bi-directionally alter SNA proved successful; however, the current study design failed to change brachial artery vascular resistance. We view our study design as a “double-edged sword”, as it altered SNA largely independently of hemodynamics, yet it was not a potent enough stimulus to alter brachial artery resistance, making it unclear if our study design is appropriate to investigate the effects of SNA on peripheral vascular function. Lastly, menstrual cycle was not taken into 100 consideration for our one female participant, and previous evidence indicates that brachial artery FMD changes throughout the menstrual cycle (Hashimoto et al., 1995). However, our primary research objective was to look at the within-day comparison of brachial FMD between LBNP, control, and LBPP trials, therefore, the results of these data should not be affected by differences in menstrual cycle between sea-level and high-altitude. Importantly, changes in blood viscosity between sea-level and high-altitude was not taken into account when analyzing brachial artery FMD. However, a reduction in brachial artery FMD was still observed at high-altitude, even though hematocrit, thus shear stress, was likely higher during cuff release. 4.5.6 Synopsis We used a novel experimental approach to investigate the relationship between sympathetic nervous activity and endothelial function by using mild lower-body negative pressure and lower-body positive pressure at both sea-level and high-altitude. We demonstrated for the first time using a novel, experimental design, that altering sympathetic nervous activity largely independent of hemodynamics (e.g. heart rate, stroke volume, shear stress) had no effect on brachial artery endothelial function. These findings suggest that brachial artery endothelial function may not be altered through sympathetic nervous activity associated vascular constraint. Together, our findings have implications for better understanding the consequential impact of sympathetic nervous activity on vascular function. 101 Chapter 5: Global REACH 2018: α1-adrenergic signaling impairs endothelial function in lowlanders and Andean highlanders with excessive erythrocytosis at 4300m. 5.1 Introduction The impact of high altitude on endothelial function in lowlanders is currently unclear. Some (Lewis et al., 2014, Bakker et al., 2015, Tymko et al., 2017, Tremblay et al., 2018), but not all (Tymko et al., 2016, Tremblay et al., 2017, Bruno et al., 2015) studies have demonstrated impaired endothelial function in lowlanders upon acclimatization to high altitude. The disparity in the literature is likely due to the severity and length of altitude exposure. For example, the studies that report endothelial dysfunction were all conducted at altitudes >4000m (Lewis et al., 2014, Bakker et al., 2015, Tymko et al., 2017, Tremblay et al., 2018). The mechanism(s) responsible for endothelial dysfunction after exposure to high altitude (>4000m) are multifactorial but include elevated oxidative stress (Goel et al., 2007, Silvestro et al., 2002), changes in hemodynamics such as shear stress and blood pressure (Birk et al., 2013, Dawson et al., 2008, Johnson, Mather et al., 2012, Lamping and Dole, 1987, Tremblay et al., 2018), and elevations in sympathetic nervous activity (i.e. SNA) (Tymko et al., 2016, Atkinson et al., 2015, Dyson et al., 2006, Hijmering et al., 2002, Thijssen et al., 2014, Thijssen et al., 2006). There is clear evidence that SNA is markedly elevated in lowlanders at high altitude (Hansen and Sander, 2003, Tymko et al., 2017, Duplain et al., 1999), and that acute increases in SNA are associated with impaired endothelial function (Padilla et al., 2014, Hijmering et al., 2002). 102 However, the mechanistic link between increased SNA and endothelial dysfunction at high altitude remains unknown. Endothelial function in populations who have had chronic high altitude exposure (e.g. Tibetan, Peruvian, and Ethiopian high altitude dwellers) is less clear. The Peruvian residents of the Andes mountains have been living at altitude for ~11,000 years (Beall, 2006, Beall, 2007), and a high percentage of long-term high altitude residents in the Cerro de Pasco region (~15-34%; (Monge et al., 1989, León-Velarde et al., 1993)) are prone to suffering from chronic mountain sickness. This type of sickness is characterized by an excessive production of red blood cells [i.e. excessive erythrocytosis (EE)], which can lead to severe hypoxemia and adverse cardiovascular health consequences (Villafuerte and Corante, 2016, Corante et al., 2018). Previous studies using flow-mediated dilation as a method to quantify endothelial function have reported that large conduit arterial function is reduced in Andean highlanders with EE compared to healthy Andean highlanders (Rimoldi et al., 2012, Bailey et al., 2013, Rexhaj et al., 2016), who have exaggerated plasma norepinephrine (i.e. elevated SNA) (Gamboa et al., 2006). However, it is unknown whether the observed reductions in endothelial function in Andean highlanders are mediated via heightened SNA. The primary purpose of the current investigation was to determine if high altitude induced elevations in SNA contribute to endothelial dysfunction in lowlanders at high-altitude, and in Andean highlanders with and without EE. We hypothesized that 1) high altitude related reductions in endothelial function in lowlanders, and 2) endothelial dysfunction in Andean highlanders with 103 EE compared to non-EE highlanders, would be due to elevated adrenergic SNA. To test these hypotheses we employed a novel, randomized, experimental design involving step-wise intra-arterial infusions of acetylcholine (ACH) and sodium nitroprusside (SNP) to elicit controlled blood flow responses that are mechanistically-linked to endothelial and smooth muscle function, respectively (Tousoulis et al., 2005). These vasoactive drugs (i.e. ACH and SNP) were administered before and after local adrenergic blockade (via phentolamine and propranolol). 104 5.2 Methods and Materials 5.2.1 Ethical Approval The Clinical Research Ethics Board at the University of British Columbia (ethics H17-02687 and H18-01404), and the Universidad Peruana Cayetano Heredia Comité de Ética (ethics #101686) approved all experimental procedures and protocols in adherence with the principles of the Declaration of Helsinki (except registration in a database). All participants provided written informed consent before participation in this study. Andean participants were provided with a translated consent form and a Spanish translator thoroughly explained the experimental protocol prior to consent. This investigation was part of a larger research expedition conducted between March and July 2018. Therefore, several participants volunteered for multiple studies conducted at the Unviersity of British Columbia (Kelowna, British Columbia; 344m) and during ~three weeks at a high altitude laboratory located in Cerro de Pasco, Peru (4300m). However, the a priori, research questions in the current investigation were exclusively addressed. An overview of the Global REACH expedition to Peru is detailed elsewhere (Tymko et al. (2019) currently under review) 5.2.2 Participants Recruited lowlander participants (n=11; all male) were normotensive (systolic blood pressure <140 and diastolic blood pressure <90 mmHg) at rest, and were asked to complete a medical history questionnaire. Participants with history of smoking, cardiovascular, cerebrovascular, or respiratory disease were avoided. One lowlander participant was excluded from data analysis due to equipment malfunction at sea-level, and a second lowlander declined to participate in post testing at high altitude. All lowlander participants arrived in Cerro de Pasco, Peru (4300m) within 105 a three day time-frame, and were tested between 14-21 days after arrival. Upon ascent, all participants refrained from prophylactic use of oral acetazolamide (i.e., Diamox), however, two participants were instructed by medical personnel to take acetazolamide briefly for the treatment of acute mountain illness symptoms within the first week of arrival in Cerro de Pasco. In these two cases, experimentation was completed well outside of the drug elimination time of acetazolamide. In addition, participants avoided using aspirin, non-steroidal anti-inflammatory drugs, and phosphodiesterase-5 inhibitors at least 24 hours prior to experimentation. Andean participants who were born, permanently living in the Cerro de Pasco region, and avoided working in the mining industry were recruited for the study using a pre-existing data base. Andean participants (n=15; 6 diagnosed with EE) were not taking any medications, and after consent was obtained, a venous blood sample was obtained to diagnose EE, which was defined as having a [Hb] of >21 g/dl. Blood pressure data at rest, but not during ACH and SNP infusion was obtained in two Andean participants (one EE, and one non-EE) due to equipment malfunction; however, an estimate of their blood pressure response from baseline during ACH and SNP infusion was calculated based on the group mean response. Interpolating these data points did not alter our study findings. 5.2.3 Experimental Design To address our research question(s) we conducted two similar protocols: Lowlander and Highlander investigations (protocols A and B, respectively; Figure 5.1). Prior to each experiment, all participants avoided exercise, alcohol, and caffeine for 12 hours. Additionally, participants were asked to consume a light meal at least two-hours prior to experimentation. 106 5.2.4 General procedures. Participants were instructed to lie motionless on a semi-recumbent bed and rest after brachial artery catheterization ~30 minutes to ensure participants were at a steady rest period and positional related shifts in blood volume was complete prior to experimentation. Participants were also instrumented with a three-lead electrocardiogram connected to an amplifier to calculate heart rate (i.e. HR; ADInstruments, FE231, Colorado Springs, USA). At both sea level and high altitude in lowlanders and highlanders the protocol began with a two-minute rest period to collecting resting measures. Immediately after rest, in a randomized fashion, an intra-arterial infusion of either ACH (to assess endothelial dependent vasodilation) or SNP (to assess endothelial independent vasodilation) was administered at three separate doses using 60 cc syringes secured onto a syringe pump (Harvard apparatus, PHD Ultra, Holliston, MA, USA). In lowlanders (i.e. protocol A; Figure 5.1), ACH was infused at 4.0, 8.0, 16.0 µg/100 mL of forearm tissue/min, and SNP at 1.0, 2.0, 4.0 µg/100 mL forearm tissue/min. In Andean highlanders (i.e. protocol B; Figure 5.1) ACH was infused at 4.0, 8.0, 16.0 µg/100 mL of forearm tissue/min, and SNP at 1.0, 2.0, 4.0 µg/100 mL forearm tissue/min in highlanders. In both lowlanders and highlanders each infusion stage was three-minutes in duration. To determine the appropriate drug infusion rates for each participant, forearm volume was estimated using measurement techniques (Thornton et al., 1992, Watenpaugh et al., 2001). In between ACH and SNP trials, participants had a 10-minute recovery period for drug washout. After both ACH and SNP trials were completed (i.e. control trial), phentolamine and propranolol [1000 μg over 5-minutes (loading dose), and 50 μg/min (maintenance dose)] were infused and the ACH and SNP infusion procedures were repeated (Richards et al., 2014). 107 Figure 5.1 Schematic of experimental design. 5.2.5 Experimental measurements 5.2.5.1 Brachial artery catheterization After local anesthesia (2% lidocaine), a 20-gauge catheter (Arrow International, Reading, PA) was inserted into the brachial artery using ultrasound guidance and a modified Seldinger technique. The catheter was connected to a commercially available arterial blood sampling kit (VAMP Adult, Edwards Lifescience, Irvine, CA), which allowed for repeated blood sampling and flushing with 0.9% saline. The catheter and VAMP blood sampling kit was also connected to a blood pressure amplifier (ADInstruments, FE117, Colorado Springs, USA), and zeroed at the level of the heart, for the collection of continuous blood pressure recording. Resting arterial blood samples (~1.5 ml) were collected in preheparanized syringes (safePICO syringes, Radiometer, Copenhagen, Denmark). Air bubbles were immediately evacuated from the syringe, and blood gas analysis was performed within 30-minutes of sampling with a gas analyzer (ABL90 FLEX, Radiometer). The 108 blood gas analyzer was calibrated at a minimum of every 8 hours using manufacturer's standard internal quality checks. Reported variables that were calibrated and analyzed included PaO2, PaCO2, and SaO2. 5.2.5.2 Brachial artery imaging The participants left arm was extended perpendicular and was fixed into position on a table at the level of the heart. Brachial artery image acquisition was obtained using a linear array probe attached to a high-resolution ultrasound machine (Vivid 7, General Electric, Milwaukee, WI, USA) to determine brachial artery man blood velocity and diameter. The probe was placed over the brachial artery proximal to the catheter insertion site as previously described (Crecelius et al., 2010). All brachial artery images were performed by the same experienced ultrasonographer (C.M.H). The probe insonation angle was maintained at <60 degrees and frequency was set at 5 MHz. Brachial artery diameter was measured at the end of rest and at the end of each drug infusion stage. Forearm blood flow was calculated as: forearm blood flow (ml/min) = mean blood velocity (cm/s) × π [brachial artery diameter (cm) / 2]2 × 60. Forearm vascular conductance was calculated as [blood flow (ml/min) / mean arterial pressure (MAP)] × 100, and expressed as ml/min/100 mmHg. In Andean highlanders, vascular conductance was normalized to estimated forearm volume by dividing forearm vascular conductance by forearm volume (in liters), and expressed as ml/min/100mmHg/l. 5.2.6 Statistics All statistical analyses were performed using SigmaStat V13 (Systat, Chicago, IL) and were reported as means ± SD. Statistical significance was set at P<0.05. Paired and un-paired t-tests 109 were used to detect changes in resting variables in lowlanders (sea level vs high altitude) and highlanders (EE vs non-EE), respectively (Table 5.1). Two-way mixed and two-way repeated measures analysis of variance were used to detect any differences in cardiovascular and brachial artery variables. When significant F-ratios were detected, post hoc comparisons were made using Tukey post hoc test for pairwise comparisons. No comparisons between lowlander and highlander data were conducted. 110 5.3 Results 5.3.1 Participants Lowlander and Andean highlander participant demographics are presented in Table 5.1. In lowlanders, PaO2, PaCO2, and SaO2 were all reduced at high altitude compared to sea level (P<0.001 for all). Between EE and non-EE Andean highlanders, no differences in age (P=0.92), PaO2 (P=0.65), and PaCO2 (P=0.06) were detected; however, Andeans with EE were taller (P=0.03), heavier (P=0.01), reduced SaO2 (P=0.02), had a lower forearm volume (P=0.02), elevated Hb (22.5 vs 18.7 g/dl; P<0.001), and had elevated CMS scores (P<0.001). Table 5.1 Lowlander and highlander participant demographics LL Sea level LL High altitude EE Highlanders Non-EE Highlanders Age (yrs) 26.3 ± 2.9 - 47.0 ± 7.8 43.8 ± 15.3 Height (cm) 177.3 ± 6.1 - 164.2 ± 3.4┼ 158.8 ± 4.4 Weight (kg) 76.0 ± 9.2 - 80.4 ± 7.0┼ 62.0 ± 6.9 Forearm volume (ml) 871.4 ± 158.9 - 1040.8 ± 118.3┼ 791.0 ± 146.8 Hb (g/dl) 15.1 ± 0.8 17.8 ± 0.6* 22.5 ± 1.2┼ 18.7 ± 1.5 PaO2 (mmHg) 96.6 ± 5.4 56.1 ± 5.0* 46.0 ± 2.4 48.0 ± 7.4 PaCO2 (mmHg) 37.8 ± 1.8 27.0 ± 3.6* 32.3 ± 1.0 29.2 ± 3.9 SaO2 (%) 98.0 ± 0.4 89.7 ± 2.2* 81.3 ± 2.3┼ 86.0 ± 3.5 CMS Score - - 6.0 ± 1.8┼ 1.6 ± 1.0 List of Abbreviations: cm, centimeters; CMS, chronic mountain sickness; dl, deciliter; EE, excessive erythrocytosis; g, grams; kg, kilograms; LL, lowlander; ml, milliliter; mmHg, millimeters of mercury; PaCO2, partial pressure of arterial carbon dioxide; PaO2, partial pressure of arterial oxygen; SaO2, arterial hemoglobin saturation of oxygen; yrs, years. *P<0.05, lowlanders sea level vs high altitude. ┼P<0.05, non-EE vs. EE highlanders 111 5.3.2 Lowlanders 5.3.2.1 Cardiovascular response During the SNP infusion protocol at sea level, MAP was reduced during SNP2 and SNP3 compared rest (P<0.001 and P=0.006, respectively; Table 5.2); however, at high altitude, MAP did not change with SNP infusion (P=0.013). At both sea level and high altitude, HR stay relatively constant throughout the infusion. During the ACH infusion protocol, both MAP and HR stayed relatively constant at both sea level and high altitude with the exception of HR being slightly elevated at ACH3 at sea level (P=0.017), and MAP being reduced at ACH3 at high altitude (P=0.014). 5.3.2.2 Forearm vascular response As expected, during both SNP and ACH infusion protocols mean blood flow velocity, arterial diameter, mean blood flow, and forearm vascular conductance all increased (Table 5.3). After 14 days of high altitude exposure absolute forearm vascular conductance was reduced at ACH1 (52.7 ± 19.6%; P=0.003), ACH2 (25.4 ± 38.7%; P=0.020), and ACH3 (35.1 ± 34.7%; P<0.001), compared to sea-level, and this effect was abolished after autonomic blockade (P=0.988; Figure 5.2). No differences between sea level and high altitude were detected in relative vascular forearm conductance during ACH infusion during both control and blockade trials (P=0.886 and P=0.981, respectively; Figure 5.3). During SNP infusion, no differences between sea level and high altitude were detected in absolute forearm vascular conductance and relative forearm vascular conductance during both control (P=0.397 and P=0.145, respectively), and blockade trials (P=0.601 and P=0.641, respectively; Figure 5.3). 112 Table 5.2 Systemic hemodynamics in lowlanders at sea level and high altitude. Control Blockade Rest SNP1 SNP2 SNP3 Rest SNP1 SNP2 SNP3 LL Sea level MAP 89.3 ± 11.8 86.1 ± 9.6 85.0 ± 9.1* 81.5 ± 7.8* 89.9 ± 8.5 88.2 ± 10.6 85.9 ± 10.7* 82.2 ± 11.8* HR 64.9 ± 11.2 66.0 ± 12.7 67.5 ± 12.0 69.3 ± 10.9* 55.2 ± 9.1┼ 57.4 ± 10.3┼ 57.4 ± 12.2┼ 63.5 ± 10.8┼* LL High Altitude MAP 94.1 ± 9.2 89.7 ± 9.2 90.4± 8.2 87.8 ± 7.7* 92.3 ± 13.9 93.5 ± 12.1 92.3 ± 11.3 91.8 ± 9.4 HR 75.2 ± 12.7 75.9 ± 15.0 75.3 ± 14.2 76.8 ± 12.4 66.3 ± 8.9┼ 69.5 ± 9.4┼ 71.2 ± 10.2┼ 68.9 ± 13.6┼ Rest ACH1 ACH2 ACH3 Rest ACH1 ACH2 ACH3 LL Sea level MAP 88.8 ± 9.1 87.2 ± 8.0 88.2 ± 8.5 87.2 ± 8.3 88.8 ± 8.8 87.3 ± 9.8 86.8± 8.3 86.6 ± 9.1 HR 66.0 ± 12.7 67.5 ± 12.7 67.8 ± 11.5 70.4 ± 11.3* 55.3 ± 12.8┼ 55.9 ± 12.2┼ 55.8 ± 11.4┼ 59.9 ± 9.5┼* LL High Altitude MAP 94.1 ± 9.0 93.2 ± 9.3 91.7 ± 7.8 89.6 ± 8.2* 91.8 ± 12.3 90.0 ± 13.9 92.2 ± 11.9 89.5 ± 11.6* HR 78.6 ± 7.5 78.3 ± 12.4 81.2 ± 15.4 79.9 ± 10.3 66.1 ± 8.7┼ 63.7 ± 9.1┼ 67.6 ± 11.8┼ 69.7 ± 9.0┼ List of Abbreviations: ACH, acetylcholine; HR, heart rate; LL, lowlander; MAP, mean arterial pressure; SNP, sodium nitroprusside. *P<0.05, vs Rest. ┼P<0.05, vs control. 113 Table 5.3 Systemic hemodynamics in lowlanders at sea level and high altitude. Control Blockade Rest SNP1 SNP2 SNP3 Rest SNP1 SNP2 SNP3 LL Sea level Vel. 6.6 ± 2.3 19.8 ± 4.4* 22.3 ± 5.9* 27.3 ± 6.4* 20.1 ± 7.3┼ 34.9 ± 12.8┼* 38.3 ± 10.3┼* 46.1 ± 10.2┼* Dia. 0.35 ± 0.03 0.37 ± 0.03* 0.37 ± 0.02* 0.38 ± 0.02* 0.37 ± 0.03┼ 0.38 ± 0.03┼* 0.39 ± 0.02┼* 0.41 ± 0.03┼* MBF 38.9 ± 13.7 125.6 ± 34.1* 142.9 ± 38.5* 188.0 ± 46.8* 125.4 ± 40.7┼ 239.3 ± 84.6┼* 275.0 ± 81.3┼* 359.8 ± 85.2┼* Cond. 43.4 ± 13.8 147.5 ± 44.7* 169.7 ± 49.1* 233.3 ± 66.0* 141.3 ± 51.4┼ 276.7 ± 108.4┼* 324.2 ± 103.6┼* 442.6 ± 109.8┼* LL High altitude Vel. 5.58 ± 2.5 14.1 ± 7.9* 23.2 ± 5.4* 28.5 ± 7.4 * 20.2 ± 7.7┼ 34.0 ± 9.5┼* 40.3 ± 6.9┼* 46.8 ± 9.3┼* Dia. 0.33 ± 0.03 0.35 ± 0.03* 0.36 ± 0.02* 0.39 ± 0.03* 0.36 ± 0.03┼ 0.37 ± 0.02┼* 0.39 ± 0.02┼* 0.41 ± 0.02┼* MBF 38.7 ± 11.8 83.2 ± 45.6* 144.0 ± 41.4* 201.1 ± 60.2* 126.4 ± 58.8 ┼ 226.1 ± 67.9┼* 293.2 ± 51.8┼* 365.9 ± 73.7┼* Cond. 30.8 ± 13.1 93.6 ± 52.4* 160.3 ± 45.2* 231.2 ± 72.3* 143.7 ± 78.7┼ 251.0 ± 94.8┼* 324.0 ± 75.5┼* 408.1 ± 114.7┼* Rest ACH1 ACH2 ACH3 Rest ACH1 ACH2 ACH3 LL Sea level Vel. 6.2 ± 2.6 19.2 ± 5.4* 23.8 ± 12.0* 34.6 ± 14.9* 19.6 ± 6.7┼ 32.1 ± 10.3┼* 38.0 ± 15.2┼* 45.3 ± 18.0┼* Dia. 0.36 ± 0.03 0.37 ± 0.03 0.37 ± 0.03 0.38 ± 0.03* 0.36 ± 0.02 0.37 ± 0.03 0.37 ± 0.04 0.39 ± 0.04* MBF 38.2 ± 17.7 127.6 ± 57.1* 156.0 ± 93.2* 247.6 ± 132.2* 116.3 ± 35.5┼ 209.6 ± 92.4┼* 263.7 ± 160.1┼* 339.1 ± 169.6┼* Cond. 42.6 ± 18.1 145.4 ± 56.8* 173.1 ± 89.8* 276.4 ± 125.7* 132.7 ± 46.0┼ 240.2 ± 97.8┼* 301.0± 167.5┼* 390.5 ± 185.7┼* LL High altitude Vel. 4.5 ± 1.7 10.2 ± 4.2* 17.1 ± 5.7* 21.0 ± 7.2* 20.7 ± 6.9 ┼ 32.4 ± 9.8┼* 36.8 ± 9.1┼* 46.4 ± 9.5┼* Dia. 0.35 ± 0.03 0.35 ± 0.03 0.36 ± 0.03 0.37 ± 0.03* 0.36 ± 0.03 0.37 ± 0.03┼ 0.38 ± 0.03┼* 0.40 ± 0.03┼* MBF 24.5 ± 6.4 59.5 ± 23.8 102.2 ± 35.8* 137.5 ± 54.0* 124.2 ± 45.1┼ 209.0 ± 78.2┼* 247.9 ± 60.4┼* 347.6 ± 68.0┼* Cond. 26.2 ± 7.0 65.9 ± 33.2 113.6 ± 47.3* 157.1 ± 73.5* 141.8 ± 68.6┼ 245.0 ± 127.4┼* 274.3 ± 81.9┼* 401.3 ± 124.4┼* List of Abbreviations: ACH, acetylcholine; Cond., absolute forearm vascular conductance; Dia, brachial artery diameter; LL, lowlander; MBF, brachial artery mean blood flow; SNP, sodium nitroprusside. *P<0.05, vs Rest. ┼P<0.05, vs control. 114 Figure 5.2 Absolute forearm vascular conductance in lowlanders at sea level and high altitude. Individual data for absolute forearm vascular conductance during acetylcholine infusion (panel A) and sodium nitroprusside infusion (panel B) before and after autonomic blockade at sea level and high altitude. The mean of the individual data is represented by the gray data point. *P<0.05, sea level vs high altitude. 115 Figure 5.3 Relative forearm vascular conductance in lowlanders at sea level and high altitude. Individual data for relative forearm vascular conductance during acetylcholine infusion (panel A) and sodium nitroprusside infusion (panel B) before and after autonomic blockade at sea level and high altitude. The mean of the individual data is represented by the gray data point. 116 5.3.3 Highlanders 5.3.3.1 Cardiovascular response In highlanders diagnosed with EE, MAP was reduced at SNP2 and SNP3 during the control trial (P=0.006 and P<0.001, respectively); however, no differences in MAP were detected during the blockade trial (P=0.07). Non-EE highlanders had a reduced MAP response during SNP infusion during both control and blockade trials compared to rest (All P<0.02). Consequently, both EE and non-EE highlanders experienced elevations in HR at SNP2 and SNP3 compared to rest during both control and blockade trials (P<0.05). In contrast, during the ACH infusion, MAP and HR stayed relatively stable in both EE and non-EE participants (Table 5.4). 5.3.3.2 Forearm vascular response As expected, during both SNP and ACH infusion protocols mean blood flow velocity, arterial diameter, mean blood flow, and forearm vascular conductance all increased (Table 5.5). Absolute forearm vascular conductance was 36% lower at ACH3 (P=0.007), in EE compared to non-EE participants, and this effect was abolished after autonomic blockade (P=0.971; Figure 5.4). Similarly, relative forearm vascular conductance was 80% lower at ACH3 in EE compared to non-EE participants (P=0.005), and this effect persisted after autonomic blockade as relative forearm vascular conductance remained reduced at ACH2 and ACH3 by 72% and 65%, respectively, in EE compared to non-EE participants (P=0.016 and P=0.002, respectively; Figure 5.5). When accounting for forearm volume, participants with EE had reduced forearm vascular conductance at ACH2 and ACH3 by 43% and 52%, respectively, compared to non-EE participants (P=0.023 and P<0.001, respectively;), and after autonomic blockade this EE participants remained lower compared to non-EE at ACH3 by 37% (P=0.038; Figure 5.6). There were no differences detected 117 between EE and non-EE participants during SNP infusion for absolute forearm vascular conductance (control: P=0.085; blockade: P=0.326), relative forearm vascular conductance (control: P=0.305; blockade: P=0.401), and absolute forearm vascular conductance corrected for forearm volume (control: P=0.280; blockade: P=0.562). 118 Table 5.4 Systemic hemodynamics in highlanders at high altitude. Control Blockade Rest SNP1 SNP2 SNP3 Rest SNP1 SNP2 SNP3 EE MAP 91.4 ± 4.5 87.8 ± 5.0 83.0 ± 4.0* 76.5 ± 4.4* 87.5 ± 10.4 83.2 ± 7.7 83.6 ± 6.6 82.5 ± 7.0 HR 81.0 ± 13.5 80.9 ± 14.4 86.3 ± 12.4* 91.3 ± 9.0* 71.9 ± 11.8┼ 75.1 ± 11.5┼ 82.2 ± 10.0┼* 84.0 ± 8.5┼* Non-EE MAP 95.5 ± 12.3 91.2 ± 9.6* 88.0 ± 11.4* 85.4 ± 8.1* 95.9 ± 12.1 91.3 ± 11.9* 88.3 ± 11.0* 84.9 ± 10.7* HR 79.6 ± 11.3 81.3 ± 12.1 85.0 ± 11.2* 87.5 ± 11.9* 69.1 ± 12.5┼ 70.4 ± 11.1┼ 76.6 ± 14.2┼* 78.8 ± 15.1┼* Rest ACH1 ACH2 ACH3 Rest ACH1 ACH2 ACH3 EE MAP 91.5 ± 3.8 90.9 ± 4.6 91.6 ± 3.0 90.0 ± 2.9 86.0 ± 9.6 89.1 ± 10.1 88.0 ± 10.5 88.3 ± 10.1 HR 81.6 ± 10.7 81.0 ± 10.4 79.9 ± 11.3 78.6 ± 10.6* 70.6 ± 9.0┼ 73.1 ± 9.9┼* 72.1 ± 10.6┼ 71.1 ± 12.1┼ Non-EE MAP 96.3 ± 12.6 95.1 ± 12.9 95.3 ± 13.6 94.2 ± 10.1 96.3 ± 11.9 96.1 ± 12.9 95.7 ± 12.3 96.5 ± 11.5 HR 82.1 ± 11.2 78.4 ± 10.9* 80.6 ± 10.4* 79.3 ± 10.2* 70.8 ± 10.7┼ 69.9 ± 11.1┼* 68.0 ± 10.8┼* 68.0 ± 11.4┼* List of Abbreviations: : ACH, acetylcholine; EE, Excessive erythrocytosis; HR, heart rate; MAP, mean arterial pressure; SNP, sodium nitroprusside. *P<0.05, vs Rest. ┼P<0.05, vs control. 119 Table 5.5 Systemic hemodynamics in highlanders Control Blockade Rest SNP1 SNP2 SNP3 Rest SNP1 SNP2 SNP3 EE Vel. 8.6 ± 2.8 22.8 ± 11.8* 26.7 ± 10.2* 25.7 ± 7.7* 15.9 ± 3.8┼ 34.0 ± 9.2┼* 33.1 ± 5.2┼* 33.7 ± 4.9┼* Dia. 0.42 ± 0.07 0.45 ± 0.09* 0.48 ± 0.09* 0.51 ± 0.10* 0.43 ± 0.06┼ 0.48 ± 0.07┼* 0.51 ± 0.07┼* 0.52 ± 0.09┼* MBF 67.9 ± 17.0 230.9 ± 161.0* 294.1 ± 147.6* 324.8 ± 158.0* 139.7 ± 41.6┼ 383.5 ± 166.7┼* 411.7 ± 131.3┼* 437.3 ± 135.6┼* Cond. 74.5 ± 19.2 268.2 ± 195.9* 354.4 ± 176.7* 425.3 ± 207.4* 161.9 ± 53.1 458.9 ± 188.5┼* 491.7 ± 150.8┼* 531.5 ± 170.3┼* Non-EE Vel. 6.8 ± 3.1 22.9 ± 8.8* 24.9 ± 10.3* 27.2 ± 7.9* 19.4 ± 7.9┼ 40.6 ± 16.2┼* 43.7 ± 14.9┼* 44.5 ± 17.2┼* Dia. 0.36 ± 0.04 0.38 ± 0.03* 0.43 ± 0.03* 0.44 ± 0.03* 0.36 ± 0.03 0.39 ± 0.04* 0.44 ± 0.04* 0.45 ± 0.04* MBF 42.5 ± 17.8 158.9 ± 65.8* 210.0 ± 78.5* 249.9 ± 67.0* 121.6 ± 54.8 299.0 ± 153.7┼* 395.6 ± 149.6┼* 419.8 ± 165.3┼* Cond. 43.9 ± 16.6 172.5 ± 64.1* 235.8 ± 74.9* 292.4 ± 76.3* 123.7 ± 48.6 320.4 ± 141.8┼* 442.7 ± 139.4┼* 490.9 ± 166.5┼* Rest ACH1 ACH2 ACH3 Rest ACH1 ACH2 ACH3 EE Vel. 6.6 ± 2.9 9.8 ± 3.5 10.5 ± 3.3* 12.4 ± 4.6* 17.6 ± 6.9┼ 20.2 ± 4.2┼ 21.3 ± 7.5┼* 25.3 ± 9.6┼* Dia. 0.44 ± 0.07 0.43 ± 0.08 0.44 ± 0.07 0.44 ± 0.07 0.45 ± 0.09 0.45 ± 0.07 0.45 ± 0.07 0.46 ± 0.07 MBF 55.0 ± 13.2 80.9 ± 17.6 87.5 ± 16.0* 101.6 ± 24.3* 155.8 ± 48.6┼ 187.0 ± 42.4┼ 193.2 ± 61.2┼* 241.0 ± 73.3┼* Cond. 60.1 ± 14.0 89.2 ± 19.3 95.4 ± 17.1* 112.9 ± 26.5* 183.4 ± 67.8┼ 210.7 ± 46.8┼ 220.2 ± 65.4┼* 273.9 ± 81.0┼* Non-EE Vel. 7.1 ± 3.9 13.0 ± 8.4* 16.5 ± 8.2* 22.5 ± 9.5* 20.5 ± 8.7┼ 26.9 ± 14.1┼* 32.2 ± 14.3┼* 40.2 ± 21.2┼* Dia. 0.38 ± 0.04 0.39 ± 0.04 0.40 ± 0.03* 0.40 ± 0.03* 0.38 ± 0.05 0.38 ± 0.05 0.39 ± 0.04* 0.41 ± 0.04* MBF 47.2 ± 23.5 88.9 ± 48.2 122.1 ± 59.7* 168.5 ± 67.7* 139.2 ± 64.8┼ 185.2 ± 104.2┼ 239.6 ± 125.1┼* 322.5 ± 180.2┼* Cond. 48.7 ± 23.1 94.2 ± 46.9 126.8 ± 56.3* 177.8 ± 65.5* 140.1 ± 56.0┼ 187.2 ± 91.4┼ 244.0 ± 109.1┼* 323.7 ± 156.8┼* List of Abbreviations: List of Abbreviations: ACH, acetylcholine; Cond., absolute forearm vascular conductance; Dia, brachial artery diameter; EE, Excessive erythrocytosis; MBF, brachial artery mean blood flow; SNP, sodium nitroprusside. *P<0.05, vs Rest. ┼P<0.05, vs control. 120 Figure 5.4 Absolute forearm vascular conductance in Andean highlanders at high altitude. Individual data for absolute forearm vascular conductance during acetylcholine infusion (panel A) and sodium nitroprusside infusion (panel B) before and after autonomic blockade between EE and non-EE highlanders. The mean of the individual data is represented by the gray data point. *P<0.05, EE vs non-EE. 121 Figure 5.5 Relative forearm vascular conductance in Andean highlanders at high altitude. Individual data for relative forearm vascular conductance during acetylcholine infusion (panel A) and sodium nitroprusside infusion (panel B) before and after autonomic blockade between EE and non-EE highlanders. The mean of the individual data is represented by the gray data point. P<0.05, EE vs non-EE. 122 Figure 5.6 Absolute forearm vascular conductance normalized to forearm volume in Andean highlanders at high altitude. Individual data for absolute forearm vascular conductance after accounting for forearm volume during acetylcholine infusion (panel A) and sodium nitroprusside infusion (panel B) before and after autonomic blockade between EE and non-EE highlanders. The mean of the individual data is represented by the gray data point. P<0.05, EE vs non-EE. 123 5.4 Discussion The primary novel findings were the following: 1) in lowlanders, endothelial function, but not smooth muscle function, was impaired at high altitude compared to sea level, and this impairment was abolished after administration of adrenergic blockade, and 2) endothelial function, but not smooth muscle function, in Andean highlanders with EE was lower compared to non-EE highlanders, and this impairment was partially abolished after adrenergic blockade. These data indicate that heightened adrenergic signaling at high altitude is a key mechanism contributing to endothelial dysfunction in lowlanders and highlanders. 5.4.1 Effect of high altitude on endothelial function in lowlanders Previous reports that investigated the effects of high altitude on endothelial function are inconsistent. For example, resting endothelial function has been shown to be unaltered at altitude in some [ascent to 3842m via cable car (Bruno et al., 2015); ascent via automobile to 3800m (Tremblay et al., 2017, Tymko et al., 2016)], and reduced in others [ascent to 4200m (Bakker et al., 2015), and 4371m (Tremblay et al., 2018); and ascent to 5050m by trekking (Lewis et al., 2014, Tymko et al., 2017, Tremblay et al., 2018)]. Of note, Tremblay and colleagues (2018) investigated the effects of different altitude exposure (3440m, 4371m, and 5050m) on lowlanders, and demonstrated that FMD was not reduced at 3440m, but it was reduced at 4371m and 5050m. Collectively, these previous reports indicate that there is an altitude threshold of ~4000m where endothelial function is impaired. The data in the current study provide further support this notion of an altitude threshold, since it was found that endothelial function was impaired in lowlanders at 4300m. It is possible that the length of altitude exposure and ascent profile (e.g. trekking or automobile) may also impact this endothelial dysfunction threshold; however, this remains to be 124 fully elucidated. In addition, each of these studies have been conducted using non-invasive flow-mediated dilation as an index of endothelial function (Bruno et al., 2015, Tremblay et al., 2017, Tymko et al., 2016, Bakker et al., 2015, Tymko et al., 2017, Tremblay et al., 2018, Lewis et al., 2014); this non-invasive approach cannot discern the influence of SNA to that of endothelial or smooth muscle dysfunction per se, and the only study that has assessed smooth muscle function at high altitude in lowlanders demonstrated that it was reduced (Lewis et al., 2014). An important strength to the current study was the assessment of vascular smooth muscle function using SNP, and importantly, our data indicates that the reductions in vascular function at 4300m in lowlanders was due to endothelial dysfunction, and not smooth muscle function. The mechanism(s) responsible for altitude-related endothelial dysfunction are unclear, but there is strong evidence that SNA may be responsible. For example, at sea level, acute elevations in SNA (via lower body negative pressure) leads to transient reductions in endothelial function as assessed using FMD (Hijmering et al., 2002, Thijssen et al., 2014), and phentolamine reverses this response (Hijmering et al., 2002). Since high altitude is known to elevate SNA (Duplain et al., 1999, Hansen and Sander, 2003, Tymko et al., 2017), our findings extend those by Hijmering et al. (2002), and demonstrate that reductions in endothelial function at high altitude are also due to adrenergic vasoconstriction. 5.4.2 Endothelial function between EE and non-EE highlanders In the mining town of Cerro de Pasco, Peru, chronic mountain sickness is a severe health crisis in both young and aging males. The disease is characterized by EE, exacerbated hypoxemia, pulmonary hypertension, which can evolve into severe cardiovascular disease (e.g. heart failure) (Villafuerte and Corante, 2016). Surprisingly, there have only been a few investigations that have 125 investigated vascular function in Andean highlanders with EE compared to healthy Andean highlanders (Rimoldi et al., 2012, Bailey et al., 2013, Rexhaj et al., 2016). Each of these studies demonstrated that endothelial function, assessed via non-invasive flow-mediated dilation, was impaired in Andeans diagnosed with EE. The studies, however, failed to account for differences in blood viscosity between the healthy and non-healthy Andean cohorts, which directly impact the flow-mediated dilation response through differences in shear stress, therefore, it is unclear if differences in endothelial function between EE and non-EE Andeans existed. Utilizing the intra-arterial infusion method, we confirmed these previous findings and observed that endothelial function was impaired in Andean highlanders with and without EE, and importantly, smooth muscle function was the same between Andean cohorts. Furthermore, after administration of local autonomic blockade, absolute, but not relative forearm vascular conductance was normalized between EE and non-EE Andeans indicating that SNA at least in-part contributes to the observed endothelial dysfunction in Andeans with EE. This is consistent with previous findings that have illustrated that Andeans with EE have heightened SNA compared to non-EE Andeans (Gamboa et al., 2006). Since differences in relative forearm vascular conductance to ACH persisted between EE and non-EE Andeans after autonomic blockade other vascular mechanism(s) that contribute to endothelial dysfunction might be present in Andeans with EE. One explanation is related to the substantially high concentrations of [Hb] in the EE compared to the non-EE Andeans. Hemoglobin, a nitric-oxide scavenger (Azarov et al., 2005, Salazar Vazquez et al., 2008), may be responsible for reduced nitric-oxide bioavailability in Andeans with EE (Bailey et al., 2018), resulting in reduced endothelial function. 126 5.4.3 Methodological considerations There are some methodological considerations that warrant further comment. First, the drug doses for ACH, SNP, and for autonomic blockade (i.e. phentolamine and propranolol) were kept the same between sea level and high altitude. It is unknown if the drug efficacy of these substances are similar between altitudes (Bailey et al., 2018). Second, the Andean highlanders had a higher (i.e. double) drug dosage of ACH and SNP at high altitude compared to the lowlanders. The experimental rationale for this approach was two-fold: 1) we anticipated an older patient cohort compared to the recruited lowlanders; therefore, direct comparison between lowlander and highlander groups was not possible and we wished to focus on the Andeans with and without EE; 2) based on previous research (Rimoldi et al., 2012, Bailey et al., 2013, Rexhaj et al., 2016), we expected to observe reduced endothelial function in the Andeans compared to lowlanders; therefore, increased the administered doses of each vasoactive drug in order to avoid a “basement” effect. Third, we did not re-measure forearm volume at high altitude in lowlander participants. Although high altitude exposure (>5000m) results in muscle wasting (Kayser, 1992), it was likely that only a little (if any) muscle wasting occurred within 14-21 days at altitude (especially in Cerro where food availability was high). Hypothetically, if forearm volume was reduced at altitude, it would potentially result in an increased forearm blood flow response due to the drug dosing remaining constant between sea level and high altitude. Despite this, we still found that endothelial function was reduced at high altitude in lowlanders. Lastly, blood pressure was slightly reduced during SNP infusion in both lowlanders and highlanders, however, the reduction in SNP during control trials was comparable in lowlanders (sea level vs high altitude) and Andean highlanders (EE vs non-EE); thus, such an influence, if any, likely didn’t alter our results. This caveat was 127 likely due to the participants being in semi-recumbent as opposed to supine position. Despite this positional difference, blood pressure during the ACH protocols remained unchanged. 5.5 Synopsis Our data confirms previous findings that high altitude exposure above ~4000m results in endothelial dysfunction in lowlanders, and the observed endothelial dysfunction was mediated through heightened adrenergic vasoconstrictor signaling. In addition, we Andean highlanders with excessive erythrocytosis have reduced endothelial function compared to healthy highlanders and, similar to lowlanders, this impairment was partially mediated through adrenergic vasoconstrictor signaling. Collectively, our data highlights the impact of sympathetic nervous activity on endothelial function in both lowlanders and highlanders at high altitude. Beyond the clinical implications this study has on patients suffering from excessive erythrocyctosis, it is well known that chronically elevated sympathetic nervous activity is associated with a myriad of disease states such as obesity (Grassi et al., 1998), sleep apnea (Narkiewicz et al., 1999), hypertension (Anderson et al., 1989), and heart failure (Grassi et al., 2007). The current data provides convincing evidence that chronically elevations in sympathetic nervous activity in disease also contributes to reduced endothelial health. These findings suggest that adrenergic-related treatment strategies for cardiovascular disease will also yield positive effects on endothelial-mediated vasomotion. 128 Chapter 6: Conclusion 6.1 Overview of research objectives and study findings The primary research objectives of the current thesis were to: 1) determine the impact of high altitude on endothelial function, and 2) investigate the role of SNA on endothelial function. Study #1 (Chapter 3) investigated the effects of exercise on endothelial function (assessed via brachial artery FMD) at sea level and high altitude. Similar to previous findings (Atkinson et al., 2015), conducted at sea-level (344m; Kelowna, BC), it was found that endothelial function was impaired immediately after 30-minutes of moderate intensity exercise, and this effect was abolished after administration of prazosin (oral α1-adrenergic blockade). To extend these findings to a chronic exposure of hypobaric protocols experimental protocols included in study #1 were repeated at 3800m after a ~7 hour ascent to White Mountain, California. Interestingly, we found that 30-minutes of moderate-intensity exercise had no effect on endothelial function, and oral α1-adrenergic blockade had no effect on endothelial function before and after exercise. Additionally, we found that endothelial function was not different between sea-level and high-altitude. It was concluded from this study that exercise related increases in SNS activity reduce endothelial function immediately following moderate-intensity exercise, but not at high-altitude. Study #2 (chapter 4) investigated the effects of transient alterations in SNA via lower-body differential pressure on endothelial function (assessed via brachial artery FMD) at both sea level and high altitude. Mild levels of LBNP (-10 mmHg) and LBPP (+10 mmHg) have been shown to increase and reduce SNA, respectively (Fu et al., 1998, DeBeck et al., 2010). For this study, a custom-built, light-weight, portable LBNP/LBPP chamber was designed and constructed. The experiments were conducted at sea level in Kelowna, BC (344m) and then repeated in Nepal at 129 5050m in the Ev-K2- Pyramid laboratory in Nepal after a 7-8 day trek. The transient LBNP and LBPP resulted in increases and decreases in SNA at sea level, respectively. Despite these changes in SNA our study found that endothelial function was not impacted by LBNP or LBPP at sea-level or high altitude. In addition, we found that endothelial function at rest was reduced at high altitude compared to sea level. It was concluded from this study that brachial artery endothelial function may not be acutely altered by SNA. The final study in this thesis (study #3) investigated the effects of high altitude on endothelial and smooth muscle vascular function in both lowlanders and Andean highlanders with and without EE via intra-arterial infusions of ACH and SNP. In addition, we examined the role of heightened SNA at high altitude on vascular function by assessing forearm vascular blood flow responses before and after administration of SNA adrenergic blockade (via phentolamine and propranolol). The experimental approach was employed as a gold-standard approach to isolated the key mechanisms of endothelial and smooth muscle vascular function. The experimental protocol for study #3 was conducted both at sea level (344m; Kelowna, BC) and high altitude (Cerro de Pasco, Peru; 4300m) following 14-21 days of altitude exposure. We found that endothelial function was reduced in lowlanders; however, this reduction was abolished after administration of SNA blockade. These data indicate that heightened SNA at high altitude is a key mechanism related to reductions in endothelial function at high altitude. In addition, we found Andean highlanders with EE had reduced endothelial function compared to Andean highlanders without EE, and the difference in endothelial function between EE and non-EE Andean highlanders was in-part normalized after administration of SNA blockade. These data confirm previous findings that high altitude exposure 130 above ~4000m resulting in endothelial dysfunction in lowlanders (Lewis et al., 2014, Bakker et al., 2015, Tymko et al., 2017, Tremblay et al., 2018, Bruno et al., 2015). The remaining portion of this thesis chapter aims to: 1) parsimoniously discuss the overall findings of these three research projects; 2) outline the challenges and nature of conducting high altitude field research; 3) highlight potential topics of future research within the context of the current thesis and findings. 6.2 Overall interpretation 6.2.1 Endothelial function after exercise induced elevations in sympathetic nervous activity One of the first studies to clearly demonstrate that SNA can alter endothelial function was conducted using LBNP (-40 mmHg) (Hijmering et al., 2002). Here, the authors assessed endothelial function via FMD in the brachial artery before and during LBNP. During LBNP, when SNA is markedly elevated (Victor and Leimbach, 1987, Scherrer et al., 1988), the authors found that endothelial function was reduced by ~50%; however, this reduction was abolished after administration of autonomic blockade with phentolamine. This study demonstrated that the SNS plays a significant role mediating endothelial-dependent smooth muscle relaxation. The first study in this thesis utilized exercise as a modality to increase SNA instead of LBNP. There have been several studies to date that have demonstrated that endothelial function is reduced immediately post-exercise (Goel et al., 2007, Dawson et al., 2008, Jones et al., 2010, Johnson, Mather et al., 2012, Birk et al., 2013), and the observed response is thought to be exercise intensity 131 dependent (Birk et al., 2013). Atkinson and colleagues investigated whether the increases in SNA during exercise are responsible for post-exercise endothelial impairment (Atkinson et al., 2015). The authors found that immediately after 30-minutes of moderate intensity exercise (75% heart rate max), endothelial function was reduced; however, this effect was abolished after administration of oral prazosin (α1-adrenergic blockade). We extended these findings by conducting a similar protocol and repeating it at high altitude (3800m; White Mountain, California), where SNA is chronically elevated (study #1). In contrast to Atkinson et al. (2015), the participants in our study conducted a maximal exercise test and their workload was set to 50% of their individual peak workload. Moreover, we assessed endothelial function after maximal exercise, which is something only few studies have done at sea level (Thijssen et al., 2006, Hwang et al., 2012, McClean et al., 2015). Similar to previous findings, endothelial function was not altered at sea level following maximal exercise, which opposes the notion that post-exercise related decreases in FMD are inversely related to exercise intensity (Birk et al., 2013). Even though SNA increases in an exercise dependent manner (Ichinose et al., 2008), An explanation for this finding is that the exercise duration of the maximal exercise test was too short in duration (i.e. exercise volume) to induce a reduction in FMD (Johnson, Padilla et al., 2012, Dawson et al., 2013). Additionally, in agreement with the findings reported by Atkinson et al. (2015), we found that endothelial function was reduced immediately after 30-minutes of moderate intensity exercise via α1-adrenergic pathway at sea level. This experimental design (among with many others) was then repeated after traveling via automobile to high altitude (3800m; White Mountain, California). The outcome from these studies at high altitude revealed two interesting findings: 1) in contrast to our sea level data we observed 132 no post-exercise reductions in endothelial function after 30-minutes of moderate intensity exercise, and 2) administration of prazosin resulted in an overall increase in FMD at across all time points. The latter finding suggests that a degree of vascular restraint may exist at 3800m; however, endothelial function was not reduced compared to sea level, which opposes some studies (Lewis et al., 2014), but not all (Tremblay et al., 2017). Overall, the findings from this study suggest that endothelial function is not altered by exercise-induced elevations in SNA at altitude; therefore, to extend these observations, we performed a follow-up study (Study #2, Chapter 4) using more direct methodological approaches to provide further support for these findings. 6.2.2 Endothelial function during lower body differential pressure In addition to increasing SNA, exercise also alters systemic hemodynamics (e.g. HR, SV, shear stress, and blood pressure), which may impact endothelial function. Elevations in SNA also lead to increases in retrograde shear (Hijmering et al., 2002, Thijssen et al., 2014), which can cause endothelial impairment (Thijssen et al., 2009). Whether SNA directly governs reductions in endothelial function, or indirectly through increases in retrograde shear rate, remains unclear. Thijssen et al. (2014) attempted to tease apart these two mechanisms (SNA vs retrograde shear) responsible for changes in endothelial function. Similar to previous work (Hijmering et al., 2002), Thijssen et al. (2014) employed LBNP (-35 mmHg) to elicit an increase in SNA. Here, the authors found that endothelial function assessed via brachial artery FMD was reduced during 10-minutes of LBNP, but this was accompanied by increases in retrograde shear stress (Thijssen et al., 2014). The authors then repeated the LBNP protocol while using forearm heating to normalize retrograde shear patterns and found that endothelial function was not affected by the LBNP-induced elevations in SNA. This finding provides evidence that SNA may not reduce endothelial function 133 directly, but indirectly via changes in blood flow patterns (e.g. retrograde shear). The authors also increased retrograde shear rate during LBNP by placing a blood pressure cuff on the forearm and inflating to 75 mmHg. However, the authors found that despite substantially increasing retrograde shear rate during LBNP endothelial function was not altered compared to the control trial. Due to the conflicting findings by Thijssen et al. (2014) the second study of this thesis was to determine the impact of SNA independent of changes in hemodynamics. To address this gap in the literature we employed an experimental design similar to previous work (Millar et al., 2013), which involved mild LBNP (-10 mmHg) and mild lower-body positive pressure (LBPP; +10 mmHg); these interventions alter both cardiopulmonary and arterial baroreflex activity (Millar et al., 2013), leading to mild, but global changes in SNA. The distinct advantage of employing a mild LBNP/LBPP model is that both modalities alter SNA that are independent of changes in heart rate (Fu et al., 1998, Rea and Wallin, 1989), stroke volume (Fu et al., 1998), blood pressure (Fu et al., 1998, Cui et al., 2004, Rea and Wallin, 1989), and brachial artery vessel diameter (Padilla et al., 2010). In supine position at rest, mild LBNP (-5 to -10 mmHg) has demonstrated to significantly increase SNA by ~30-60% (Cui et al., 2004, Scherrer et al., 1988, Rea and Wallin, 1989), while LBPP (+10 to +20 mmHg) decreases SNA by ~30% (Fu et al., 1998), in healthy individuals. Consistent with these observations, we found that SNA was elevated by 60.3 ± 25.5% during -10 mmHg LBNP, and reduced by -37.2 ± 12.7% during LBPP. Despite significantly altering SNA we found no differences in endothelial function at both sea level and high altitude. These findings indicate that SNA does not directly alter endothelial function, but the indirect physiological consequences of heightened SNA (e.g. changes in blood 134 flow patterns and shear rates) are responsible for reduced endothelial function. However, it is also possible that we did not manipulate SNA activity enough in order to influence endothelial function. Both studies #1 and #2 suggest that altering SNA at high altitude does not impact endothelial function assessed via FMD. However, an important finding from study #2 was that endothelial function was reduced at rest at 5050m, which was in contrast to our findings in study #1 (at 3800m). These comparisons suggest that the high altitude related reductions in endothelial function may be dependent upon the magnitude of altitude exposure (see Table 6.1 and Figure 6.1). Therefore, for the final thesis research project we sought to investigate whether endothelial function was reduced at an altitude >4000m, and to mechanistically determine if this reduction in endothelial function was due to heightened SNA. This led our international research team, Global REACH, to Cerro de Pasco, Peru, where we conducted ~15 human physiology experiments in both lowlanders and Andean highlanders with and without EE. 135 Table 6.1 Summary of studies that investigated the impact of high altitude on brachial artery FMD. Study Location and altitude Ascent Technique Participants Main Findings (Lewis et al., 2014) Nepal (5050m) Trekking Brachial artery FMD 12 lowlanders FMD was reduced at altitude (Bakker et al., 2015) Nepal (4200m) Trekking Brachial artery FMD 11 lowlanders FMD was reduced at altitude (Bruno et al., 2015) France (3842m) Cable car Brachial artery FMD 22 AMS- lowlanders; 12 AMS+ lowlanders FMD was reduced in participants with AMS, but not in AMS- participants (Tymko, et al., 2016) White Mountain, CA (3800m) Automobile Brachial artery FMD 9 lowlanders FMD was not reduced at altitude (Tymko et al., 2017) Nepal (5050m) Trekking Brachial artery FMD 14 lowlanders FMD was reduced at altitude (Tremblay et al., 2017) White Mountain, CA (3800m) Automobile Brachial artery FMD 12 lowlanders FMD was not reduced at altitude (Tremblay et al., 2018) Nepal (3400m, 4200m, and 5050m) Trekking Brachial and Femoral FMD 22 lowlanders; 12 Sherpa FMD was reduced at 4200m and 5050m, but not at 3400m 136 -6 -4 -2 0 2 4Overall EffectLewis et al. 2014Bakker et al. 2015Bruno et al. 2015 (AMS-)Bruno et al. 2015 (AMS+)Tremblay et al. 2017Tremblay et al. 2018Tymko et al. 2016Tymko et al. 2017Study Figure 6.1 Updated forest plot of studies that have investigated the impact of high altitude on brachial artery flow mediated dilation. Each point represents mean reduction in FMD with 95% confidence intervals. The blue line represents the mean reduction across all studies. The arrows highlight the studies that found endothelial function is reduced at high altitude and NC represents the studies that found that high altitude had no impact (i.e. No Change) on endothelial function. NC NC NC 137 6.2.3 Endothelial function during changes in sympathetic nervous activity at high altitude in lowlanders and Andean highlanders The final thesis study investigated the effects of altitude >4000m on endothelial function, SNA and altitude pathology on endothelial function. For the first time at high altitude, we employed the intra-arterial forearm infusion model to assess endothelial function. To date, all vascular function research at high altitude (see table 6.1 and figure 6.1) have been completed using non invasive methods such as FMD (both brachial and femoral artery) and arterial stiffness. This is due to: 1) arguably more robust measures of endothelial function (e.g. forearm arterial infusion technique) requires trained medical personnel and more sophisticated experimental setup, 2) sourcing pharmaceutical agents in foreign countries is challenging, and 3) obtaining ethical approval to conduct invasive measures on lowlanders and/or highlander dwellers is arduous. In March 2018, our international research team conducted ~4 weeks of baseline testing near sea level (Kelowna, BC; 344m), and then repeated these experiments at high altitude in Peru in July 2018. Fifteen separate studies, all with unique research questions were successfully completed. Among these, was the third study of the current thesis, which involved brachial intra-arterial infusions of ACH and SNP, before and after full adrenergic blockade. These studies were conducted in lowlanders and Andean highlanders with and without EE. As anticipated, high altitude exposure resulted in a reduction in endothelial function at rest, which supports the theory that endothelial function is impaired at altitudes >4000m; however, this exact threshold remains to be determined. Importantly, the forearm blood flow response to SNP - a smooth muscle relaxant - was unchanged between sea level and high altitude meaning that the 138 reductions in forearm blood flow to ACH were not due to smooth muscle impairment, but rather were indeed due to endothelial dysfunction. After administration of local autonomic blockade, the forearm blood flow response to ACH was normalized to sea-level values. These findings clearly demonstrate that high altitude related reductions in endothelial function are due to increased adrenergic signaling causing vascular constraint in lowlanders. In addition to testing lowlanders at high altitude, we also recruited a cohort of Andean highlanders. In the mining town of Cerro de Pasco, Peru, CMS is a severe health crisis in both young and aging males. The disease is characterized by EE, exacerbated hypoxemia, pulmonary hypertension, which can evolve into severe cardiovascular disease (e.g. heart failure) (Villafuerte and Corante, 2016). The studies that have assessed endothelial function between Andean highlanders with EE compared to healthy Andean highlanders demonstrated that endothelial function - assessed via non-invasive FMD - was impaired in Andeans diagnosed with EE (Rimoldi et al., 2012, Bailey et al., 2013, Rexhaj et al., 2016). Utilizing the forearm intra-arterial infusion approach, we confirmed these previous findings and observed that endothelial function was impaired in Andean highlanders with and without EE. In addition to this finding, we found that such differences were potentially, only in-part, normalized between EE and non-EE highlanders after adrenergic blockade. Expanding on this latter point, absolute forearm vascular conductance was, but relative forearm vascular conductance was not, normalized after adrenergic blockade. The discrepancy between these findings indicates that there are other mechanism(s) beyond SNA mediated vascular constraint that are responsible for the observed differences between EE and non-EE Andeans. However, previous reports have demonstrated that Andeans with EE have heightened SNA compared to non-EE Andeans (Gamboa et al., 2006). Moreover, these data are also consistent with 139 unpublished observations from our research group that found MSNA was elevated in EE vs non-EE Andean highlanders (Steinback et al. 2019, unpublished findings). Since the adrenergic blockade did not completely abolish the differences in endothelial function between Andean highlander cohorts, there must be other mechanism(s) that contribute to endothelial dysfunction in Andeans with EE. One explanation could be related to the substantially high concentrations of hemoglobin in the EE Andeans compared to the non-EE. Hemoglobin is an NO scavenger (Azarov et al., 2005, Salazar Vazquez et al., 2008), and recent findings indicate that Andean highlanders with EE have reduced NO bioavailability (Bailey et al., 2018). 6.2.4 Thesis key points The reported series of experiments determined several novel findings: 1) Endothelial function was not impacted immediately after moderate intensity nor maximal exercise at high altitude (Study #1). 2) Endothelial function was not altered by acute and mild changes in SNA at high altitude (Study #2). 3) A threshold exists for endothelial impairment at high altitude, and this threshold seems to occur at ~4000m (Studies #1-3). 4) Reductions in endothelial function at altitude >4000m in lowlanders was mediated through increased adrenergic signaling (Study #3) 5) Andean highlanders with EE have reduced endothelial function compared to healthy Andean highlanders and this reduction was partly mediated through increased adrenergic signaling (Study #3). 140 6.3 The challenge of high altitude research For each of these high altitude research expeditions (White Mountain, Nepal, Peru) ~3-4 metric tons of equipment and consumable items (e.g. needles, syringes, saline), amounting to ~one million dollars (USD), were packed in a combination of pelican cases, duffle bags, suitcases, and waterproof barrels. Back-up equipment for nearly every research device was packed in case of equipment failure at high altitude. The equipment used on these expeditions were travelled as checked-baggage on multiple international flights with each Global REACH team member. More fragile equipment, such as Duplex ultrasound machines and laptops, accompanied team members as carry-on luggage. One of the major challenges for each of these high altitude expeditions is that appropriate documents for legal importation of all equipment and consumable items had to be obtained prior experimentation. Surprisingly, during our research expeditions to the United States and Nepal we had few issues gaining entry for the equipment; however, despite our efforts, ~15% of our equipment was rejected entry into Peru upon arrival. Through sustained perseverance by both Global REACH members and our local Peruvian collaborators, these items were released from Peru customs within ~14 days. During these substantial logistical hurdles, the research expedition carried on and was largely successfully due to teamwork, equipment sharing, and schedule re-organization. 6.4 Future directions This thesis describes the role SNA on endothelial function at sea level and high altitude using a variety of novel experimental approaches. Future research should focus on a number of themes, including: 141 6.4.1 Targeting the magnitude of hypoxemia required to reduce endothelial function The current thesis provides strong evidence that a hypoxemia threshold exists before a reduction in endothelial function is observed. Collectively, it seems that this threshold is ~4000m, which is equivalent to a fraction of inspired oxygen (FIO2) of ~0.14%. In order to test this hypothesis, exposure to step-wise reductions in FIO2 within a normobaric hypoxia chamber (e.g. FIO2 = 0.21, 0.18, 0.14, 0.12) can be utilized to determine the threshold where endothelial function is reduced. A recent study examined the effects of graded isocapnic hypoxia on endothelial function in healthy humans and demonstrated that endothelial function was reduced after only 30 minutes of exposure to mild isocapnic hypoxia (PETO2 = 75 mmHg; FIO2 = ~0.16) and severe hypoxia (PETO2 = 50 mmHg; FIO2 = ~0.12) (Lewis et al., 2017). However, during this particular study PETCO2 was maintained via dynamic end-tidal forcing, which does not truly simulate high altitude. During altitude exposure, early respiratory alkalosis (i.e., hypocapnia) occurs rapidly via hyperventilation, followed by metabolic compensation. 6.4.2 Exploring the impact of arterial carbon dioxide on endothelial function Surprisingly, very little research has been completed on the effects of carbon dioxide on peripheral artery endothelial function. Elevations in PaCO2 (i.e. hypercapnia) can result in elevations in brachial artery blood flow (Vantanajal et al., 2007, Norcliffe-Kaufmann et al., 2008), while reductions in PaCO2 (i.e. hypocapnia) can result in reductions in brachial artery blood flow (Norcliffe-Kaufmann et al., 2008). Changes in blood flow patterns can lead to alterations in shear stress that may impact endothelial function. In addition, elevations and reductions in PaCO2 can elevate and reduce SNA, respectively (Shoemaker et al., 2002). Since there is evidence that 142 changes in SNA can impact endothelial function (including the current series of thesis studies) (Hijmering et al., 2002), changes in PaCO2 may have a direct impact on endothelial function. In order to test this hypothesis experimentally a dynamic end-tidal forcing system can be employed to alter PETCO2 (Tymko et al., 2015, Tymko et al., 2016), a surrogate of PaCO2, independent from changes in PETO2 in order to isolate the effects of PCO2 on endothelial function. In addition, isolating the effects of pH independent of PCO2 is another area of study that should be addressed. This isolation could be achieved through PETCO2 clamping during bicarbonate infusion. 6.4.3 Endothelial function across different high altitude populations Although not presented in the current thesis, our research team, Global REACH, has also conducted a series of vascular function experiments on Sherpa highlanders that reside within the Khumbu valley in Nepal. Currently, our research team is organizing a research expedition to travel to Ethiopia so that we can, for the first time, compare and contrast peripheral vascular function (in addition to other experimental questions) data across the Andean, Nepalese, and Ethiopian highlanders. We know significantly less about adaptations of Ethiopian Amhara and Oromo highlanders from the Simien and Bale mountains, respectively. The Amhara have [Hb] concentrations similar to lowlanders, yet higher than expected SaO2, potentially due to an increased affinity of their [Hb] for oxygen (Beall et al., 2002, Beall, 2006). Only few sophisticated physiological data sets exist on Ethiopian highlanders. Conceivably, the Sherpa, Quechua, and Amhara phenotypes represent distinct evolution-driven strategies that permit survival and performance at high altitude. Ethiopia will be the final installment in Global REACH’s trilogy of high altitude adaptation studies. 143 6.5 Clinical implications of high altitude research Research on high altitude physiology offers complementary insight into biological adaptation to hypoxia. Ways to apply results from these expeditions are multifaceted, with implications for military deployment to high altitude (e.g. Afghanistan), for the growing numbers (>1 million) of lowlanders vacationing at high altitude destinations, and for commercial flight personnel who are hypoxic during flight (mild hypoxia). These data can have potential translational impact for patients in critical care and in other clinical situations of hypoxia (e.g. lung disease, heart failure, circulatory shock). Importantly, the findings may provide novel insight into the current cardiovascular health status of local highlanders (Tibetan, Peruvian, and Ethiopian), which will directly benefit these populations. An obvious clinical aspect to the current project was that the manifestations of EE that mirror the complications in other diseases associated with chronic hypoxic (e.g., pulmonary hypertension and right-sided heart failure), such as chronic obstructive pulmonary disease and heart failure. The results from this study will be a valuable step toward effective treatments for the cardiopulmonary consequences of EE, and beyond to more effective treatments for other hypoxic disorders that affect individuals who live at lower altitudes. In particular, understanding the pathophysiological mechanism of pulmonary hypertension will benefit the thousands of diagnoses with the disorder worldwide each year, along with providing information on how to improve treatment avenues for the high altitude Peruvian dwellers that suffer from EE. 144 6.6 Final thoughts The endothelium consists of a single layer of highly sensitive cells that are critical to vascular health. 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Science362, 1049-1051, DOI: 10.1126/science.aat8824 [doi]. 165 Appendices Appendix A The 2018 Global Research Expedition on Altitude-related Chronic Health (REACH) to Cerro de Pasco, Peru: An Experimental Overview Tymko, M.M., Hoiland, R.L., Tremblay, J.C., Stembridge, M., Dawkins, T., Coombs, G., Patrician, A., Howe, C.A., Gibbons, T., Moore, J., Steinback, C.D., Stacey, B.S., Bailey, D.M., MacLeod, D., Gasho, C., Anholm, J., Bain, A., Lawley, J., Hansen, A.B., Villafuerte, F., Vizcardo, G., Ainslie, P.N. (2018). The 2018 Global Research Expedition on Altitude-related Chronic Health (REACH) to Cerro de Pasco, Peru: An Experimental Overview. 166 Abstract Background: In 2016, the international research team - Global Research Expedition on Altitude-related Chronic Health (REACH) - was established. The team consists of ~45 students, principal investigators, and physicians with the common objective to conduct a similar series of sophisticated experiments focusing on high altitude adaptation in both lowlanders (i.e. born <1500m altitude) and local highlanders of Nepal (Sherpa), Peru (Quechua), and Ethiopia (Amhara and Oromo). The experimental objectives, organization and characteristics, and novel select data from Global REACH’s latest research expedition to Peru are outlined herein. Methods: The expedition involved 15 major studies with two principal objectives: 1) to identify the physiological differences to high altitude acclimatization between lowlanders and Andean born highlanders with and without Chronic Mountain Sickness (CMS); and 2) to compare the physiological results obtained in Andean highlanders to the Sherpa highlanders from our recent 2016 expedition to Nepal. After baseline testing in Kelowna, BC, Canada (344m), Global REACH travelled to Lima, Peru (~150m), and then ascended by automobile to Cerro de Pasco, Peru (~4300m) where experiments were conducted for 25 days. The core studies focused on the mechanism(s) underpinning cerebral and peripheral vascular function, cardiopulmonary regulation, exercise performance, intra-ocular pressures, and autonomic control. Results: Despite encountering serious logistical challenges, each of the proposed studies were completed at both sea level and high altitude amounting to ~780 study sessions and >3000hrs of experimental testing in lowlanders (n=30) and Andean highlanders with (n=22) and without (n=45) 167 CMS. Participant demographics, exercise capacity, arterial blood gases, and acid-base balance between the different groups are presented. Conclusions: The collective findings will contribute to our understanding of how lowlanders and Andean highlanders have adapted under the stress of residence at high altitude. Future goals for Global REACH are to conduct a similar series of experiments to compare results from the Sherpa and Quechua highlanders to the Amhara and Oromo highlanders of Ethiopia. Key words: High-altitude, hypoxia, Andean highlanders, Global REACH, Chronic Mountain Sickness 168 Introduction Indigenous populations of the high plateaux of Tibet (Sherpa), Peru (Quechua), and Ethiopia (Amhara and Oromo) have developed separate physiological adaptations to thrive in their respective harsh hypoxic environments (Beall, 2006). Although there is considerable debate on when these plateaux were inhabited, the consensus is that the Old World plateaux has been settled longer (Ethiopian ~50,000 to ~70,000 y and Tibetan ~25,000 y) compared to the Altiplano in the New World (Peruvian ~11,000 y; Beall et al., 2002; Beall, 2006; Beall, 2007; Alkorta-Aranburu et al., 2012; Zhang et al., 2018). The majority of these Indigenous populations show impressive function at high altitude compared to lowlanders – function that arises from fundamental changes in oxygen delivery and utilization. In some ways, the Sherpa resemble an unacclimatized lowlander at high altitude, with lower hemoglobin concentration and oxygen saturation at similar altitudes compared to other highlanders such as the Peruvian Quechua (Claydon et al., 2004; Beall, 2006). Some (Gilbert-Kawai et al., 2014; Tremblay et al., 2018), but not all studies (Liu et al., 2016; Wang et al., 2018), indicate that Sherpa rely on enhanced blood flow and endothelial function to sustain convective oxygen delivery and cellular metabolic adaptations to more efficiently use oxygen. The high altitude Andeans rely on increased hemoglobin concentration, but they are prone to chronic mountain sickness (CMS) – a genetic maladaptation that results in excessive red blood cell production, exaggerated vascular resistance (i.e. sluggish blood flow), elevated systemic oxidative-inflammatory-nitrosative stress (Bailey et al., 2013; Bailey et al., 2018), and profound hypoxemia (Monge, 1943; Villafuerte and Corante, 2016). Less is known about the adaptations of Ethiopian highlanders (Amhara and Oromo populations) from the Simien and Bale mountains, respectively. The Amhara have hemoglobin concentrations similar to lowlanders, yet higher than expected oxygen saturation, potentially due to an increased affinity of 169 oxygen for their hemoglobin (Beall et al., 2002; Beall, 2006). Conceivably, the Sherpa, Quechua, and Amhara phenotypes represent distinct evolutionarily-driven strategies that permit survival and performance at high altitude (Beall et al., 2002; Beall, 2006; Beall, 2007). In 2016, our international research team, Global Research Expedition on Altitude-related Chronic Health (REACH) was formed with the objective to conduct a series of experiments on the Tibetan, Peruvian, and Ethiopian highlanders. High altitude physiology research offers valuable insight on the mechanistic adaptation to hypoxia, which is translatable to a myriad of clinical diseases characterized by hypoxemia (Levett et al., 2010). The timing of this research is urgent, as migration and modernization are rapidly changing traditional ways of life and altitude exposure of high altitude dwellers (Beall, 2013). In October 2016, our research team successfully conducted a series of experiments in lowlanders and Sherpa at the Ev-K2 Pyramid International Laboratory/Observatory near Mt. Everest basecamp (5050m; Willie et al., 2018), and in July of 2018, Global REACH traveled to Cerro de Pasco, Peru (4300m) to conduct a similar series of experiments in the Quechua highlanders with and without chronic mountain sickness (CMS). Chronic mountain sickness is a significant public health concern for high altitude dwellers; it profoundly affects quality of life, mental and physical performance (Bailey et al., 2018), and leads to premature death (Rivera-Ch et al., 2007). An estimated 5-10% of high altitude residents have CMS, which is a major public health problem primarily in the Andes, but also in Kyrgyzstan, Northern India, and among migrants to high altitude in Tibet and the United States (Penaloza and Arias-Stella, 2007; Pei et al., 2012; Sahota and Panwar, 2013). Among the highest reported prevalence is in the mining city of Cerro de Pasco, Peru, where 15% of men aged 30-39, and 34% aged 60-69 present with excessive erythrocytosis (Monge et al., 1989). Chronic mountain sickness 170 is primarily characterized by excessive erythrocytosis (females hemoglobin levels > 19 g dl-1; males > 21 g dl-1) and severe hypoxemia (at 4300m, <83% oxyhemoglobin saturation; Monge C et al., 1992) accompanied by three or more of the following symptoms: breathlessness, palpitations, sleep disturbance, cyanosis, dilation of veins, paresthesia, headache, or tinnitus (see Qinghai CMS score; Leon-Velarde et al., 2005). Herein, we provide a summary of Global REACH’s research expedition to Cerro de Pasco, the second installment of our team’s common research objective to study and compare the Tibetan, Peruvian, and Ethiopian highlanders. The principal scientific themes and experimental details of the expedition along with the more notable aspects of its execution are discussed herein; the results of specific studies will be published as discrete papers. Finally, similar to our research group’s overview of our Nepal expedition (Willie et al., 2018), we characterize important physiological data of our experimental populations including arterial blood gases, exercise performance, and acid-base balance. 171 Method and Materials Global REACH. Our research team, consisting of 45 students, principal investigators, and physicians commenced baseline studies in March 2018, and travelled to Cerro de Pasco on June 25th 2018, where the majority of the team remained until July 22nd 2018 (refer to figure 1). The leadership for the research expedition originated from the University of British Columbia - Okanagan (Canada), Unversity of Alberta (Canada), Duke University (United States), Loma Linda University (United States), University of Innsbruck (Austria), University of Boulder Colorado (United States), Cardiff Metropolitan University (United Kingdom), University of Cambridge (United Kingdom), University of South Wales (United Kingdom), and Bangor University (United Kingdom). The research expedition, was aided by the help of local collaborator, Dr. Francisco Villafuerte, and three graduate students from the Universidad Peruano Cayetano Heredia, Peru. Ethical approval. In accordance with the Declaration of Helsinki, the lowlander and highlander based studies were approved by the UBC Clinical Research Ethics Board (H17-02687 and H18-01404, respectively), and local Peruvian ethics committee for the Universidad Peruana Cayetano Heredia (#101686). All lowlander and highlander study participants signed the approved consent form (English and Spanish forms were available) after each study was thoroughly explained in their native language. Prior to voluntary consent, we provided opportunity for questions directly with each study principal investigator. For each study involving highlanders in Cerro de Pasco, Peru, an official translator was present for the entire testing duration to ensure proper communication was maintained between the researchers and participant. All participants were free to withdraw without justification or penalty from all experiments at any time. Andean participants 172 were monetarily compensated for their time based on a payment suggested by the Universidad Peruana Cayetano Heredia research ethics board. Participants. All Global REACH members were of lowlander descent and were born and currently living below 1500m. All lowlander participants avoided visiting high altitude (>1500m) for at least six-months prior to the expedition. Each expedition study had different sample sizes pooled from the same cohort of participants according to prospective power calculations (see below). Global REACH members were included in up to 11 experiments at either sea level and/or high altitude with adequate recovery between each and close attention to non-contamination between protocols (e.g. washout following drug infusion). The Andean highlanders were only tested at high altitude. Andean highlanders were recruited through an established database compiled in-part by Dr. Villafuerte from the Universidad Peruana Cayetano Heredia, Lima, Peru. Highlanders were recruited by telephone by one of three local translators. During a familiarization visit to the high altitude laboratory, a detailed family and altitude history was collected from all Andean participants including altitudes during childhood, in adulthood, and for the 12-months preceding the studies. All recruited Andean participants (and their parents) were born at, and permanently lived at high altitude (within the Cerro de Pasco region). After providing informed consent, each recruited Andean participant was requested to fill out a pre-screening Lake Louise altitude sickness score (Savourey et al., 1995), and a Qinghai CMS questionnaire (Leon-Velarde et al., 2005). Prior to any study at either low or high altitude, participants were asked to refrain from exercise and caffeine for a minimum of 12-hours, and were fasted for a minimum of two-hours. We also obtained an antecubital venous blood sample for the measurement of hematocrit and hemoglobin. Due to the high number of proposed investigations that needed to be completed within a short 173 time-frame, six laboratories in Cerro de Pasco ran simultaneously and typically operated for 12-18 hrs/daily. Study participants were included if they were between the ages of 18-60 years old without any medical history of cardiovascular, cerebrovascular, pulmonary, metabolic disease, or history of working in the local mines (e.g. Lead and Sulphur mines). A subset of premenopausal women aged 18-30 were recruited to investigate potential sex differences within healthy Andeans. Chronic Mountain Sickness. Clinically, CMS is defined by the following criteria: 1) Excessive erythrocytosis: hemoglobin concentration [Hb] >19g/dL for women and [Hb] >21g/dL for men, 2) Clinical symptoms: Qinghai CMS score >6 (Leon-Velarde et al., 2005), 3) Clinical signs: Severe hypoxemia (SpO2 <83% at Cerro De Pasco, 4300m; Villafuerte and Corante, 2016). However, due to the day-to-day variability in the Qinghai CMS symptom scores (Leon-Velarde et al., 2005), we defined CMS participants based on excessive erythrocytosis ([Hb] >19g/dL for women and [Hb] >21g/dL for men). Low and high altitude laboratories. Between February-April 2018, baseline studies were conducted in the Cerebrovascular Physiology Laboratory at the University of British Columbia – Okanagan (Kelowna, BC, Canada). There, the large laboratory (PB = ~730 mmHg, humidity= ~30%, laboratory temperature = ~22ºC) was divided into five separate simultaneous testing areas, and one space was dedicated to blood analysis compliant with standard research governance guidelines. Global REACH arrived in Cerro de Pasco in late June 2018. The testing facility associated with the Universidad Peruana Cayetano Heredia in Cerro de Pasco was expansive (PB = ~450 mmHg, humidity= ~75%, laboratory temperature = ~15ºC), being able to accommodate six complex (i.e., invasive and/or pharmacological) studies running simultaneously. Electricity 174 was consistent for the majority of expedition; however, on ~5 days we encountered long-term black-outs and power surges, the latter resulted in short-lasting electrical fires. Therefore, all equipment was equipped with surge protectors, and after the first local electrical blackout (of several), a large generator served as a back-up power source for the remainder of our expedition. Another regular issue encountered was water supply shortage. The laboratory was not accustomed to housing ~45 guests at once (excluding Andean participants). Our team consistently ran out of water for the facility’s toilets and sinks, and needed to get the water reservoir filled 1-2 times per week. Equipment Logistics. Over four metric tons of equipment and consumable items (e.g. needles, syringes, saline), amounting to ~1.3 million dollars (USD) were packed in a combination of pelican cases, duffle bags, suitcases, and waterproof barrels. Back-up equipment for nearly every research device was packed in case of equipment failure at high altitude. This equipment travelled as checked-baggage on multiple international flights with each Global REACH team member. More fragile equipment, such as Duplex ultrasound machines and laptops, accompanied team members as carry-on luggage. Appropriate documents for legal importation of all equipment and consumable items were obtained prior to traveling to Peru. Despite these efforts, ~15% of our equipment was rejected entry into Peru upon arrival; however, through sustained perseverance by both Global REACH members and our local Peruvian collaborators, these items were released from Peru customs within ~14 days. In advance, 43 K- and T-size compressed gas cylinders were purchased (Linde Industrial Gas, Lima, Peru) and delivered to the high altitude laboratory in Cerro de Pasco. One small compressed gas cylinder of 100% Oxygen was acquired as a safety precaution in the event of an emergency evacuation of a team member (or Andean participant) related to 175 altitude illness – thankfully, no serious adverse health outcomes were experienced. Several studies required blood and plasma samples to be flash frozen and stored in liquid nitrogen Dewars prior to transportation back to the Canada/UK/USA for batch analyses. Ascent profile to Cerro de Pasco. Global REACH members traveled to Lima, Peru, in June ~2-7 days prior to departure to Cerro de Pasco. During this time, final equipment organization and preparations were completed, including the processing of several documents to release the equipment that remained in Peru customs. Immediately prior to ascent to Cerro de Pasco, our two liquid nitrogen Dewar’s were filled locally to flash freeze all upcoming collected blood samples. Research equipment, personal luggage, and liquid nitrogen Dewar’s were transported by a one-tonne cube truck to Cerro de Pasco (~270km; 7-8 hour drive). Global REACH team members, along with local collaborators were transported by cargo van on three separate days (June 25th, 26th, and 27th). Participants refrained from taking prophylactic medication against high altitude illness (e.g. Diamox), however, if participants requested medication to relieve symptoms while at high altitude, or were requested by our physicians to undergo treatment, it was made available to them and their health and symptoms were monitored closely. The majority of the Global REACH team spent 3-4 weeks in Cerro de Pasco completing all studies. Commonly Employed Methodologies Dynamic end tidal forcing. Several studies required the precise control of arterial blood gases for a portion, or for the entire duration of their protocol. To accomplish this, we employed a portable dynamic end-tidal forcing system (Airforce, UBC, Canada) to control the partial pressure of end-tidal oxygen and carbon dioxide (PETO2 and PETCO, respectively), as a surrogate 176 for the partial pressure of arterial oxygen and carbon dioxide (PaO2 and PaCO2, respectively; Tymko et al., 2015; Tymko, Hoiland et al., 2016). Our custom-built system uses independent gas solenoid valves for O2, CO2, and N2 and controls the volume of each gas being delivered to an inspiratory reservoir through a mixing and humidification chamber on a breath-by-breath basis. This system has been used previously to effectively control end-tidal gases during many different physiological stressors, and its use has been validated at high altitude (Tymko et al., 2015). Although this device works extremely well in participants with background knowledge in respiratory physiology, extensive training and instruction was required from our local translators to aid the Andean participants to breathe normally on the device. Respiratory and cardiovascular measurements. Nearly all respiratory and cardiovascular parameters were acquired using an analog-to-digital converter (Powerlab/16SP ML 880; ADInstruments, Colorado Springs, CO, USA) interfaced with a personal computer. Commercially available software was used to analyze ventilatory and cardiovascular variables (LabChart V7.1, ADInstruments, Colorado Springs, CO, USA). Respired gases were sampled at the mouth and analyzed for PETO2 and PETCO2 (ML206; ADInstruments, Colorado Springs, CO, USA). During the expedition, two gas analyzers failed due to pump failure and excess condensation, the latter issue was able to be resolved. Respiratory flow was also measured near the mouth using a pneumotachograph (HR 800L, HansRudolph, Shawnee, KS, USA) and a differential pressure amplifier (ML141, ADInstruments, Colorado Springs, CO, USA). One unexpected problem we encountered due to the high volume of data collection was excess condensation accumulating in the pneumotachometers resulting in respiratory “drift” and signal artifact. This issue was resolved by replacing spirometry filters more frequently (e.g. two per participant). Heart rate was 177 determined from a standard lead II electrocardiogram (ML 132, ADInstruments, Colorado Springs, CO, USA), and the majority of blood pressure measurements were conducted on a beat-by-beat basis using finger photoplethysmography (Finometer pro, Finapres Medical Systems, Netherlands). At times, due to the cold environment, a hot water bottle was required to warm the participant’s hand prior to experimentation in order to obtain a proper blood pressure waveform, and all studies recorded manual blood pressure measurements to both confirm and calibrate the finometer blood pressure waveform. Transcranial and duplex Doppler ultrasound. Several studies required the use of transcranial Doppler (TCD) and/or duplex Doppler ultrasound. These techniques function based on the same fundamental principles, however, Duplex ultrasound allows for the simultaneous acquisition of both blood vessel image and blood velocity, whereas TCD only records blood velocity of an insonated blood vessel (Thomas et al., 2015; Willie et al., 2011). The same experienced sonographers were used for all studies, and they used commercially available ultrasound machines (TCD, Spencer Technologies, PMD150B; Duplex ultrasound, uSmart 3300, Terason; Vivid, GE, Fairfield, CT, USA). For our studies, TCD ultrasound utilized a low frequency probe (2MHz) to assess blood velocity in the middle and posterior cerebral arteries using previously described techniques (Willie et al., 2011). Our peripheral duplex ultrasound machines used a higher-frequency (10MHz) linear array probe and were portable with a short battery life (~90 minutes), therefore, routine access to power supplies were needed. The peripheral duplex ultrasound machines were used to measure blood flow through the brachial, renal, common carotid, internal carotid, external carotid, and vertebral arteries using previously established guidelines and principles (Thomas et al., 2015). Data backups were performed daily to multiple portable 178 encrypted solid-state hard drives. In previous high altitude expeditions, our team has encountered several failures with older spinning hard drives likely due to the reduction in barometric pressure. Therefore, we ensure that we only use solid-state external and internal hard drives while at high altitude. Transthoracic Echocardiography. For studies assessing cardiac function and/or pulmonary pressure, a portable, battery-powered, cardiac ultrasound machine was employed (Vivid Q, GE, Fairfield, CT, USA). Ultrasound images were acquired and analyzed using a range of echocardiographic techniques including two-dimensional, Doppler and speckle-tracking modalities in accordance with published guidelines (Rudski et al., 2010; Lang et al., 2015). Pulmonary artery systolic pressure (PASP) was a primary outcome variable for a number of studies, and was measured by Doppler echocardiography based upon measurement of the maximum velocity of the tricuspid regurgitation jet (Bertini et al., 2009). The peak systolic pressure gradient of the right ventricle (∆ P max) to the right atrium was calculated by the simplified Bernoulli equation (4*V2), where V is the peak systolic velocities of the tricuspid regurgitate. Pulmonary artery systolic pressure was then determined by adding the right atrial pressure. Right atrial pressure was estimated by evaluation of the inferior vena cava diameter and response to a deep inspiration (Aessopos et al., 2000). Blood sampling. Blood samples were obtained following placement of an indwelling cannula located in either a radial/brachial artery, or forearm antecubital vein as required. Blood was drawn directly into a safePico syringe (Radiometer, Copenhagen, Denmark) for immediate blood gas analysis (outlined in detail below) and into Vacutainers® (Becton, Dickinson and Company, 179 Oxford, UK) containing either K2-ethylenediaminetetraacetic acid (K2-EDTA), serum separation gel or sodium citrate, before centrifugation at 600g (4°C) for 10-minutes. Plasma, serum and red blood cell slurry were decanted into 2mL cryogenic vials (Simport™, Fischer Scientific Ltd, UK) and immediately snap frozen and stored in liquid nitrogen (-196 °C) prior to international transportation back to the United Kingdom, USA, and Canada for specialist batch analyzes. Worth noting, due to the reduced barometric pressure in Cerro de Pasco, and volume percent of plasma in individuals with excessive erythrocytosis, ~twice as many vacutainers were required (compared to typical low altitude studies) to account for the reduced blood volume collected. Blood gas analysis. Both venous and arterial blood samples were collected and analyzed by either a stationary commercial blood gas (ABL90 Flex, Radiometer Canada), or a portable blood analyzer (i-STAT, Abbot Point of Care, Princeton, New Jersey). The Radiometer ABL90 analyzer aspirates blood samples into a chamber containing electrodes that are selective for the variables of interest. The ABL90 analyzer, although reliable and functional at altitude, required constant calibration, and encountered numerous blood clots likely due to a combination of the high volume of blood samples analyzed (~1000 samples) and the abnormally viscous blood encountered in the CMS+ Andean participants. In general, throughout the expedition our research team consistently encountered higher than expected blood clotting incidents in both venous and arterial catheters, especially in the CMS+ Andeans. It was interesting to note that several samples obtained from the CMS+ patients were hemolyzed (red tinge in plasma/serum), which was resolved following isovolemic hemodilution, implicating excessive polycythemia and increased red blood cell “fragility” as potential contributory factors. The portable i-STAT devices were used as a back-up in case the ABL90 required flushing or calibration, and were used for a few select studies that 180 were measuring either venous or arterial blood (~250 samples in total). Despite the apparent electrical demise of our hot water bath early in the expedition, we successfully acquired novel measures of blood viscosity after some technical ingenuity, which involved removing, dismantling, and mending the equipment’s electrical motor. Following collection into lithium-heparin vacutainers, whole blood viscosity was measured in duplicate at a shear rate of 225 s-1 and was body temperature corrected using a cone and plate viscometer (DV2T Viscometer, Brookfield Amtek, USA; Gnasso et al., 2001; Baskurt et al., 2009; Tremblay et al., 2018). Blood volume testing. Blood volume, plasma volume, and hemoglobin mass was determined using the modified carbon monoxide rebreathing method, as described previously (Schmidt and Prommer, 2005), and has been successfully used by our research team (Williams et al., 2016; Stembridge et al., 2018). Briefly, the protocol consisted of a venous blood draw for the determination of hemoglobin concentration and the percentage of carboxyhaemoglobin (HbCO) via co-oximetry (ABL 90, Radiometer, Denmark). Subsequently, the participants began rebreathing 100% oxygen via a closed circuit (Bloodtec, GbR, Germany) whilst carbon monoxide was added to the gas mixture (1.0 ml kg-1). Following two-minutes of rebreathing, a second venous blood draw was taken for the assessment of the same hematological parameters. Hematocrit was assessed via centrifugation and mirco-hematocrit reader. This technique has previously used successful at high altitude (Ryan et al., 2014), and proven to be reliable against gold standard labelling techniques (Siebenmann et al., 2017). Arterial cannulation and pharmacological infusion. After local anesthesia (2% lidocaine) a 4.45-cm, 20G catheter (Arrow, Markham, ON, Canada) was inserted under aseptic conditions into the 181 brachial artery of the non-dominant arm for local pharmacological infusions and measurement of mean arterial pressure (MAP). Pharmacology was infused using a standard infusion only syringe pump (PhD Ultra Syringe Pumps, Harvard Apparatus, Holliston, MA, USA). Cognitive function. Cognitive function was assessed using cognitive performance assessment software (Cogstate Ltd., Melbourne VIC, Australia). This standardized and automated computerized battery of neuropsychological tests (Cole et al., 2013), required participants to respond to playing cards, which assesses different aspects of cognitive function (e.g. attention, psychomotor performance, working memory). Male and female Andeans who were asked to complete these neuropsychological tests were provided the Spanish version after the test was thoroughly explained by one of our local translators. Microneurography. Muscle sympathetic nerve activity was obtained in either the radial or peroneal nerve (dependent upon the study) by inserting a Tungsten microelectrode into a muscle nerve fascicle of a sympathetic nerve bundle and a reference electrode subcutaneously 2–3 cm from the recording electrode. This technique was conducted by the same experienced microneurographers. Neural signals were collected using commercially available recording systems (662C-3, Bioengineering of University of Iowa, Iowa City, IA; Neuro AMP EX FE185, ADInstruments, Colorado Springs, CO, USA). Common electrical noise issues were encountered at both sea level and high altitude, and these issues were exaggerated when using the back-up generator as a power source. Linking the systems to an earth-ground (fence post in close proximity to the research laboratory) reduced electrical noise issues. One of the nerve traffic analyzers was part of the equipment locked in Peru customs upon arrival. This made running two 182 microneurography-based studies problematic at the high altitude laboratory. It was not until the arrival of the equipment of this equipment to the laboratory that both microneurography studies could run simultaneously as originally planned. The nerve signals were amplified (gain 70 000–160 000), band-pass filtered (700–2000 Hz), full-wave rectified, and integrated with a resistance-capacitance circuit (time constant 0.1 s). Criteria for adequate MSNA recording included: (1) pulse synchrony, (2) facilitation during the hypotensive phase of the Valsalva maneuver, and suppression during the hypertensive overshoot after release, (3) increases in response to breath holding, and (4) insensitivity to a gentle skin touch or a loud shout. Earphones (Audiotechnica ATH M40x) were used during microneurography searching to reduce distraction related to ambient noise. Exercise testing. Maximal cardiopulmonary exercise testing was performed in the semi-recumbent position on an electronically braked cycle ergometer (Corival Paediatric, Lode B.V., Groningen, Netherlands). The bicycle frame that the participant laid on was custom made in Cerro de Pasco using wood purchased from a local store, and was assembled in the laboratory using common power tools. The protocol was explained thoroughly by a local translator prior to the exercise test and participants were instructed to maintain a cadence between 70 and 80 rpm with verbal feedback given throughout the experiment. Following an initial rest period, the first stage of exercise was two-minutes in duration at 20 W with subsequent increments of 20 W every minute until the participant reached volitional exhaustion. Peak power output was calculated as the 20 W increment divided by 60, multiplied by the time into the last stage and added to the power output from the last completed stage. Respiratory measures were assessed via breath-by-breath online gas analysis (Oxycon Mobile, Carefusion, San Diego, CA, USA), heart rate was recorded using a heart 183 rate strap and transmitted to a Polar Electro RS4000 watch (Polar Electro, Kemple, Finland), and peripheral oxygen saturation measured via fingertip pulse oximetry (Choice Mmed, MD300C2, Beijing Choice Electric Technology Co Ltd, Beijing, China). Peak oxygen consumption (VO2peak) was calculated as the highest oxygen uptake (VO2) over a 30-second average. Sleep Monitoring. Sleep architecture was assessed using a wrist-worn ambulatory sleep system (WatchPat Central Plus, Itamar Medical, Israel). This system incorporates arterial pulsatile volume changes (via peripheral arterial tone signal) in the finger, pulse oximetry, and actigraphy, to algorithmically evaluate and score metrics of sleep disordered breathing (e.g. apnea-hypopnea index, oxygen desaturation index, rapid eye movements versus non-rapid eye movements stages of sleep; Yalamanchali et al., 2013). Questionnaires performed before sleep and upon awakening were also conducted to quantify sleep quality. Nitric oxide synthesis. Nitric oxide (NO) for clinical use was not available in Peru when these studies were conducted. To overcome this challenge, pure NO was produced in small quantities from the reaction of sodium nitrite (a common food additive) with hydrochloric acid (Arlin B. Blood, PhD, personal communication). This reaction produces small quantities of nitric oxide compounds (NOx) such as NO2 and N2O2 in addition to pure NO gas itself. To eliminate the other NOx compounds, the gas resulting from the initial reaction with acid was mixed with a solution of sodium hydroxide (NaOH), resulting in pure NO. All the reactions were performed in an anaerobic environment with the use of 50 ml syringes, initially flushed with nitrogen. The pure NO immediately diluted with nitrogen to reduce the concentration of NO to a few hundred parts per million (ppm). Finally, this diluted NO/N2 was mixed with oxygen immediately before it was used, 184 to produce a mixture of ~21% oxygen, 79% N2, and 40 ppm NO. After verification of the gas concentrations with analyzers for oxygen and NO, Subjects inhaled this mixture from a Douglas bag for studies #4 and #8 outlined in Table 1. Blood analysis and transport. Approximately 4000 samples were transported back to the laboratory in Lima for analysis and international shipment. Whilst ~200 samples remained in Lima for the measurement of serum concentrations of iron (trans-ferritin, ferritin, iron, etc) and plasma erythropoietin (MedLab, Lima, Peru), the majority of biological samples were immediately shipped on dry ice (-78.5°C; Marken Ltd; temperature verified) to the United Kingdom, Canada, and United States for subsequent analysis. These analyses included the following measurements of systemic endothelial microparticles [including endothelial activation (i.e., CD62e+) and apoptosis (i.e., CD31+/42b-)] and oxidative-inflammatory-nitrosative-structural (OXINOS) stress that have previously been described in detail elsewhere (Bailey et al., 2017; Bain et al., 2017; Tremblay et al., 2017; Bailey et al., 2018) 1. Oxidative Stress: Samples will be analyzed for the ascorbate free radical directly using X-band electron paramagnetic resonance spectroscopy. Serum lipid hydroperoxides will be assayed spectrophotometrically as complementary biomarkers of lipid peroxidation. Plasma ascorbic acid and lipid soluble antioxidants will be assayed by fluorimetry and high-performance liquid chromatography. 2. Inflammatory Stress: high-sensitivity (hs) C-reactive protein and tumor necrosis factor-α will be assayed by hs enzyme-linked immunosorbent assay (ELISA). 185 3. Nitrosative Stress: Plasma and red blood cell concentrations of nitrite, S-nitrosothiols and S-nitrosoHb will be determined by ozone-based chemiluminescence. Plasma 3-nitrotyrosine, a biomarker reflecting the oxidative inactivation of NO, will be measured by hs-ELISA. 4. Structural stress: S100β, neuron-specific enolase, myelin basic protein, neurofilament light, ubiquitin carboxy-terminal hydrolase-L and glial fibrillary acidic protein (biomarkers of blood-brain barrier integrity, neuronal-parenchymal damage and glial damage respectively) will be measured by hs-ELISA. Data Analysis Sample size estimates. sizes were determined a priori based on study specific effect size estimates. Moreover, based on our research team’s previous experience with high altitude expeditions, adjustments were made to account for expected participant dropout (e.g. Foster et al., 2014; Lewis et al., 2014; Smith et al., 2014; Smirl et al., 2014; Willie et al., 2014; Tymko et al., 2015; Stembridge et al., 2016; Tremblay et al., 2016; Tymko et al., 2017; Tremblay et al., 2018; Busch et al., 2018;). Statistical power calculations where a power of 0.8 was assumed, and an alpha value of 0.05 was set (G*power). Depending on the variability of the primary outcome of each study, 10-58 study participants were recruited. Statistics. Statistical analysis included in the current manuscript was performed using SigmaStat V13 (Systat, Chicago, IL, USA), and all data are reported as mean ± SD. Statistical significance 186 was set at P<0.05. For statistical comparisons between lowlander sea level and high altitude data, paired (e.g. table 4) and independent (e.g. table 5) t-tests were used. For statistical comparisons between lowlanders at altitude and Andean highlanders (males and females) a one-way analysis of variance design was used, and when significant F-ratios were detected, differences between means were determined using Bonferonni-corrected independent samples t-tests. 187 General Results and Characterization of Cohorts Studies conducted and participants recruited. In total, 15 separate a priori studies (see table 1), amounting to >780 study sessions were completed between sea level testing in Kelowna, BC, Canada (344m), and over ~25 days in Cerro de Pasco (4300m). This amounted to 829 experimental hours in lowlanders at sea level, 1522 experimental hours in lowlanders at high altitude, 500 experimental hours in highlanders without CMS, and 219.75 experimental hours in highlanders with CMS (refer to table 2). Table 3 highlights the participant demographics (age, height, weight and body mass index) for lowlanders (n=30), and highlanders with (n=22; all male) and without (n=45; 11 female) CMS. Arterial blood data. Table 4 displays arterial blood data obtained in 13 male lowlanders at sea level (Lima, Peru) immediately prior to ascending to Cerro de Pasco, and after 14 days of high altitude exposure. In addition, table 4 illustrates baseline arterial blood data in 16 male Andean participants with and without CMS. Compared to sea-level, lowlanders after 14 days of high altitude acclimatization had higher pH, but lower PaCO2, PaO2, SaO2 and HCO3- (all P<0.001). Lowlanders after 14 days of high altitude exposure had an elevated arterial blood pH and SaO2 compared to Andean CMS+ participants (both P<0.001), however, lowlanders on Day 14 at altitude had lower PaCO2 and HCO3- compared to Andean CMS+ participants (both P<0.001). Lowlanders at high altitude had the same arterial blood pH (P=0.116), PaCO2 (P=1.00), PaO2 (P=1.00), HCO3- (P=1.00), and SaO2 (P=1.00) compared to male Andean CMS- participants. Compared to male Andean CMS- participants, CMS+ participants had a lower arterial blood pH (P=0.011) and SaO2 (P<0.001), but an elevated PaCO2, and HCO3- (both P<0.001). There were no differences in PaO2 observed between male Andean CMS- and CMS+ participants (P=0.137). 188 Venous blood data. Table 5 displays venous blood data obtained in male lowlanders at sea-level (n=24; Kelowna, BC), and after 14 days of high altitude exposure (n=17). In addition, table 5 illustrates baseline venous blood data in male Andean participants with CMS (n=21), and males and females without CMS (n=26 and n=11, respectively). After 14 days of high altitude acclimatization, lowlanders had an increase in hemoglobin and blood viscosity compared to sea level values (both P<0.001). Lowlanders at altitude had lower hemoglobin and blood viscosity compared to male Andean CMS+ (both P<0.001), and CMS- participants (both P<0.001). Male Andean CMS+ participants had higher hemoglobin and blood viscosity compared to CMS- (both P<0.001). Notably, four participants’ (all CMS+; Hb of these participants = 24.7±1.1 g dL-1) blood viscosity exceeded the upper limit of the viscometer (>10.22 cP), thus, their blood viscosity was reported as 10.22 cP. Female CMS- Andeans had lower hemoglobin and blood viscosity compared to male CMS- Andeans (P=0.007 and P=0.004). Exercise performance. Figure 2 displays exercise performance data between lowlanders at sea level, lowlanders at high altitude, Andean male participants with and without CMS, and Andean female participants. At high altitude, lowlanders had a lower relative VO2 max (P=0.001), and peak power output (P<0.001), compared to sea level values. Lowlanders at high altitude had an elevated peak power output compared to male CMS+ (P<0.001) and CMS- Andeans (P<0.001), and female CMS- Andeans (P<0.001). Relative VO2 max was higher in lowlanders at high altitude compared to female CMS- Andean participants (P=0.032), but not male Andean participants with (P=0.619) and without CMS (P=0.492). No differences in relative VO2 max, and peak wattage were observed between male Andean CMS+ and CMS- participants (both P=1.00). Female CMS- 189 Andeans had a similar relative VO2 max (P=1.00), and a lower peak wattage compared to male CMS- Andeans (P=0.015). Comparison of blood data between lowlanders and Andeans at 4300m, to Sherpa at 5050m. Table 6 displays arterial blood data obtained in 13 male lowlanders after 14 days at 4300m, 16 male Andean participants with and without CMS at 4300m, 11 female Andean participants at 4300m, and 11 healthy male Sherpa participants at 5050m (data from our Nepal 2016 expedition). The male Sherpa were 23.7 ± 7 yrs old, with a BMI of 21± 3 kg m2. Lowlanders at 4300m had an elevated blood pH compared to Andeans with CMS and Sherpa at 5050m (both P<0.001). Andeans who were CMS- had elevated blood pH compared to the Sherpa (P=0.013). Males with CMS had an elevated PaCO2 compare to lowlanders and male Andeans without CMS (both P<0.001). As expected, Andeans with CMS and Sherpa at 5050m had a lower PaO2 compared to lowlanders (P=0.045 and P=0.004, respectively). Male Andean with CMS had an elevated HCO3- compared to lowlanders (P=0.034), CMS- Andeans (P<0.001), and Sherpa (P=0.006). Lowlanders and CMS- Andeans had elevated SaO2 compared to male CMS+ Andeans (Both P<0.001, respectively) and Sherpa (both P<0.001, respectively). Venous blood hemoglobin was elevated in Male CMS+ compared to lowlanders, CMS- Andeans, female Andeans, and Sherpa (all P<0.001). Additionally, CMS- Andeans had an elevated hemoglobin compared to Sherpa and female Andeans (P<0.001 and P=0.012, respectively). Blood viscosity in Andean CMS+ and CMS- participants was elevated compared to lowlanders (both P<0.001), female Andeans (P<0.001 and P=0.005, respectively), and Sherpa (both P<0.001). Also, Andean CMS+ had an elevated blood viscosity compared to Andean CMS- participants (P<0.001). As shown in Figure 3, an H-bicarbonate (i.e. Davenport) diagram was used to demonstrate the various acid–base compensation and comparisons in a) 190 lowlanders, Andeans with and without CMS at 4300m and b) in Sherpa at 5050m (Willie et al., 2018) 191 Discussion Despite some of the major obstacles encountered during the expedition outlined throughout this manuscript, our research team, Global REACH, collected ~90% of the data for the proposed projects. Below we outline some of the lessons learnt from this expedition, and discuss in further detail the forthcoming publications from this expedition, comparative physiological data between our participating groups, a comparison to our latest Nepal expedition in 2016, and clinical translation. Finally, a brief discussion of the future goals of Global REACH is provided. Major challenges. The success of large-scale field research expeditions typically depends on establishing appropriate and adequate local support. Through a historical connection, our research team was capable of organizing contact with local Peru collaborators: Dr. Francisco Villafuerte and his laboratory team consisting of graduate students and administrators. Six months prior to the research expedition, two members of Global REACH traveled to Cerro de Pasco to collect some preliminary data and construct initial planning for our core studies with our local collaborators. Without the local Peruvian team and their unfailing help and support, several aspects of the expedition would not have been possible including: 1) recruitment of Andean highlanders using their established database, 2) organizing transport to the high altitude laboratory in Cerro de Pasco, and 3) local ethical approval for all proposed studies. Our team had full-time access to multiple graduate students of Dr. Villafuerte who handled nearly all recruitment and screening of participants and offered their skills of being a translator between the study principal investigators and Andean study participants. 192 During our Nepal expedition in 2016, our research team collected data upon ascent to high altitude (5050m) without the confounding influence of participants taking prophylactic acetazolamide. In order for our data to remain consistent between our Nepal and Peru high altitude expeditions, we aimed to refrain from taking any high altitude medications. However, if participants fell severely ill from altitude and/or non-altitude related sickness, our physicians had adequate medication available for treatment. Unlike our expedition in Nepal, which involved a gradual ascent to 5050m over a period of ~7-10 days, we were transported to altitude by automobile over a 7-8 hour rapid ascent to Cerro de Pasco. Due to this, nearly all participants reported symptoms of acute mountain sickness (via Lake Louise scoring system), with several having to be treated with Diamox after arrival (n=6). No participants had to be evacuated due to altitude related illness; however, these individuals were either removed from specific studies due to illness or were rescheduled to participate once they were fully recovered and endured a complete drug washout time (i.e. at least five half-lives). Lastly, the biggest challenge of this expedition was the logistical aspect of transportation and importation of research equipment. Unfortunately, despite a substantial effort to organize and submit importation documents for legal entry into Peru, a large portion of our equipment was initially rejected entry into Peru. Rectifying this issue required countless hours and financial assurances from the local university to release this equipment. This unforeseen complication resulted in the delay of multiple research projects; however, through equipment sharing and working excessive hours, our research team was able to cope with the temporary equipment loss until it arrived ~14 days after arrival to Cerro de Pasco. For these reasons, we recommend future 193 expeditions to bring back-up and duplicate equipment whenever possible in case of any equipment issues related to foreign country importation. Forthcoming Publications. In addition to an enormous effort from all team members, successful large-scale research expeditions require logistical planning ~1-2 years in advance and adequate funding ($150-200K USD, excluding individual salaries). Due to this extensive time commitment, it is of the utmost importance to maximize research output in these unique environments. As such, high altitude field studies often result in multiple publications with overlapping data (e.g. participant demographics, blood analysis, acute mountain sickness and CMS scores); however, this is justifiable and encouraged given the complexity of this global undertaking underpinned by stand-alone investigations defined by separate working hypotheses (see table 1). The research expedition by Global REACH to Peru is expected to submit ~15-20 publications to both clinical and physiology based journals. Exercise performance. Similar to previous reports, our data indicate that exercise performance (both relative VO2 max and peak wattage) is reduced with high altitude exposure in lowlanders (Cymerman et al., 1989; Wehrlin and Hallen, 2006; Smith et al., 2014; Tymko et al., 2015; Tremblay et al., 2016). Our exercise data in male Andean highlanders supports previously published data that indicate that CMS+ participants have a similar VO2 max and peak wattage compared to CMS- participants despite substantially elevated Hb and reduced SaO2 (Groepenhoff et al., 2012; Swenson, 2012). 194 Arterial blood gases and acid-base balance. When a lowlander travels to high altitude, hyperventilation occurs as a result from hypoxemia associated peripheral chemoreceptor stimulation, the PaCO2 falls, and the arterial blood pH rises in accordance with the Henderson–Hasselbach equation: Equation 1: Henderson-Hasselbach equation where [HCO3−] is the bicarbonate concentration in millimoles per liter and the PaCO2 is in mmHg. However, the kidney responds by eliminating bicarbonate, which is prompted via reductions in PCO2 in the renal tubular cells. This results in a more alkaline urine due to decreased reabsorption of bicarbonate ions. The decrease in plasma bicarbonate then moves the bicarbonate/PaCO2 ratio back towards its normal equilibrium. This relationship is known as metabolic compensation for respiratory alkalosis. The compensation may be complete, in which case the arterial pH returns to ~7.40 or, more often, incomplete with a steady-state pH that exceeds ~7.40. There are two noteworthy and novel observations from the arterial blood gas data (Table 4). First, two-weeks of acclimatization at 4300m in lowlanders resulted in comparable changes in SaO2, PaO2, PaCO2, and HCO3- when compared to healthy Andean highlanders who have resided at this elevation for many years. In contrast, despite the comparable changes in PaCO2 (and hence likely stimulus to reduce HCO3-), pH remained elevated in lowlanders. Thus, despite comparable oxygenation and metabolic compensation in the Andeans – as indexed via HCO3- – it failed to compensate for the respiratory alkalosis (i.e., elevated pH; refer to figure 6). It is possible that these results could be 195 directly due to differences in strong base ions since differences in standard base excess can alter blood pH for a given PaCO2 (Morgan, 2009). Unfortunately, we did not acquire standard base ion in lowlanders at this time-point since these blood samples were analyzed using iSTAT cartridges that did not output these data. Also of note was that the CMS- female highlanders had a lower blood Hb and viscosity compared to CMS- male highlanders. These results align with previous findings demonstrating that premenopausal female highlanders have lower incidences of CMS, which may be related to elevated levels of progesterone during their menstrual cycle resulting in elevated ventilation, thus SaO2 – a primary stimulus for red blood cell production (Leon-Velarde et al., 1997; Leon-Velarde et al., 2001). Albeit over a shorter period, the time course of the change in arterial pH when normal subjects ascend abruptly to high altitude has been studied by several investigators (Severinghaus et al., 1963; Lenfant et al., 1971; Dempsey et al., 1978). In one study, lowlanders were taken within five hours from sea level to an altitude of 4509m (PB = 446 mmHg) and remained there for four days. The arterial pH rose to a mean of about 7.47 within 24-hours and then gradually declined but was still ~7.45 at the end of the four-day period (Lenfant et al., 1971). In another study, four normal subjects were taken abruptly to 3800m for eight days. The arterial pH rapidly rose from a mean of 7.424 at sea level to 7.485 after two days, and remained constant, being 7.484 at the end of eight days (Severinghaus et al., 1963). In a further study, 11 lowlanders traveled to 3200m altitude where they remained for 10 days (Dempsey et al., 1978). The arterial pH rose by 0.03–0.04 units within two days, and then remained essentially unchanged. In all instances, the PaCO2 continued to decline as did the plasma bicarbonate concentration. Consistent with the current data, it appears that the return of the arterial pH to (or near to) its sea level value is very slow and may not occur 196 even after years of exposure to high altitude (as confirmed by the elevated pH observed in healthy Andeans). Another interesting observation consistent with previous reports is the relative hypoventilation in the CMS+ Andeans, as reflected in the arterial hypoxemia and attenuated reductions in PaCO2 compared to the healthy Andean and lowlander groups. The mechanism(s) driving this relative hypoventilation has been explained in detail elsewhere (Villafuerte and Corante, 2016). Nevertheless, it is interesting to note that despite this greater hypoxemia and attenuated reductions in PaCO2, the greater reductions in HCO3- resulted in full metabolic compensation for the respiratory alkalosis i.e., comparable pH in CMS+ to lowlanders at sea level. Lastly, we compared our data obtained in lowlanders and Andean highlanders at 4300m to Sherpa at 5050m from our Nepal 2016 expedition (refer to table 6 and figure 3). Interestingly, despite being at a higher altitude compared to the lowlanders and Andeans, the Sherpa cohort had the lowest hemoglobin and blood viscosity values. Sherpa also had a similar blood pH compared to male Andeans with CMS, but lower compared to healthy Andeans without CMS. Furthermore, as illustrated in the Davenport diagram (Figure 3), when compared to the lowlanders at sea level, the Sherpa had metabolic compensation for the marked respiratory alkalosis. This is a remarkable observation giving the even higher altitude (5050m) of the Sherpa compared to male Andeans with and without CMS, and lowlanders at altitude (4300m). Such data further highlight another pathway of effective adaptation in the Sherpa who are well-known for their exceptional tolerance to hypoxia and outstanding performance at extreme altitude on the highest Himalayan peaks (Hochachka et al., 2002; Gilbert-Kawaii et al., 2014). 197 Comparison to Nepal 2016 expedition. Although the fundamental goals of our international research teams expeditions to Nepal and Peru were similar, there are distinct differences between these two expeditions that warrant further comment. First, there were a large number of “ascent” studies conducted in both lowlanders and Sherpa over a gradual ascent to a slightly higher altitude (5050m vs 4300m). However, we tested a large cohort of Sherpa at 5050m that did not descend to lower altitude in a number of studies. Another consideration is the difference in lifestyles and environment between Nepal and Peru. Our Sherpa cohort, by nature, were likely to be more physically active compared to the Andeans, having to walk long distances on a daily basis and serving as guides in nearby mountains. Nevertheless, previous reports indicate similar rates of obesity between Sherpa and Andean highlanders (~10% vs ~8%, respectively; Sherpa et al., 2010; Woolcott et al., 2016). It is also worth acknowledging that our Andean highlanders had access to a greater variety of food in Cerro de Pasco compared to the Sherpa in the Khumbu valley. Clinical Translation. Research on high altitude physiology offers complementary insight into biological adaptation to hypoxia. Ways to apply results from these expeditions are multifaceted, with implications for military deployment to high altitude (e.g. Afghanistan), for the growing numbers (>1 million) of lowlanders vacationing at high altitude destinations, and for commercial flight personnel who are hypoxic during flight (mild hypoxia). These data can have direct translational impact for patients in critical care and in other clinical situations of hypoxia (e.g. lung disease, heart failure, circulatory shock), and importantly, it will give us novel insight into the current cardiovascular health status of local highlanders (Tibetan, Peruvian, and Ethiopian), which will directly benefit these populations. 198 An obvious clinical aspect to the current project was that the manifestations of CMS that mirror the complications in other diseases associated with chronic hypoxic, such as chronic obstructive pulmonary disease and heart failure. The results from this study will be a valuable step toward effective treatments for the cardiopulmonary consequences of CMS, and beyond to more effective treatments for other hypoxic disorders that affect individuals who live at lower altitudes. In particular, understanding the pathophysiological mechanism of pulmonary hypertension will benefit the thousands of diagnoses with the disorder worldwide each year, along with providing information on how to improve treatment avenues for the high altitude Peruvian dwellers that suffer from CMS. The future of Global REACH. Due to the numerous publications, the local cultural benefit (e.g. CMS research), the international scientific relationships gained (e.g. Dr. Villafuerte), and the opportunity for training highly qualified personnel with each expedition (e.g. graduate and medical student training), our research team, Global REACH, intends to organize future large-scale high altitude research expeditions. We know significantly less about adaptations of Ethiopian Amhara and Oromo highlanders from the Simien and Bale mountains, respectively. The Amhara have hemoglobin concentrations similar to lowlanders, yet higher than expected oxygen saturation, potentially due to an increased affinity of their hemoglobin for oxygen (Beall et al., 2002; Beall, 2006). Only few sophisticated physiological data sets exist on Ethiopian highlanders. Conceivably, the Sherpa, Quechua, and Amhara phenotypes represent distinct evolution-driven strategies that permit survival and performance at high altitude. Ethiopia will be the final installment in Global REACH’s trilogy of high altitude adaptation studies. 199 Conclusion The 2018 Global REACH expedition to Peru was comprised of 15 independent studies on three distinct cohorts: lowlanders (n=30), healthy Andeans (n=46), and Andeans diagnosed with CMS (n=23). Studies were conducted at sea level (Kelowna, BC; 344mm) and after ascending to Cerro de Pasco (4300m) from Lima (~150m) over a 7-8 hour automobile ride, which focused on cardiovascular, cerebrovascular, cardiopulmonary, autonomic and neurocognitive aspects of human physiological responses to hypobaric hypoxia acclimatization. The findings from this study will be reported in several forthcoming publications according to their respective a priori hypotheses. 200 Acknowledgements: The success of this expedition is directly attributed to the team members impressive problem solving abilities and resiliency. We would like to give thanks to our local Peru collaborators for their support in the weeks leading up to, and during data collection in Peru. The authors are grateful to all other members and participants of the 2018 Global REACH expedition to Peru: Tyler Vermeulen, Alexander Hansen, Courtney Clark, Andrew Steele, Florian Hofstatter, Simon Rainer, Joseph Donnelly, Lisa Wong, Victoria Meah, Kaitlyn Tymko, Rachel Stone, Lydia Simpson, Christopher Hearon, Sachin Amin, Suman Thapamagar, Gilberto Moralez, Aimee Drane, John Atkins, Alex Williams, Antoinette Santoro, Daniela Bermudez Pumasunco, and Romulo Joseph Figueroa Mujica. Conflict of Interest: We have no conflict of interests to declare Author contributions: M.M.T. and P.N.A were responsible for the concept of the manuscript. All authors contributed to the analysis, interpretation of the data, along with drafting the article or critically revising it for important intellectual content. All authors approved the final version of the manuscript and all person designated as authors qualify for authorship, and all those who qualify for authorship are listed. 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Science 362, 1049-1051, DOI: 10.1126/science.aat8824. 208 Table 1 Overview of experimental studies Study Study Title Study Aim Participants Techniques used 1 The common carotid artery vasomotor response to the cold pressor test at sea level and high altitude in lowlanders and Andeans: the role of oxygen To determine the effects of altitude on the common carotid artery vasomotor response to the cold pressor test at sea level and high altitude Lowlanders (N =14) CMS- (N = 12) Duplex ultrasound; finger photoplethysmography 2 Nitric oxide-mediated endothelium-dependent vasodilation in lowlanders at sea level and high altitude To determine the influence of oxidative stress on endothelial dependent vasodilatory function in lowlanders with chronic exposure to high altitude. Lowlanders (N =11) Venous occlusion plethysmography with intra-brachial infusions of acetylcholine and sodium nitroprusside 3 Investigating the role of haemoglobin concentration, plasma volume and absolute blood volume on cardiac function and exercise capacity in high altitude natives To explore whether hemoglobin mass or absolute blood volume is associated with exercise performance in the Andean population, and whether differences in performance are related to cardiac structure and function Lowlanders (N =12) CMS- (N = 42) CO rebreathing; venous blood sampling; Duplex ultrasound; maximal exercise test 4 Sympathetic function in lowlanders and high altitude Andean’s with and without chronic mountain sickness To investigate sympathetic nervous activity in Peruvian highlanders and identify a specific link between sympathetic hyperactivity and elevated pulmonary arterial pressure Lowlanders (N =18) CMS- (N = 10) CMS+ (N=10) Microneurography to assess muscle sympathetic nervous activity; Duplex ultrasound; finger photoplethysmography 5 The effects of oxidative stress on cutaneous vasodilation at sea level and high altitude To determine the role of oxidative stress on cutaneous vascular function at sea level and high altitude Lowlanders (N =11) CMS- (N = 11) Microdialysis; laser-Doppler flowmetry 6 The effects of venesection on peripheral and central To quantify the influence of reductions in hematocrit (via blood removal) on CMS+ (N=10) Duplex ultrasound; arterial/venous blood 209 vascular function in Andeans with chronic mountain sickness central and peripheral vascular function in Andean highlanders suffering from elevated hematocrit and CMS sampling; finger photoplethysmography 7 The factors effecting resting and active (exercising) skeletal muscle blood flow through the process of acclimatization, adaptation and maladaptation to high altitude To provide a integrative assessment of the factors influencing skeletal muscle blood flow during adaptation and maladaptation to high altitude Lowlanders (N =11) CMS- (N = 10) CMS+ (N = 8) Exercise tests; Microneurography to assess muscle sympathetic nervous activity; Brachial catheterization; Duplex ultrasound; local arterial pharmacological infusions 8 Pulmonary vascular changes to acute and chronic high altitude hypoxia To study pulmonary vascular responses at rest and during exercise, at both low and high altitude in lowlanders and in Andeans with and without CMS Lowlanders (N =11) CMS- (N = 13) CMS+ (N = 9) Duplex ultrasound 9 Endothelial function and shear stress in high altitude Andeans with and without chronic mountain sickness To characterize resting shear stress patterns and assess endothelial function via flow-mediated dilation in response to transient and sustained elevations in shear stress CMS- (N = 33) CMS+ (N = 20) Duplex ultrasound; handgrip exercise 10 The global cerebral blood flow and intracranial pressure response to hypobaric hypoxia in high-altitude Andeans with and without chronic mountain sickness To determine the effects of high altitude on cerebral blood flow and intracranial pressure in high-altitude Andeans with and without CMS CMS- (N = 33) CMS+ (N = 20) Duplex ultrasound 11 The effects of iron supplementation on vascular function between lowlanders and high altitude Andeans To determine the effect of iron sucrose intravenous supplementation on peripheral vascular function between healthy lowlanders and Andean highlanders Lowlanders (N = 24) CMS- (N = 24) Duplex ultrasound; venous blood sampling; 210 12 The effect of a positive expiratory pressure mask with dead space on sleep, acute mountain sickness and cognitive function during normobaric and hypobaric hypoxia To assess the combined effect of positive expiratory pressure and dead space on sleep, acute mountain sickness and cognitive function during normobaric and hypobaric hypoxia. Lowlanders (N = 15) Sleep monitoring 13 Redox-regulation of cerebrovascular function during acute exposure to environmental hypoxia To examine to what extent free radical-mediated alterations in nitric oxide bioavailability contribute towards systemic vascular impairment following acute exposure to environmental hypoxia and to what extent this translates to the cerebrovasculature and corresponding implications for cognition. Lowlanders (N = 12) CMS- (N = 18) Duplex ultrasound; Transcranial Doppler ultrasound; finger photo-plethsmography; venous blood sampling; electron paramagnetic resonance spectroscopy; ozone chemiluminescence; hs-ELISA; fluorimetry; HPLC; neurovascular coupling; cognition 14 The effect of high altitude exposure on the regulation of cerebral blood flow during heat and cold stress To understand how high-altitude acclimatization impacts the regulation of cerebral blood flow during heat and cold challenges. Lowlanders (N =11) Duplex ultrasound; esophageal and rectal temperature monitoring; arterial blood sampling; 15 Renal reactivity at high altitude To determine the effects of acute and chronic altitude exposure on kidney function in lowlanders and Andean highlanders Lowlanders (N = 30) CMS- (N = 17) CMS+ (N = 11) Duplex ultrasound; urine sampling; Definition of abbreviations: CMS, chronic mountain sickness. 211 Table 2 Experimental hours for each study LL at sea level LL at high altitude Andeans CMS - Andean CMS+ Study #1 14 13 12 0 Study #2 30 27 0 0 Study #3 6 10 42 0 Study #4 0 54 30 30 Study #5 80 44 44 0 Study #6 0 0 0 70 Study #7 88 80 80 64 Study #8 22 44 26 18 Study #9 0 0 49.5 30 Study #10 0 0 8.25 5 Study #11 0 154 168 0 Study #12 456 999 0 0 Study #13 48 18 36 0 Study #14 77 66 0 0 Study #15 8 13 4.25 2.75 Total 829 1522 500 219.75 Definition of abbreviations: CMS, chronic mountain sickness. LL, lowlanders. 212 Table 3 Participant Demographics Lowlanders Males CMS(-) Males CMS(+) Females CMS(-) N 30 (3F) 35 22 10 Age (yrs) 29.9 ± 7.8 30.2 ± 11.2 44.1 ± 12.8 25.5 ± 3.1 Stature (cm) 175.3 ± 5.8 162.6 ± 4.4 159.8 ± 5.8 152.1 ± 5.0 Mass (Kg) 72.5 ± 7.9 63.8 ± 8.1 66.7 ± 9.9 53.6 ± 4.1 BMI (Kg/m2) 23.5 ± 1.6 24.2 ± 3.2 26.1 ± 3.3 23.2 ± 2.1 Definition of abbreviations: yrs, years; cm, centimeter; kg, kilogram; m, meter; CMS, chronic mountain sickness. 213 Table 4 Arterial Blood Data Male LL sea level Male LL Day 14 Male CMS+ Male CMS- N 13 13 16 16 pH 7.42 ± 0.02 7.47 ± 0.03* 7.42 ± 0.02† 7.45 ± 0.03†‡ PaCO2 (mmHg) 40.8 ± 2.1 28.6 ± 1.5* 35.0 ± 3.2† 29.2 ± 3.6‡ PaO2 (mmHg) 98.7 ± 7.4 50.2 ± 5.6* 44.2 ± 3.7 49.42 ± 7.1 HCO3 (mmol/l) 26.2 ± 1.5 20.7 ± 1.3* 22.5 ± 1.5† 20.0 ± 2.0‡ SaO2 (%) 97.8 ± 0.7 87.8 ± 2.6* 79.0 ± 4.7† 86.4 ± 4.0‡ Definition of abbreviations: mmHg, millimeters of mercury, mmol, millimoles; l, liters; CMS, chronic mountain sickness. LL, low-landers. *P<0.05, low-landers sea level vs low-landers Day 14. †P<0.05, vs low-landers Day 14. ‡CMS+ vs CMS- 214 Table 5 Venous Blood Male LL sea level Male LL Day 14 Male CMS+ Male CMS- Females CMS- N 24 17 21 26 11 Hb (g/dl) 14.9 ± 1.0 18.0 ± 1.1* 22.6 ± 1.7†‡ 18.9 ± 1.7† 16.8 ± 1.3┼ Blood viscosity (cP) 4.3 ± 0.7 4.9 ± 0.6* 8.5 ± 1.2†‡ 6.0 ± 0.8† 4.9 ± 0.7┼ Definition of abbreviations: g, grams; l, liters; CMS, chronic mountain sickness. LL, low-landers. *P<0.05, low-landers sea level vs low-landers Day 14. †P<0.05, vs low-landers Day 14. ‡P<0.05, male CMS+ vs male CMS-. ┼P<0.05, male CMS- vs female CMS-. 215 Table 6 Blood data between lowlanders and Andeans at 4300m, and Sherpa at 5050m Male LL Day 14 (4300m) Male Andean CMS+ (4300m) Male Andean CMS- (4300m) Female Andean CMS- (4300m) Male Sherpa (5050m) N 13 16 16 11 11 Blood pH 7.47 ± 0.03 7.42 ± 0.02* 7.45 ± 0.03‡† - 7.41 ± 0.02* PaCO2 (mmHg) 28.6 ± 1.5 35.0 ± 3.2* 29.2 ± 3.6† - 31.9 ± 2.4 PaO2 (mmHg) 50.2 ± 5.6 44.2 ± 3.7*‡ 49.42 ± 7.1 - 41.7 ± 6.2* HCO3- (mmol/l) 20.7 ± 1.3 22.5 ± 1.5†‡* 20.0 ± 2.0 - 20.3 ± 1.6 SaO2 (%) 87.8 ± 2.6 79.0 ± 4.7* 86.4 ± 4.0†‡ - 77.4 ± 6.4* Hb (g/dl) 18.0 ± 1.1 22.6 ± 1.7*†‡┼ 18.9 ± 1.7‡┼ 16.8 ± 1.3 15.9 ± 1.0 Blood viscosity (cP) 4.9 ± 0.6 8.5 ± 1.2*†‡┼ 6.0 ± 0.8*‡┼ 4.9 ± 0.7 4.6 ± 0.2 Definition of abbreviations: mmHg, millimeters of mercury, mmol, millimoles; l, liters; g, grams; l, liters; CMS, chronic mountain sickness. LL, low-landers. *P<0.05, vs lowlanders Day 14. †P<0.05, CMS- vs CMS+. ‡P<0.05 vs Sherpa. ┼P<0.05, vs female Andeans 216 Figure 1 A timeline of Global REACH’s latest research expedition to Peru, and a schematic of the laboratory facility used in Cerro de Pasco. 217 SL LL HA LL M CMS+ M CMS- F CMS-Relative VO2 max (ml/kg/min)10203040506070Peak Watts50100150200250300350400450A.B. Figure 2 Exercise performance between male lowlanders and Andean highlanders. Panel A, individual data for relative VO2 max (ml/kg/min). Panel B, individual data for peak wattage obtained during the exercise test. Definition of abbreviations: CMS, chronic mountain sickness; F, female; HA, high altitude; kg, kilograms; LL, lowlanders; M, male; min, minutes; ml, milliliters; SL, sea level. Brackets represent differences between data sets (P<0.05). Horizontal solid line represents mean value. 218 Figure 3 Davenport diagram illustrating acid-base balance between male lowlanders at sea level (●), male lowlanders after 14 days at 4300m (♦), Andean highlanders diagnosed with CMS at 4300m (▲), Andean highlanders without CMS at 4300m (■), and Sherpa highlanders at 5050m (▬) Definition of abbreviations: CMS, chronic mountain sickness; HCO3-, bicarbonate; PaCO2, partial pressure of arterial carbon dioxide. 219 Appendix B How to build a lower-body differential pressure chamber integrated on a tilt-table: a pedagogy tool to demonstrate the cardiovagal baroreflex Tymko, M.M. (2016). How to build a lower-body differential pressure chamber integrated on a tilt-table: a pedagogy tool to demonstrate the cardiovagal baroreflex. Facets. 1: 225-244. ABSTRACT The cardiovagal baroreflex is an important physiological reflex that is commonly taught in health-related university physiology courses. This reflex is responsible for the rapid maintenance of blood pressure through dynamic changes in heart rate and vascular resistance. The use of lower-body negative (LBNP) and positive (LBPP) pressure can manipulate these stretch sensitive baroreceptors. High performance, and relatively inexpensive homemade LBNP and LBPP chambers can be easily constructed providing a valuable tool for both research and teaching purposes. There have been previous documentations on how to build a LBNP chamber; however, the information available usually lacks appropriate construction details, and there is currently no literature on how to build a chamber that can accommodate both LBNP and LBPP. In addition, a recently developed novel LBNP/LBPP chamber situated on a 360 degree tilt-table provided the unique utility of superimposing both LBNP/LBPP and body position as independent or combined stressors to alter central blood volume. The primary purposes of this manuscript are to 1) provide step-by-step instructions on how to build a tilt-table LBNP/LBPP chamber, and 2) demonstrate the effectiveness of a tilt-table LBNP/LBPP chamber to facilitate undergraduate and graduate learning in the laboratory via effectively demonstrating the cardiovagal baroreflex. 220 Key Words: Lower-body negative pressure, Lower-body positive pressure, Cardiovagal baroreflex INTRODUCTION The cardiovagal baroreflex is a bi-directional, pressure/stretch-sensitive, negative feedback response to changes in mean arterial pressure (MAP) mediated by the autonomic nervous system (Mancia et al., 1984, Fadel and Raven, 2012). This reflex is critical for moment-to-moment regulation of heart rate (HR) and vascular resistance in humans, and is often a fundamental concept taught in physiology and medical-related courses. The additional value of laboratory demonstrations of physiological functions are well-known (Berg et al., 2012, O'Donaughy et al., 2002), as they are critical for student learning and development in the physiological sciences. There are several well established methods that can be used to demonstrate baroreflex responses in a laboratory setting in humans, including: 1) sitting and standing protocols (Zhang et al., 2009), 2) steady-state changes in body position (i.e. head-down tilt; (Tymko et al., 2015)), 3) thigh cuff release (Ogoh et al., 2010), 4) neck suction (Ebert et al., 1984, Eckberg et al., 1975), 5) the modified Oxford technique (Ebert and Cowley, 1992), and 6) lower body negative pressure (LBNP; (Stevens and Lamb, 1965)). From a teaching perspective, sustained or progressive LBNP is a reliable method to elicit a cardiovagal baroreflex response, as it is non-invasive, reproducible (Howden et al., 2001), easy to operate, and the pressure stimulus can be easily adjusted and controlled on an individual basis. Lower-body negative pressure experimentation requires the participant to lie in a vacuum sealed chamber, and with the use of suction provided by a vacuum source, the pressure the LBNP chamber decreases relative to atmospheric pressure. Exposure to 221 negative pressure results in blood translocating from the upper body and into the lower extremities, causing a reduced venous return and MAP which changes the afferent nervous outflow from baroreceptors. The baroreflex involves a cardiac and vascular arm, with responses mediated by the sympathetic and parasympathetic nervous system. The cardiac response can be easily monitored using recordings of HR and stroke volume (SV), while the vascular response can be monitored with measures of muscle sympathetic nervous activity (MSNA) or vascular ultrasound, or both. These latter techniques, however, require expensive equipment and trained personnel. Both of these responses simultaneously contribute in the maintenance of MAP which can be monitored on a beat-by-beat basis. Although LBNP has been used extensively for the past several decades, there has recently been an emergence of the utility of lower-body positive pressure (LBPP), particularly with rehabilitation (Takacs et al., 2013). Conversely to LBNP chambers, LBPP chambers have been employed to investigate the effects of increased venous return and MAP, which has profound effects on the cardiovascular and cerebrovascular systems (Nishiyasu et al., 2007, Perry et al., 2014). For example, LBPP chambers have been employed to investigate the regulatory responses of the brain to transient increases in blood pressure (Perry et al., 2013). Ideally, having access to both LBNP and LBPP chambers would be highly useful to demonstrate the integrated cardiovascular responses to simulated central volume loading (using LBPP), and unloading (using LBNP). As previously mentioned, both steady-state changes in body position such as head-up tilt (HUT) and head-down tilt (HDT) and lower-body differential pressure (negative and positive) can have profound effects on venous return, cardiac output (CO; the product of HR and SV) and MAP 222 (Stevens and Lamb, 1965, Tymko et al., 2015). In light of this, a novel innovation recently developed is a suspended LBNP/LBPP chamber fastened to a 360 degree tilt-table. Although there has been previous research using a combination of upright tilt and LBNP (Deegan et al., 2010, Thomas et al., 2009), this apparatus is unique as it allows the manipulation of HUT and HDT, concomitantly with LBNP or LBPP. As such, the apparatus has great utility as both a research tool to improve the current understanding on the mechanisms involved in blood pressure regulation, along with being a valuable teaching instrument. Lower-body differential pressure chambers can be purchased commercially, however, these are usually expensive ($10,000 to $20,000 USD) and have a similar performance compared to homemade chambers, which are relatively easy to construct. Helpful documentation on how to construct a basic LBNP chamber exists (Esch et al., 2007); however, other literature has lacked an appropriate amount of detail (Hisdal et al., 2003, Lategola and Trent, 1979, Verghese and Prasad, 1993, Russomano et al., 2005). In addition to LBNP, the use of LBPP can be beneficial to show the bi-directionality of the cardiovagal baroreflex. Yet, there are no published details on how to build a chamber that can accommodate both LBNP and LBPP, nor one that combines LBNP/LBPP and steady-state tilt. Ideally, a constructed lower body differential pressure chamber should be able to achieve both negative pressures and positive pressures while having a capacity to manipulate body position, to maximize research and teaching potential. The purpose of this manuscript is to 1) for the first time, outline a strategy to construct a LBNP/LBPP tilt-table apparatus, and 2) demonstrate how this apparatus can be effectively used in the laboratory setting for teaching purposes using sample data traces. 223 After building a LBNP/LBPP tilt-table apparatus the students (undergraduate and/or graduate students) will accomplish the following learning objectives after conducting the proposed laboratory exercises. 1) Be able to describe the specific cardiovagal baroreflex-mediated mechanisms that is responsible for the maintenance of blood pressure during LBNP and LBPP in supine position, 2) be able to recognize cardiovascular collapse, a potential response when using LBNP, in order to ensure participant safety, 3) be able to describe the specific cardiovagal baroreflex-mediated mechanisms that are responsible for blood pressure regulation during changes in body position (e.g. 45 degrees HDT, supine, and 45 degrees HUT) at rest, and 4) understand the differences in cardiovascular response to LBNP in different body positions. MATERIALS AND METHODS Overview. The additional value of laboratory demonstrations of physiological functions are well known (Berg et al., 2012, O'Donaughy et al., 2002), as they are critical for student learning and development in the physiological sciences. However, the use of LBNP or LBPP is usually restricted to research purposes, and use as a teaching instrument is often overlooked. Herein, we provide an example on how the constructed chamber (see results section for construction details) can be used as a demonstration tool to aid undergraduate student learning of the cardiovagal baroreflex, and we quantify the effectiveness of the laboratory demonstration with an anonymous, voluntary student survey. Laboratory demonstrations involving LBNP or LBPP, or both should be performed under supervision by experienced, and knowledgeable personnel. Improper use of LBNP/LBPP could lead to participant injury. 224 Ethical approval. Appropriate ethical approval is always needed in order to perform laboratory demonstrations with human participants. The following experimental protocols were approved by a Human Ethics Committee at the University of British Columbia., and were in accordance with the Canadian Government Tri-Council Policy Statement on Human Research (TCPS2) and the Declaration of Helsinki. Participant considerations. The recruited male participant (height: 177 cm, weight: 88 kg) filled out a health history questionnaire to ensure normal pulmonary, cardiovascular, and cerebrovascular health. Participant exclusion criteria involved low resting blood pressure (e.g. <100 mmHg systolic blood pressure, and <60 mmHg diastolic blood pressure), and/or a history of syncope (i.e. fainting), as these participants are at greater risk of reaching cardiovascular collapse during LBNP. Participants were also non-smokers, had no reported previous history of respiratory, cardiovascular, cerebrovascular diseases, and were not taking any medications. Additionally, the participant was asked to refrain from vigorous physical activity, alcohol consumption, and caffeine for at least 12-hours prior to experimentation. Participant instrumentation. Upon arrival on the experimental day, the participant placed a kayak skirt, which was integrated to the lid of the chamber, around the iliac crest and positioned themselves within the chamber in the upright (i.e. 90 degrees HUT) position. Improper positioning of the kayak skirt can influence the cardiovascular responses observed during LBNP and LBPP (Goswami et al., 2008). Once proper positioning of the kayak skirt, two velcro stretchable waist belts was fastened around the participant to ensure an air tight seal around the waist. Briefly, the 225 vacuum was turned on to -50 mmHg and +40 mmHg to familiarize the participant to LBNP and LBPP stress. Cardiovascular measurements. Cardiovascular measurements were collected at 200 Hz using an analog-to-digital converter (Powerlab/16SP ML880; AD Instruments; ADI; Colorado Springs, CO, USA). Participants were instrumented with electrocardiogram (ECG) electrodes in lead II configuration in conjunction with a bioamp (ML132; ADInstruments, Colorado Springs, CO, USA) to derive instantaneous HR from the R-R interval of the ECG. Beat-by-beat arterial blood pressure, cardiac output (CO), and stroke volume (SV) were measured using finger photoplethysmography (Finometer Pro, Finapres Medical Systems, Amsterdam, NL). Prior to baseline data collection, the Finometer was calibrated using the return-to-flow function, and blood pressure accuracy was confirmed with manual sphygmomanometry. Mean arterial pressure (MAP) was calculated by taking the mean of each individual arterial waveforms outputted from the Finometer. Experimental protocols. Three protocols were utilized to effectively demonstrate the cardiovagal baroreflex in one participant: 1) LBNP vs LBPP protocol, 2) Body position protocol, and 3) LBNP in different body positions (i.e. 45 degrees HDT, supine, and 45 degrees HUT) protocol. The following protocol termination guidelines were followed to ensure participant safety: 1) the participant voluntarily terminated the test due to the onset of subjective symptoms (e.g., gray-out, nausea, dizziness or discomfort), or 2) the participant reached presyncope, which was identified in real time by the investigators by the sudden onset of bradycardia, and/or a 30% reduction of baseline systolic blood pressure (calculated in advance). 226 Protocol #1 - LBNP and LBPP After instrumentation, the participant was instructed to lie motionlessly in supine position and to breathe normally for approximately 10-minutes to ensure that orthostatic-related changes in blood volume distribution were stable. Afterwards, an initial five-minute quiet baseline period was recorded in supine position, and then the pressure within the chamber was immediately lowered to -50 mmHg (LBNP) for 10-minutes. After exposure to 10-minutes of LBNP, the vacuum was disengaged and the participant rested quietly for approximately 10-minutes of recovery. Immediately after the 10-minute recovery period, the pressure within the chamber was increased to +40 mmHg (LBPP) for 10-minutes. Protocol #2 – Body Position After instrumentation, the participant was instructed to lie motionlessly in supine position and to breathe normally for approximately 10-minutes to ensure that orthostatic-related changes in blood volume distribution were stable. Immediately afterwards, 10-minutes of normal resting data was collected. This protocol was repeated in two other randomized body positions: 45 degrees HDT and 45 degrees HUT. Protocol #3 - LBNP in different body positions After instrumentation, the participant was instructed to lie motionlessly and to breathe normally for approximately 10-minutes to ensure that orthostatic-related changes in blood volume distribution were stable. The protocol consisted of an initial five-minute quiet baseline period in the supine position, after which the pressure inside the LBNP chamber was immediately lowered 227 to -50 mmHg (LBNP) for 10-minutes. Immediately after LBNP termination, the participant completed a 10-minute recovery period, and then repeated the same protocol (5-minute baseline, LBNP, and recovery period) in the remaining two randomized tilt positions: 45 degrees HDT and 45 degrees HUT. Student Survey In order to quantify the effectiveness of the laboratory demonstration for undergraduate student learning of the cardiovagal baroreflex, an anonymous survey was conducted at the end of the laboratory session. The following questions were asked: 1) The LBNP demonstration aided my learning, 2) I have a better understanding of key cardiovascular physiology concepts from the LBNP demonstration, 3) The LBNP demonstration encouraged me to think critically about cardiovascular physiology, and cardiovascular collapse, 4) I recommend the implementation of the LBNP demonstration in future classes. The students were asked to rate their answers to these questions from 1 to 10, with 1 being considered “not useful” and 10 being “very useful”. Data Analysis. All recorded cardiovascular data was exported from our data acquisition software (ADI LabChart Pro software V7.2), and averaged into time bins (15 and 30 seconds) using Microsoft Excel. The averaged data SEM was plotted using SigmaStat (Systat, Chicago, IL, USA, V11.5,). 228 RESULTS Chamber Construction There are several different lower-body differential chambers that are in use today, each with unique designs. The following section provide information on the vacuum source and variable transformer required, along with building instructions on how to construct a LBNP/LBPP chamber integrated on a tilt-table. Vacuum source. A commonly used inexpensive device to manipulate pressure within a differential pressure chamber is an industrial vacuum. For the chamber described below, a 120 volt, powerful industrial vacuum (six horsepower), with a 12 amperage rating was used. This vacuum provided a pressure range of approximately -90 to +40 mmHg with our airtight chamber (Tymko et al., unpublished findings), and other research groups found a similar performance with this type of vacuum (Esch et al., 2007). Real time differential pressure was monitored during experimentation using a digital manometer (DigiMano 1000, 200-200IN, Netech Corporation, Farmingdale, NY, USA). This range of differential pressure is adequate for both laboratory demonstrations and for research projects (for a detailed review see, (Goswami et al., 2008)). Variable transformer. Some commercial vacuums have power settings allowing the user to change the power output, but a more versatile way to incrementally change vacuum power output through a variable transformer. A variable transformer provides the operator with the unique advantage of fine tuning the pressure stimulus on an individual basis. The variable transformer had the following criteria 1) appropriate electrical current rating (15 amperage), 2) input voltage rating of 120, and 229 3) was able to control power output on a volt-by-volt basis. For this chamber, we used a 120V input, 0-140V output variable transformer (Variac, 3PN1510B, ISE, inc., Cleveland, OH, USA). LBNP/LBPP chamber and tilt-table design. The design of the LBNP/LBPP chamber and tilt-table will be presented in the following order: I) the supporting frame, II) LBNP/LBPP chamber inner frame, III) LBNP/LBPP outer frame, and IV) the participant opening. Building a structurally sound support frame is essential to ensure participant safety, however, due to the size of the supporting frame it must be collapsible for transportation. In order to achieve this, three separate metal frames were constructed. Each of these frames were built in the same fashion, and comprised of two pieces of 48-in. long, 3-in. wide X 1-in. C-channel steel uprights connected by one 50-in. long, 3-in. X 3-in. rectangular steel tubing (see figure 1A). Each of the three supporting frames were welded together. After welding these individual supporting frames together, they were connected using four 3 ½-in. X 3 ½-in. pieces of wood (two on each side – only left side shown in figure 1A), each being 54-in. in length. The wood braces and metal frames were joined together using 6-in. long, ½-in. diameter, hex bolts with ½-in. washers and ½-in. nuts (see figure 1A). Once the supporting frame structure was complete, two 1 ¾-in. diameter holes were drilled near the top of the middle metal frame to allow for a 60-in. long, 1 ½-in. diameter metal axle to be suspended across and through the holes of the supporting frame (see figure 1A). The LBNP/LBPP chamber and table was bolted to this axle using three 5-in. long, ½-in. diameter hex bolts with ½-in washers and ½-in nuts (see the next section on the chamber construction for more details on this specific step). 230 The inner frame of the LBNP/LBPP chambers was built with 1 ½-in. X 5 ½-in. lumber. The dimensions of our differential pressure chamber is: 18-in. height X 25-in. width X 42-in. length (see figure 1B and 1C). The inner frame of the LBNP chamber is fastened together using 2 ½-in. wood screws, wherever necessary. Once the inner frame was completed, the chamber outer panels were made from ¾-in. thick plywood, and cut to size to fit over top of the inner frame. The two side plywood panels of the chamber were 18-in. X 42-in., the front panel was 25-in. X 42-in., and the bottom panel was 25-in. X 18-in. (see figure 1B and 1C). Once the outer panels of the chamber were cut to size, a bead of acoustic sealant (PL Acousti-Seal, Lepage, Mississauga, ON, CA) was run along the inner frame prior, to fastening the ¾-in. plywood outer panels to the inner frame. The inner frame of the LBNP/LBPP chamber was adhered to a long table piece build out of ¾-in. plywood that was used for participant upper body support. Additionally, two metal supporting A-frames were bolted within the chamber to increase its performance in regards to handling positive pressure (see figure 2B). The table piece was 25-in. width X 75.in length, cut from ¾-in. plywood. The upper-most portion of the table piece (where the participant head rests) was cut in a semi-circular shape with a jigsaw (see figure 1B, 1C, and 2). To provide further structural support, three 1 ½-in. X 3 ½-in., 75-in. long pieces of lumber were fastened onto the rear side of the table piece using 2-in. long wooden screws, without this support the apparatus would undoubtedly break due to the overall load and weight distribution of the apparatus (lower half being much heavier than the upper half). The chamber and tilt-table was mounted to an axle (see figure 1A and 2A) using three 5-in. long, ½-in. diameter hex bolts, tightened with ½-in. nuts and ½-in. washers. These hex bolts were inserted through ½-in. diameter, pre-drilled holes, which were located at the mid-point 231 of each 1 ½-in. X 3 ½-in. piece of wood (mid-point at 37 ½-in.) located at the rear side of the tilt-table. A neoprene kayak skirt (see figure 1 and 2) was used to create an airtight seal between the participant and the chamber. The neoprene kayak skirt was fastened inside a 17-in. in diameter hole cut within the top outer panel of the chamber (18-in. X 25-in.). To fasten the neoprene kayak skirt to the chamber, a combination of a heavy duty staples and construction adhesive was used. Once the last outer panel (with kayak skirt) was fastened to the chamber frame, silicon sealant was applied to the inside perimeter of the panel. The negative pressure itself causes the kayak skirt to collapse upon itself, creating a seal. Nevertheless, if the neoprene skirt fails to seal properly, or is too big, a high potential exists for the chamber to equilibrate with the atmospheric pressure, reducing the intentional stimulus. The outer (top) plywood panel containing the neoprene kayak skirt (see figure 1B and 2) was built in a fashion so that it was removable, allowing the participant to enter the kayak skirt prior to entering the chamber while being in the upright position. After the participant is placed inside the chamber (see figure 2B), the front outer panel of the chamber was able to shut tightly using ratchet straps. An airtight seal between the front outer panel of the chamber, and the rest of the chamber was created using adhesive sealant tape (see figure 2B). After the LBNP/LBNP chamber was complete, it was attached to the metal axle from the support stand using three 5-in. long, ½-in. diameter hex bolts, with ½-in. nuts and ½-in. washers. The current dimensions provided for the LBNP/LBPP tilt-table chamber allow for participants to be placed in any HUT and HDT body position desirable (see figure 1 and 2). Changing the body 232 position of the chamber is done manually, and the chamber is held in place using steel chain or cable connected from the chamber to the bottom of the supporting frame (see figure 2A). Additional features. Importantly, participants that are placed in HDT positions with this apparatus risk falling out from the chamber. In order to prevent this, a 30-in. long, 1-in. diameter safety bar was placed through the bottom half of the chamber, and allowed the participant to hook into the axle using specifically designed boots (Teeter Hang-ups, Tacoma, WA, USA) (see figure 2A and 2B). This was not only a safety feature, but also served utility by holding participants in position during LBPP. In addition to this feature, it is also recommended to add some sort of padding (i.e. foam, blanket) to table portion of the chamber to aid with subject comfort (see figure 2B). Experimental Results LBNP and LBPP protocol Figure 3 illustrates the cardiovascular responses to -50 mmHg LBNP and +40 mmHg LBPP in one participant. During LBNP, HR progressively increased, while SV progressively decreased. This resulted in CO being rather stable during the beginning of the LBNP protocol, but as LBNP progressed, CO decreased near the end of LBNP protocol. Regardless of these cardiovascular changes during LBNP, MAP remained relatively unchanged during the entire LBNP protocol. During LBPP, HR, SV, and CO remained similar throughout the entire protocol, but as expected, MAP increased by ~10 mmHg. Figure 4 represents data from an individual who reached presyncope during -50 mmHg LBNP, and the protocol was terminated using our safety guidelines (see experimental protocols section above). The participant experiences a rapid decrease in MAP, followed abruptly by relative bradycardia. 233 Body position protocol Figure 5 illustrates the cardiovascular responses to steady-state 45 degrees HUT, supine, and 45 degrees HDT in one participant. In the 45 degree HUT position, HR and CO were elevated compared to supine and 45 degree HDT. In contrast, SV was lower during 45 degree HUT compared to supine and 45 degree HDT. As expected, in the volume loading (i.e. 45 degree HDT) position, MAP was elevated compared to both 45 degree HUT and supine body positions. LBNP in different body positions Figure 6 illustrates the cardiovascular responses to steady-state 45 degrees HUT, supine, and 45 degrees HDT combined with -50 mmHg LBNP in one participant. The HR response to LBNP is dose dependent, with 45 degree HUT being the largest and 45 degree HDT being the smallest. The SV response to LBNP and body position is the opposite to HR, where the SV was reduced the most in the 45 degree HUT position, and reduced by the least amount in the 45 degree HDT position. Cardiac output during LBNP was highest during the 45 degree HUT position, while it was similar in both supine and 45 degree HDT position. Despite different baseline MAP between body positions, MAP during LBNP was similar between each body position. Student Survey Fourth year undergraduate students (n=22) were present for the laboratory demonstration described above. Each of these students anonymously participated in a short written survey in order to provide feedback information on the effectiveness of the experimental protocol. Below are the questions provided in the survey, and the students were asked to rate their answers to these 234 questions from 1 to 10, with 1 being considered “not useful” and 10 being “very useful”, reports of these values are mean  SD. (1) The LBNP demonstration aided my learning. Answer: 8.0  1.7 (2) I have a better understanding of key cardiovascular physiology concepts from the LBNP demonstration. Answer: 8.0  1.5. (3) The LBNP demonstration encouraged me to think critically about cardiovascular physiology, and cardiovascular collapse. Answer: 7.8  1.8 (4) I recommend the implementation of the LBNP demonstration in future classes Answer: 8.9  1.0 235 DISCUSSION The primary purposes of this manuscript is to 1) outline a strategy to construct a LBNP/LBPP tilt-table apparatus, and 2) demonstrate how this apparatus can be effectively used in the laboratory setting for teaching purposes. After constructing a purpose built LBNP/LBPP tilt-table apparatus, three protocols were conducted in one participant during a laboratory demonstration to undergraduate students. The laboratory demonstrations aided the students articulation of the following learning objectives: 1) be able to describe the specific cardiovagal baroreflex-mediated mechanisms that are responsible for the maintenance of blood pressure during LBNP and LBPP in supine position, 2) be able to recognize cardiovascular collapse, a potential response when using LBNP, in order to ensure participant safety, 3) be able to describe the specific cardiovagal baroreflex-mediated mechanisms that are responsible for blood pressure regulation during changes in body position (45 degrees HDT, supine, and 45 degrees HUT) at rest, and 4) understand why there are differences in the cardiovascular response to LBNP in different body positions (45 degrees HDT, supine, and 45 degrees HUT). Cardiovascular responses to lower-body differential pressure Lower-body negative pressure. The use of LBNP is an effective, non-pharmacological approach to alter central blood volume. During LBNP, blood in the upper torso is temporarily accumulated in the lower limbs, resulting in a decreased central blood volume, venous return, cardiac preload, and SV (See figure 3) (Cooke et al., 2004). In order to compensate for these physiological changes, the baroreflex is activated (sympathoexcitatory response) to increase HR and vascular peripheral resistance in order to maintain MAP (see figure 3). The magnitude of the compensatory response to LBNP is directly related to the duration of LBNP, and LBNP magnitude (i.e. degree of negative 236 pressure). For example, approximately 400-500ml of blood is displaced with LBNP between -10 and -20 mmHg (Thompson et al., 1990, Hanson et al., 1998), and another study concluded that -30 to -50 mmHg of LBNP results in a translocation of 500-1000ml of blood (Wolthuis et al., 1975). The absolute amount of blood volume translocated for a given LBNP is quite variable and it is dependent on the effectiveness an individual’s cardiovagal baroreflex response. There is large inter-individual variability to LBNP as it can vary due to gender (Convertino, 1998, Kelly et al., 2004), age (Rutan et al., 1992), height (Ludwig and Convertino, 1994, Pavy-Le Traon et al., 1999), pathology (e.g. hypertension) (Kuwajima et al., 1991), and fitness level (Convertino, 1987, Convertino, 1993). However, after accumulation of approximately 1000ml of blood into the lower limbs during LBNP, signs of syncope are usually present (i.e. cardiovascular collapse). This occurs when the cardiovagal baroreflex associated physiological responses are no longer able to effectively maintain MAP, resulting in hypotension (see figure 4). At this stage during LBNP, MAP and HR rapidly plummet due to sympathetic nervous activity withdrawal, and the individual likely experiences nausea, light-headedness, tunnel vision, and even syncope (see figure 4). Lower-body positive pressure. Similar to LBNP, LBPP is an effective experimental method to alter central blood volume, but in contrast to LBNP, LBPP increases central blood volume by displacing fluid from the lower limbs to the central cavity (Hinghofer-Szalkay et al., 1988, Nishiyasu et al., 1998), and this results in an increase in central venous pressure, cardiac filling, and MAP (see figure 3) (Nishiyasu et al., 1998, Fu et al., 1998), but despite these physiological changes, CO and HR remain relatively unchanged during LBPP (see figure 3) (Rubal et al., 1989). However, during long durations of LBPP (e.g. >30 minute), HR and CO have been shown to increase (Geelen et al., 1992). The absolute amount of blood displacement that occurs during 237 LBPP is unclear, however, the volume of blood displaced would inversely proportional to LBNP (as described in the section above). Similar to LBNP, the magnitude of the response to LBPP (e.g. increase in MAP) is directly related to the duration of LBPP, and LBPP magnitude (i.e. degree of positive pressure). For example, +20 mmHg and +40 mmHg of LBPP have been demonstrated to increase MAP by 7 mmHg and 13 mmHg, respectively (Perry et al., 2013). Data from the laboratory demonstration (see figure 3), clearly shows an increase in MAP with +40 mmHg, with a magnitude of ~10 mmHg, similar to results found by Perry et al., (2013). Cardiovascular responses to steady-state changes in body position. Body position is often overlooked as a profound physiological stimulus, but in fact, when moving from supine into the upright position, gravity draws a substantial volume of blood down into the lower extremities causing a decrease in central blood volume (similar effect as LBNP), while an increase in central blood volume occurs during HDT position (similar effect as LBPP) (Bundgaard-Nielsen et al., 2009, Murrell et al., 2011). To compensate for changes in body position, the cardiopulmonary and arterial baroreceptors detect changes in venous return and MAP via changes in mechanical stretch, and alter HR and vascular resistance in order to maintain CO, MAP, and thus, cerebral perfusion pressure (Wehrwein and Joyner, 2013, Cooke et al., 2004). Figure 5 illustrates the steady-state cardiovascular responses to body positions (45 degree HUT, supine, and 45 degree HDT). In the 45 degree HUT position, when central blood volume is decreased, CO and MAP are maintained by elevating HR in order to compensate for the gravity-associated decrease in venous return, thus a reduction in SV. While in the 45 degree HDT position, when central blood volume is increased, MAP is slightly elevated compared to supine and 45 238 degree HUT body position (similar to LBPP), likely due to increased peripheral resistance. These results are similar to previous findings from our research group (Tymko et al., 2015). Cardiovascular responses during LBNP in different body positions. The tilt-table LBNP/LBPP apparatus, built by the author, has the unique advantage of eliciting a cardiovagal baroreflex in different body positions, giving undergraduate students an important demonstration of real-time integrative human physiology. As illustrated in figure 6, the cardiovascular responses to LBNP is body position dependent, and this was due to the different hydrostatic pressure gradients between the three body positions. The hydrostatic pressure gradient that is important in the context of the cardiovagal baroreflex is the vertical distance between the head and the heart of the individual. Hydrostatic pressure can be calculated using the following equation: equation one: p = gh*C Where p = pressure in mmHg,  (rho) = the density of blood (~1.04 g/cm3), g = the acceleration due to gravity (-9.81 m/s2), h = the absolute height in which the head is above or below the heart in meters, and C = the conversion factor from kilopascals to mmHg (7.5). In the HUT position, the hydrostatic pressure is negative (i.e. head above the heart), meaning that the heart must overcome greater gravitational forces in order for adequate blood flow to return to the heart. In the HDT position, the hydrostatic pressure gradient is positive (i.e. head below the heart), meaning that gravitational forces are aiding venous return to the heart (see figure 7 for an example of these calculations). Due to the reduced hydrostatic pressure associated with HUT, central blood volume is reduced compared to supine and HDT positions. This resulted in a decreased SV and elevated 239 HR response to LBNP in HUT, compared to supine and HDT (see figure 6). The observed cardiac responses to LBNP are partly responsible for the maintenance in MAP. Mean arterial pressure is usually maintained near resting levels during LBNP (see figure 6). Additional Physiological Assessments during LBNP/LBPP Respiratory measures. Respiratory measurements that should be considered are respiratory flow, and the end-tidal partial pressure of carbon dioxide (PETCO2) and oxygen. Having these measurements could allow for the assessment of respiratory variables of the participant, as it is common to observe an increase in ventilation in response to LBNP. Respiratory gas measurements, particularly PETCO2, are important when measuring blood flow intracranially (see section below), as blood flow through the cerebrovasculature is sensitive to changes in PETCO2. During hypercapnia (high PETCO2) blood flow through the brain increases due to downstream vasodilation of arterioles, and during hypocapnia (low PETCO2) blood flow through the brain decreases due to arteriolar vasoconstriction (Kety and Schmidt, 1948). The strong effects of PETCO2 on cerebral blood flow is an important principle when measuring intracranial blood flow (Willie et al., 2012). Intracranial blood flow. Transcranial Doppler ultrasound (TCD) can be employed to measure the intracranial cerebral blood flow velocity within the large conduit arteries within the cerebral vasculature. The most commonly insonated cerebral arteries are the middle cerebral artery (MCA; located anteriorly) and posterior cerebral artery (PCA), which supply the brain with approximately 70% and 30% of the cerebral circulation, respectively (Willie et al., 2011). The TCD system uses a 2 MHz ultrasound probe connected to a headset, which emit sound waves through the temporal window and registers the frequency of the sound reflected from moving red blood cells within the 240 vessel of interest. The resulting frequency shift (i.e. Doppler shift) can provide the instantaneous velocity of the moving red blood cells. Since cerebral blood flow is affected by vessel diameter, it is important for diameter to remain constant in order for blood velocity to reflect the underlying changes in cerebral blood flow (Willie et al., 2011). Previous literature reports that cerebral blood velocity via TCD can be used as a surrogate measurement of cerebral blood flow during both orthostatic stress (Serrador et al., 2000). During LBNP, it has been established that cerebral blood flow is reduced (Levine et al., 1994), but during LBPP, cerebral blood flow remains relatively unchanged (Perry et al., 2013, Perry et al., 2014). Muscle sympathetic nervous activity. Muscle sympathetic nerve activity (MSNA) is a direct measurement of multi-unit postganglionic activity and can obtained from fascicles of the right peroneal nerve at the popliteal fossa using a common microneurographic technique that has been used extensively since the 1960’s (Vallbo et al., 2004). This technique can be time consuming and requires that subject comfort be of utmost importance. Muscle sympathetic nerve recordings provides insight of the activation of the peripheral vascular system during LBNP/LBPP. However, this technique is difficult, and it requires someone with extensive experience to perform correctly (Vallbo et al., 2004). Muscle sympathetic nervous activity linearly increases with progressive LBNP until cardiovascular collapse is reached (Rea and Wallin, 1989, Convertino et al., 2004), where MSNA rapidly decreases (Hayoz et al., 1996, Sanders and Ferguson, 1989), which is responsible for the decrease in peripheral vascular tone (vasodilation) and HR. During LBPP, MSNA is supressed at positive pressures of +10 mmHg and +20 mmHg, however, MSNA linearly increases with more severe levels of LBPP (>30 mmHg). Reasoning for this is currently unclear, 241 but it is suggested that MSNA is increased due to activation of intramuscular pressure-sensitive receptors (Fu et al., 1998). Student project application. In the current manuscript, the tilt-table LBNP/ LBPP chambers described in detail above have also been effectively used for group and/or individual student research projects. For this purpose, students developed their own experimental design, recruited appropriate participants, collected and analyzed data, wrote a lab report presenting their findings, and presented their findings to the University faculty members. This pedagogical approach is impactful for student learning and critical thinking, but caution must be advised when using LBNP as they have the potential of eliciting adverse effects from participants (e.g. syncope, vomiting, nausea) as illustrated in figure 4. It is recommended that an appropriately trained investigator be present whenever collecting data with this chamber. Conclusion The custom built tilt-table LBNP/LBPP device has proven beneficial for student learning by demonstrating the cardiovagal baroreflex. After completing the proposed laboratory protocols, students had a clear understanding of the following learning objectives: 1) the cardiovagal baroreflex-mediated mechanisms that are responsible for the maintenance of blood pressure during LBNP and LBPP in supine position, 2) recognize cardiovascular collapse, a potential response when using LBNP, in order to ensure participant safety, 3) the cardiovagal baroreflex-mediated mechanisms that are responsible for blood pressure regulation during changes in body position (e.g. 45 degrees HDT, supine, and 45 degrees HUT) at rest, and 4) the differences in cardiovascular 242 response to LBNP in different body positions. The cardiovagal baroreflex is an important concept taught in physiology related courses, and demonstrating it using LBNP, LBPP, and body position in real-time aids student learning and development. 243 Appendix: Key references for instructors 1. Cooke WH, Ryan KL & Convertino VA (2004). Lower body negative pressure as a model to study progression to acute hemorrhagic shock in humans. J Appl Physiol (1985)96, 1249-1261. 2. Esch BT, Scott JM & Warburton DE (2007). Construction of a lower body negative pressure chamber. Adv Physiol Educ 31, 76-81. 3. Fu Q, Sugiyama Y, Kamiya A, Shamsuzzaman AS & Mano T (1998). Responses of muscle sympathetic nerve activity to lower body positive pressure. Am J Physiol2 75, H1254-9. 4. Goswami N, Loeppky JA & Hinghofer-Szalkay H (2008). LBNP: past protocols and technical considerations for experimental design. Aviat Space Environ Med 79, 459-471. 5. Perry BG, Schlader ZJ, Raman A, Cochrane DJ, Lucas SJ & Mundel T (2013). Middle cerebral artery blood flow velocity in response to lower body positive pressure. Clin Physiol Funct Imaging3 3, 483-488. 6. Rea RF & Wallin BG (1989). Sympathetic nerve activity in arm and leg muscles during lower body negative pressure in humans. J Appl Physiol (1985) 66, 2778-2781. 7. Stevens PM and Lamb LE (1965). Effects of lower-body negative pressure on the cardiovascular system. Am J Cardiol 16(4) 506-515. 8. Zoller RP, Mark AL, Abboud FM, Schmid PG, Heistad DD (1972). The role of low pressure baroreceptors in reflex vasoconstrictor responses in man. J Clin Invest 51(11): 2967-72. 244 Author Contributions: MMT was responsible for the conception and design of the tilt-table LBNP/LBPP chamber, along with collecting data, making figures, and drafting the manuscript. Acknowledgements: I would like to thank Drs. Philip Ainslie, Trevor Day and Greg duManoir, along with Tony Bain, Josh Tremblay, and Ryan Hoiland for their critical insight and aid in the manuscript preparation. Funding: None. Disclosures: The author has no conflict of interest. 245 References Berg RM, Plovsing RR & Damgaard M (2012). Teaching baroreflex physiology to medical students: a comparison of quiz-based and conventional teaching strategies in a laboratory exercise. 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Am J Physiol Regul Integr Comp Physiol 297, R116-23. 250 List of Tables Table 1 List of tools required to construct the tilt-table LBNP/LBPP chamber. Tool Description Metal saw Used during the cutting of the C-channel support frames Mig Welder For welding the C-channel support frames together Angle grinder Only used for cleaning the metal surfaces of the C-channel support frames prior to welding Hand drill Used for drilling in wood screws, and drilling through the holes in the metal support frame Table saw Used to cut through the ¾-in plywood (outer panels), and inner frame lumber Silicon Used along all edges of the chamber to provide an air tight seal Jig saw Used to cut the hole in the top outer panel for the kayak skirt, and to cut the semi-circular end of the table piece. 251 Figure 1 Wire sketch of the tilt-table supporting frame (Panel A) and LBNP/LBPP chamber (Panel B and C). White dots (Panel A) on the supporting frame represent where the 6-in. hex bolt were drilled and fastened. Note, that two of the 3 ½-in. X 3 ½-in. wood pieces are not represented on the right side in Panel A. 252 Figure 2 Picture of the LBNP/LBPP chamber with tilt-table during a HDT LBNP protocol (Panel A) and the inside of the LBNP/LBPP chamber (Panel B). The three black circles high-light the three ratchet straps that were used to tighten the front chamber door to the chamber frame. 253 Figure 3 Raw data tracing of the cardiovascular responses during -50 mmHg LBNP and +40 mmHg LBPP in one subject. Black circles () represent LBNP data, and white circles () represent LBPP data. The first dashed line represents initiation of lower-body differential pressure, the second dashed line represents termination of lower-body differential pressure, on each plot. Each data point represents a 30 second average  SEM. Each protocol consisted of a two-minute eupneic breathing baseline period, immediately following baseline the vacuum pump was turned on and pressure was sustained for a total of 10-minutes. After 10-minutes of lower-body pressure stimulus, the vacuum was turned off allowing for the chamber to equilibrate with atmospheric pressure. 254 Figure 4 Raw data tracing of one participant reaching presyncope during -50 mmHg LBNP in 45 degree HUT position. The dashed line represents initiation of lower-body negative pressure, the solid line represents termination of lower-body differential pressure due to the participant reaching presyncope. Each data point represents a 15 second average  SEM. Each protocol consisted of a two-minute eupneic breathing baseline period, immediately following baseline the vacuum pump was turned on to generate -50 mmHg of LBNP. The protocol was terminated (i.e. vacuum pump turned off) when the participant reached cardiovascular collapse, as indicated by a relative drop in heart rate and mean arterial pressure. 255 Figure 5 Raw data tracing of the cardiovascular responses during 45 degrees HUT, supine, and 45 degrees HDT in one subject. Black circles () represent 45 degree HUT data, grey circles () represents steady-state supine data, and white circles () represents 45 degree HDT data. Each data point represents a 30 second average  SEM. Each protocol consisted of 10-minutes of normal eupneic breathing in each body position. 256 Figure 6 Raw data trace of the cardiovascular responses during -50 mmHg LBNP in 45 degrees HUT position, supine position, and 45 degrees HDT position in one subject. Black circles () represent 45 degree HUT data, grey circles () represents steady-state supine data, and white circles () represents 45 degree HDT data. The dashed line represents initiation of lower-body differential pressure, the second dashed line represents termination of lower-body differential pressure, on each plot. Each data point represents a 30 second average  SEM. Immediately after the eupneic breathing baseline period the vacuum pump was turned on and pressure was sustained for a total of 10-minutes in each body position. After 10-minutes of lower-body pressure stimulus, the vacuum was turned off allowing for the chamber to equilibrate with atmospheric pressure. 257 Figure 7 Illustration of the relative hydrostatic pressure gradients present during 45 degrees HUT position, supine position, and 45 degrees HDT position. The following equation can be used to calculated hydrostatic pressure: p = gh*C. Assuming that the density of blood is 1.04 g/cm3, and the acceleration due to gravity is constant at -9.81 m/s2, the following is an example calculation of hydrostatic pressure in HUT and HDT, where the head is 0.3 m above or below the heart. In HUT, p = ((1.04 g/cm3)*(-9.81 m/s2)*(0.3 m))*(7.5), which equals -23.0 mmHg. In HDT, p = ((1.04 g/cm3)*(-9.81 m/s2)*(-0.3 m))*(7.5), which equals +23.0 mmHg. Therefore, the hydrostatic pressure is reduced in HUT (-23 mmHg) compared to HDT (+23 mmHg).
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The impact of high altitude on endothelial function in humans
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