Health and Social Development, Faculty of (Okanagan)
Health and Exercise Sciences, School of (Okanagan)
DSpace
UBCO
Carr, Jordan Mark John Ramone
Doctor of Philosophy - PhD
University of British Columbia
During hypercapnia (elevated partial pressure of arterial CO₂; PaCO₂) cerebral blood flow (CBF) is elevated in order to maintain pH in the cerebral tissues; mean arterial pressure (MAP), minute ventilation (Vᴇ), and heart rate also increase. The shearing force caused by blood flowing through arteries also influences haemodynamics in the vasculature throughout the body, in a regionally varied manner. These PaCO₂, MAP, and shear stress-mediated responses are becoming relatively well understood, though the integration of such responses in both cerebral and peripheral vasculatures simultaneously has mostly been ignored. As hypercapnia causes both cerebral and systemic effects, which are inextricably connected, this thesis compared and contrasted hypercapnic-mediated responses between the cerebral and peripheral vasculatures, with recourse to the various influences of shear stress, MAP, and Vᴇ. Study one examined vascular responses to shear stress in cerebral and peripheral conduit arteries, comparing these against the hypercapnia-mediated response in the cerebral vasculature. The findings revealed that neither brachial artery shear-mediated vasodilation nor CBF reactivity to CO₂ were correlated with cerebral shear-mediated function. In Study two, via retrospective analyses, we assessed the within-test stability of CBF responses to CO₂ using dynamic end-tidal forcing, demonstrating that CBF responses to CO₂ do not fluctuate unless the CO₂ stimulus does. Study three investigated the influence of acute reductions in arterial oxygen (O₂) content on CBF responses to PaCO₂. Here, acute anemia elevated basal CBF which maintained cerebral O₂ delivery and elevated cerebral CO₂ washout. Study 4A assessed the peripheral vascular response to hypercapnia while accounting for the influences of shear stress and sympathetic nervous adrenergic activity (which controls MAP). We found that peripheral vascular responses to hypercapnia are suppressed by adrenergic innervation. Study 4B then extended these findings to address the influence of adrenergic activity on cerebral shear-mediated and hypercapnic responses, and found that, unlike peripheral vascular responses, α1-adrenoreceptors do not influence the cerebral blood flow response to CO₂, but may cause internal carotid artery (ICA) vascular restraint. In summary, the regionally heterogenous blood flow and vasodilator responses to both hypercapnia and shear stress are indicative of the differential balance of end-organ perfusion requirements and conduit artery vasodilator reserve. This dissertation advances our knowledge of the integration of hypercapnia, shear stress, and sympathetic nervous activity in healthy humans.
Cerebral and peripheral vascular responses to hypercapnia by Jordan Mark John Ramone Carr MLitt, University of Aberdeen, 2017 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The College of Graduate Studies (Kinesiology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) August 2022 © Jordan Mark John Ramone Carr, 2022 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled: Cerebral and peripheral vascular responses to hypercapnia submitted by Jordan Mark John Ramone Carr in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Kinesiology Dr Phil Ainslie, School of Health and Exercise Sciences Supervisor Dr Chris McNeil, School of Health and Exercise Sciences Supervisory Committee Member Dr Greg DuManoir, School of Health and Exercise Sciences Supervisory Committee Member Dr Chris West, School of Kinesiology, UBC Vancouver University Examiner Dr James Duffin, University of Toronto External Examiner iii Abstract During hypercapnia (elevated partial pressure of arterial CO2; PaCO2) cerebral blood flow (CBF) is elevated in order to maintain pH in the cerebral tissues; mean arterial pressure (MAP), minute ventilation (V̇E), and heart rate also increase. The shearing force caused by blood flowing through arteries also influences haemodynamics in the vasculature throughout the body, in a regionally varied manner. These PaCO2, MAP, and shear stress-mediated responses are becoming relatively well understood, though the integration of such responses in both cerebral and peripheral vasculatures simultaneously has mostly been ignored. As hypercapnia causes both cerebral and systemic effects, which are inextricably connected, this thesis compared and contrasted hypercapnic-mediated responses between the cerebral and peripheral vasculatures, with recourse to the various influences of shear stress, MAP, and V̇E. Study one examined vascular responses to shear stress in cerebral and peripheral conduit arteries, comparing these against the hypercapnia-mediated response in the cerebral vasculature. The findings revealed that neither brachial artery shear-mediated vasodilation nor CBF reactivity to CO2 were correlated with cerebral shear-mediated function. In Study two, via retrospective analyses, we assessed the within-test stability of CBF responses to CO2 using dynamic end-tidal forcing, demonstrating that CBF responses to CO2 do not fluctuate unless the CO2 stimulus does. Study three investigated the influence of acute reductions in arterial oxygen (O2) content on CBF responses to PaCO2. Here, acute anemia elevated basal CBF which maintained cerebral O2 delivery and elevated cerebral CO2 washout. Study 4A assessed the peripheral vascular response to hypercapnia while accounting for the influences of shear stress and sympathetic nervous adrenergic activity (which controls MAP). We found that peripheral vascular responses to hypercapnia are suppressed by adrenergic innervation. Study 4B then extended these findings to address the influence of adrenergic activity on cerebral shear-mediated and hypercapnic responses, and found that, unlike peripheral vascular responses, 𝛼1-adrenoreceptors do not influence the cerebral blood flow response to CO2, but may cause internal carotid artery (ICA) vascular restraint. In summary, the regionally heterogenous blood flow and vasodilator responses to both hypercapnia and shear stress are indicative of the differential balance of end-organ perfusion requirements and conduit artery vasodilator reserve. This dissertation advances our knowledge of the integration of hypercapnia, shear stress, and sympathetic nervous activity in healthy humans. iv Lay Summary Arteries supply all tissues in the body with blood, which conveys the fuels for - and conveys away the products of - metabolism. The primary by-product of metabolism is CO2, which, if not properly cleared from the body, can affect the balance of acidity in tissues. Changes in acidity can be detrimental to the brain, so it is profoundly reactive to changes in arterial CO2. Additionally, movement of blood through arteries produces shearing forces on the inner vessel lining, and arteries throughout the body are sensitive to this force. This dissertation details four studies comparing and contrasting responses in the brain and limb arteries to changes in CO2 and shear rates. We provide evidence regarding the different nature of these responses in the two areas, showing that the brain’s sensitivity to CO2 outweighs the influences of shear stress, and in limb arteries, CO2 responses are restrained by mechanisms controlling blood pressure. v Preface The University of British Columbia Clinical Research Ethics Board approved all experiment chapter in this dissertation (CREB IDs: H16-01028, H17-00929, H17-01810, H19-02355, H20-02716). Chapter 1 Written by Jay Carr, and edited by Professor Philip Ainslie. Chapter 2 Written by Jay Carr, and edited by Professor Philip Ainslie. Chapter 3 Published in The Journal of Physiology (Carr et al., 2020). Author contributions: J.M.J.R.C., R.L.H., H.G.C., C.A.H., G.B.C., J.C.T., and P.N.A. were involved in data collection. J.M.J.R.C., R.L.H., J.C.T., and P.N.A. were involved in data analyses, subsequent interpretation, and drafting the manuscript. All authors critically reviewed the manuscript. R.L.H., D.J.G., and P.N.A. conceived the study design. All authors approved the final version of this manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. Chapter 4 Published in the Experimental Physiology (Carr et al., 2021b) Author contributions: R.L.H., H.C., K.S., D.J.G., and M.M.T., were involved in data collection. J.M.J.R.C. and R.L.H. conceived the study hypotheses and methods, and analyzed the data. J.M.J.R.C., H.G.C., P.N.A., and R.L.H. subsequently interpreted and drafted the article. All authors critically reviewed the manuscript. All authors approved the final version of this article and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. vi Chapter 5 Accepted in The Journal of Cerebral Blood Flow and Metabolism at the time of dissertation submission. Author contributions: R.L.H., D.B.M., J.C.T., D.N-F. C.A.H., M.S., A.P., G.B.C., B.S., D.M.B., D.J.G., and P.N.A. were involved in data collection. J.M.J.R.C., R.L.H., and P.N.A. were involved in data analyses, subsequent interpretation, and drafting the article. All authors critically reviewed the article. All authors approved the final version of this article and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. Chapter 6 In review in Experimental Physiology at the time of dissertation submission. Author contributions: J.M.J.R.C. conceived the study rationale, J.M.J.R.C. and J.C.T conceived the study design. J.M.J.R.C., J.C.T., C.A.H., G.V-G., R.L.H., H.G.C., C.V.B., A.R.S., were involved in data collection. J.M.J.R.C., analyzed all data. J.M.J.R.C., J.C.T., and P.N.A. all contributed to interpretation. J.M.J.R.C., drafted the manuscript. All authors critically reviewed the final manuscript draft. All authors approved the final version of this manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. Chapter 7 In review in Journal of Applied Physiology at the time of dissertation submission. Author contributions: J.M.J.R.C. conceived the study rationale, J.M.J.R.C. and P.N.A. conceived the study design. J.M.J.R.C., J.C.T., C.A.H., G.V-G., M.M.T., A.R.S., T.D.G., were involved in data collection. J.M.J.R.C., analyzed all data. J.M.J.R.C., J.C.T., and P.N.A. all contributed to interpretation. J.M.J.R.C., drafted the manuscript. All authors critically reviewed the final manuscript draft. All authors approved the final version of this manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. Chapter 8 Written by Jay Carr, and edited by Professor Philip Ainslie vii Table of Contents Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface ............................................................................................................................................ v Table of Contents ........................................................................................................................ vii List of Tables ................................................................................................................................. x List of Figures ............................................................................................................................... xi List of Abbreviations .................................................................................................................. xv Acknowledgements .................................................................................................................. xviii Dedication ................................................................................................................................... xix Chapter 1: Introduction .......................................................................................................................... 1 1.1 Aims and hypotheses.............................................................................................................................. 2 1.2 Individual study aims and hypotheses: .................................................................................................. 2 1.2.1 Study 1 – Internal carotid artery and brachial artery shear-dependent vasodilator function in young healthy humans ................................................................................................................................................ 2 1.2.2 Study 2 – Stability of cerebrovascular CO2 reactivity following attainment of physiologic steady-state 2 1.2.3 Study 3 – Cerebral O2 and CO2 transport in isovolumic haemodilution: compensation of cerebral delivery of O2 and maintenance of cerebrovascular reactivity to CO2 ............................................................ 2 1.2.4 Study 4A – Brachial artery responses to acute hypercapnia: the roles of shear stress and sympathetic tone .............................................................................................................................................. 3 1.2.4.1 Study 4B – Adrenergic nervous activity in cerebral endothelium-dependent function and reactivity to hypercapnia ............................................................................................................................. 3 Chapter 2: Literature Review ................................................................................................................ 4 2.1 Atmospheric CO2: history and projections ............................................................................................ 4 2.2 CO2 toxicity ............................................................................................................................................ 6 2.3 Fundamental circulatory anatomy and vascular constitution ................................................................. 7 2.3.1 Tunica externa ................................................................................................................................... 8 2.3.2 Tunica media ..................................................................................................................................... 9 2.3.3 Tunica intima ..................................................................................................................................... 9 2.3.3.1 Historical sidebar - Andreas Vesalius.................................................................................... 10 2.4 Cerebral vascular anatomy ................................................................................................................... 12 2.4.1 The blood-brain barrier and neurovascular unit .............................................................................. 14 2.4.2 Pericytes .......................................................................................................................................... 17 2.4.3 Astrocytes ........................................................................................................................................ 18 2.4.3.1 Historical sidebar - Rudolph Virchow ................................................................................... 18 2.4.4 Endothelial cells .............................................................................................................................. 19 2.5 Regulation of cerebral blood flow........................................................................................................ 21 2.5.1 Carbon dioxide ................................................................................................................................ 21 2.5.1.1 Driving Stimuli: Hydrogen or carbon dioxide? ..................................................................... 26 2.5.1.2 [H+]: Henderson-Hasselbalch vs. Stewart’s physicochemical approach ............................... 27 2.5.1.3 CO2 bulk transport ................................................................................................................. 30 viii 2.5.2 Oxygen ............................................................................................................................................ 31 2.5.2.1 Hypoxic hypoxaemia vs anemic hypoxaemia ....................................................................... 32 2.5.2.2 Hyperoxia .............................................................................................................................. 34 2.5.3 Cerebral Autoregulation .................................................................................................................. 34 2.5.4 Neurovascular coupling ................................................................................................................... 36 2.5.5 Sympathoexcitation ......................................................................................................................... 37 2.6 Regulation of peripheral blood flow .................................................................................................... 40 2.6.1 Metabolism ...................................................................................................................................... 40 2.6.2 Sympathetic mediation of vascular tone ......................................................................................... 42 2.6.3 Shear stress ...................................................................................................................................... 43 2.7 Assessments of vascular function in research and clinical settings ..................................................... 45 2.7.1 Peripheral flow mediated dilatation ................................................................................................ 45 2.7.2 Cerebrovascular reactivity to carbon dioxide .................................................................................. 46 2.7.2.1 Influence of MAP: ................................................................................................................. 49 2.7.3 Cerebral shear-mediated dilatation .................................................................................................. 51 2.8 Summary: ............................................................................................................................................. 52 Chapter 3: Study 1 – Internal carotid artery and brachial artery shear-dependent vasodilator function in young healthy humans ....................................................................................................... 53 3.1 Background: ......................................................................................................................................... 55 3.2 Methods: ............................................................................................................................................... 57 3.3 Statistical analysis: ............................................................................................................................... 63 3.4 Results: ................................................................................................................................................. 63 3.5 Discussion: ........................................................................................................................................... 72 3.6 Experimental considerations: ............................................................................................................... 75 3.7 Conclusion: .......................................................................................................................................... 79 Chapter 4: Study 2 – Stability of cerebrovascular CO2 reactivity following attainment of physiologic steady-state ......................................................................................................................... 81 4.1 Background: ......................................................................................................................................... 82 4.2 Methods: ............................................................................................................................................... 84 4.3 Statistical analyses: .............................................................................................................................. 87 4.4 Results: ................................................................................................................................................. 88 4.5 Discussion: ........................................................................................................................................... 97 4.6 Methodological Considerations: ........................................................................................................ 101 4.7 Conclusions: ....................................................................................................................................... 103 Chapter 5: Study 3 – Cerebral O2 and CO2 transport in isovolumic haemodilution: compensation of cerebral delivery of O2 and maintenance of cerebrovascular reactivity to CO2 ....................... 104 5.1 Introduction: ....................................................................................................................................... 105 5.2 Methods: ............................................................................................................................................. 106 5.3 Statistical Analyses: ........................................................................................................................... 110 5.4 Results: ............................................................................................................................................... 111 5.5 Discussion: ......................................................................................................................................... 122 5.6 Experimental considerations: ............................................................................................................. 125 5.7 Conclusion: ........................................................................................................................................ 126 Chapter 6: Study 4A – Brachial artery responses to acute hypercapnia: the roles of shear stress and sympathetic tone........................................................................................................................... 127 6.1 Introduction: ....................................................................................................................................... 128 6.2 Methods: ............................................................................................................................................. 129 6.3 Statistical analyses: ............................................................................................................................ 133 6.4 Results: ............................................................................................................................................... 135 6.5 Discussion: ......................................................................................................................................... 143 6.6 Experimental considerations: ............................................................................................................. 145 ix 6.7 Conclusion: ........................................................................................................................................ 146 Chapter 7: Study 4B – Adrenergic nervous activity in cerebral endothelium-dependent function and reactivity to hypercapnia ............................................................................................................. 147 7.1 Introduction: ....................................................................................................................................... 148 7.2 Methods: ............................................................................................................................................. 149 7.3 Statistical analyses: ............................................................................................................................ 154 7.4 Results: ............................................................................................................................................... 155 7.5 Discussion: ......................................................................................................................................... 160 7.6 Experimental considerations: ............................................................................................................. 162 7.7 Conclusion: ........................................................................................................................................ 163 Chapter 8: Discussion ......................................................................................................................... 164 8.1 Main findings: .................................................................................................................................... 164 8.2 Contributions to the field: .................................................................................................................. 165 8.3 Integrated summary:........................................................................................................................... 167 8.4 Strengths of the dissertation: .............................................................................................................. 167 8.4.1 Cerebral endothelium-dependent function: ................................................................................... 167 8.4.2 Adrenergic restraint of peripheral vascular response to hypercapnia: .......................................... 168 8.5 Weaknesses of the dissertation/methodological considerations: ....................................................... 168 8.6 Future directions: ............................................................................................................................... 169 Chapter 9: Conclusion ........................................................................................................................ 171 Bibliography .............................................................................................................................. 172 Appendices ................................................................................................................................. 229 x List of Tables Table 1-1 – Approximate circulation blood flow values in humans at rest .................................... 1 Table 2-1. Standard vs Stewart models of acid-base balance ....................................................... 29 Table 2-2. Feline pial artery responses to sympathetic and parasympathetic stimulation ............ 39 Table 2-3. Attributes of the different experimental approaches used for the assessment of CVR 48 Table 3-1. Peripheral and cerebral vascular characteristics for endothelium-dependent and -independent tests ........................................................................................................................... 67 Table 3-2. Mean vascular and cardiorespiratory variables one minute before, and during administration of CO2 for cFMD (30 second), and CVR ............................................................. 68 Table 5-1. Cerebrovascular parameters during CVR prior to and following haemodilution. .... 113 Table 5-2. Cerebrovascular reactivity slopes in response to elevations in PaCO2. .................... 118 Table 6-1. Cardiorespiratory variables during baseline and hypercapnia in study one .............. 136 Table 6-2. Viscosity, shear stress, and plasma NO at baseline and last minute of hypercapnia in study one ..................................................................................................................................... 139 Table 7-1. Baseline cardiorespiratory variables.......................................................................... 155 Table 7-2. cFMD variables ......................................................................................................... 156 Table 7-3. Cardiorespiratory variables during CVR ................................................................... 158 Table 7-4. ICA variables during CVR ........................................................................................ 160 xi List of Figures Figure 2-1. Atmospheric CO2 concentrations within the last 800000 years ................................... 4 Figure 2-2. Atmospheric CO2 and fossil fuel related CO2 emissions ............................................. 5 Figure 2-3. Global CO2 projections ................................................................................................ 5 Figure 2-4. Constitutions of vessel walls across the circulatory system......................................... 8 Figure 2-5. Integra to tius magnae arteriae & Venarum arteriarumque cerebri............................ 11 Figure 2-6. Organization of cerebral blood flow and collateral blood flow ................................. 12 Figure 2-7. Segmental vascular resistance of the cerebrovascular tree ........................................ 13 Figure 2-8. The neurovascular unit ............................................................................................... 15 Figure 2-9. Transport across the BBB. ......................................................................................... 16 Figure 2-10. Overview of the vascular triad of the Blood-Brain Barrier...................................... 20 Figure 2-11. Relationship between cerebral blood flow and PaCO2. ........................................... 22 Figure 2-12. Regulation by CO2 across the cerebral vasculature. ................................................. 25 Figure 2-13. Signaling mechanisms underlying the cerebrovascular response to CO2 ................ 26 Figure 2-14. Relationship between cerebral blood flow and PaO2. .............................................. 32 Figure 2-15. Effects of anaemic hypoxaemia via haemodilution on CBF .................................... 33 Figure 2-16. Lassen's curve .......................................................................................................... 35 Figure 2-17. Modern cerebral autoregulation ............................................................................... 35 Figure 2-18. Adrenoceptors and calcium channels in the cerebral vasculature ............................ 38 Figure 2-19. Idealized schematic of the destination of cardiac output during rest and exercise .. 41 Figure 2-20. Adrenoreceptor densities, and sensitivity to sympathetic stimulation in the peripheral vasculature ................................................................................................................... 42 Figure 2-21. Regulation of vessel diameter by shear stress .......................................................... 44 xii Figure 2-22. CVR, cSMD, and BA FMD during saline and nitric oxide synthase inhibition ...... 51 Figure 3-1. Study 1 - Protocol schematic...................................................................................... 59 Figure 3-2. Study 1 - Diameter profiles for each test ................................................................... 65 Figure 3-3. Study 1 - Response profiles to different GTN doses.................................................. 69 Figure 3-4. Study 1 - Correlational analysis. ................................................................................ 71 Figure 3-5. Study 1 - Correlational analysis of allometrically scaled ICA and BA endothelium-dependent responses...................................................................................................................... 71 Figure 3-6. Study 1 - Correlations by sex ..................................................................................... 78 Figure 4-1. Study 2 - Cardiorespiratory response to hypercapnia. ............................................... 89 Figure 4-2. Study 2 - Cerebrovascular response to hypercapnia. ................................................. 90 Figure 4-3. Study 2 - Absolute CVR of MCAv (top), QICA (middle), and relative CVR comparing both (bottom). ............................................................................................................. 91 Figure 4-4. Study 2 - VE-CO2 sensitivity during 5-minutes of CO2 exposure. ............................. 92 Figure 4-5. Study 2 - 60 s means vs 30 s means for QICA, QICA CVR, MCAv, and MCAv CVR. ............................................................................................................................................. 93 Figure 4-6. Study 2 - Bland-Altman plots comparing 60 s and 30 s data means of QICA (top) and MCAv (bottom), at baseline and each minute. ............................................................................. 95 Figure 4-7. Study 2 - Bland-Altman plots comparing 60 s and 30 s data means of QICA CVR (top) and MCAv CVR (bottom) at each minute. .......................................................................... 96 Figure 5-1. Study 3 - Intervention parameters at baseline prior to and following haemodilution...................................................................................................................................................... 112 Figure 5-2. Study 3 - Cerebral blood flow and metabolism at baseline prior to and following haemodilution. ............................................................................................................................ 115 xiii Figure 5-3. Study 3 - The relationship between global cerebral blood flow and arterial CO2. .. 117 Figure 5-4. Study 3 - Cerebral metabolism during hypercapnia prior to and following haemodilution. ............................................................................................................................ 120 Figure 5-5. Study 3 - Cerebral CO2 tensions and contents during hypercapnia prior to and following haemodilution. ............................................................................................................ 121 Figure 6-1. Study 4A - Brachial artery diameter, flow, conductance, and shear stress/rate. ...... 138 Figure 6-2. Study 4A - Cardiorespiratory variables with Prazosin and placebo ........................ 140 Figure 6-3. Study 4A - Brachial artery diameter, flow, conductance, and shear rate with placebo and Prazosin. ............................................................................................................................... 142 Figure 7-1. Study 4B - cFMD ..................................................................................................... 157 Figure 7-2. Study 4B - Cerebrovascular reactivity to CO2 ......................................................... 159 Figure 0-1: Appendix B.1 - Internal carotid artery shear-stress mediated dilation during saline infusion and L-NMMA (eNOS blockade) infusion. ................................................................... 232 Figure 0-2: Appendix B.1: Timeline of experimental visit......................................................... 236 Figure 0-3: Appendix B.2 - Protocol. ......................................................................................... 241 Figure 0-4: Appendix B.3 - Protocol schematic ......................................................................... 244 Figure 0-5: Appendix B.3 - ICA diameter and shear rate, before, during, and following 30 second bolus of +9 mmHg PETCO2. ........................................................................................................ 244 Figure 0-6: Appendix B.5 - Timeline of experimental visit. ...................................................... 249 Figure 0-7: Appendix B.6 - Transient increases in shear stress causing internal carotid artery (A) and brachial artery (B) vasodilation. ........................................................................................... 251 Figure 0-8: Appendix B.7 - Cerebrovascular reactivity and ventilatory sensitivity to carbon dioxide with reductions in cerebral blood flow. ......................................................................... 255 xiv Figure 0-9: Appendix B.7 - Hypothetically expected findings ................................................... 256 Figure 0-10: Appendix B.7 - Illustration of the theorized relationship between CVR and VE-CO2...................................................................................................................................................... 257 Figure 0-11: Appendix B.7 - CVR and VE-CO2 using DEF ....................................................... 259 Figure 0-12: Appendix B.7 - Single stage CVR and VE-CO2 using DEF ................................... 259 Figure 0-13: Appendix B.7 - Single stage CVR and VE-CO2 using Douglas bag ...................... 260 Figure 0-14: Appendix B.7 - Cerebral flow-mediated dilation (cFMD) .................................... 260 Figure 0-15: Appendix B.7 - Experimental protocol .................................................................. 261 Figure 0-16. Supplemental Figure 1. Protocol schematic ........................................................... 265 Figure 0-17. Supplemental Figure 2. Trans-cerebral plasma nitrite exchange prior to and following haemodilution. ............................................................................................................ 265 Figure 0-18. Supplemental Figure 3. Blood gases during hypercapnia prior to and following haemodilution. ............................................................................................................................ 266 xv List of Abbreviations BA – brachial artery BBB – blood-brain barrier CA – carbonic anhydrase Ca2+ – calcium ion CaCO2 – content of arterial carbon dioxide CaO2 – arterial content of oxygen CBF – cerebral blood flow CDO2 – cerebral delivery of oxygen cFMD – cerebral flow-mediated vasodilation (also called cSMD – cerebral shear-mediated vasodilation in places) CjvCO2 – content of jugular venous carbon dioxide CMRCO2 – cerebral metabolic rate of carbon dioxide CMRO2 – cerebral metabolic rate of oxygen CO – cardiac output CO2 – carbon dioxide CVC – cerebrovascular conductance CvO2 – venous content of oxygen CVR – cerebrovascular reactivity to carbon dioxide EC – endothelial cell eNOS – endothelial nitric oxide synthase FMD – flow mediated dilation gCBF – global cerebral blood flow GTN – glyceryl trinitrate [H+] – concentration of hydrogen ions H2CO3 – carbonic anhydrase HCO3- – bicarbonate ion ICA – internal carotid artery IJV – internal jugular vein iNOS – inducible nitric oxide synthase xvi MCA – middle cerebral artery MCAv – middle cerebral artery blood velocity nNOS – neuronal nitric oxide synthase NO – nitric oxide NO2- – nitrite NOS – nitric oxide synthase NVC – neurovascular coupling NVU – neurovascular unit O2 – oxygen OEF – oxygen extraction fraction PaCO2 – arterial partial pressure of carbon dioxide PaO2 – arterial partial pressure of oxygen PbtCO2 – brain tissue partial pressure of carbon dioxide PbtO2 – brain tissue partial pressure of oxygen PCA – posterior cerebral artery PCaCO2 – plasma content of arterial carbon dioxide PCAv – posterior cerebral artery blood velocity PCjvCO2 – plasma content of jugular venous carbon dioxide PetCO2 – end-tidal partial pressure of carbon dioxide PetO2 – end-tidal partial pressure of oxygen PjvCO2 – jugular venous partial pressure of carbon dioxide PjvO2 – jugular venous partial pressure of oxygen PvCO2 – venous partial pressure of carbon dioxide PvO2 – venous partial pressure of oxygen Q – blood flow QICA – internal carotid artery blood flow ROS – reactive oxygen species SaO2 – arterial oxygen saturation of hemoglobin SNA – sympathetic nervous activity SpO2 – peripheral oxygen saturation of hemoglobin xvii SRAUC – shear rate area-under-the-curve TJ – tight junction V̇A – alveolar ventilation VA – vertebral artery VC – vascular conductance V̇E – minute ventilation V̇E-CO2 – ventilatory response to carbon dioxide VEGF – vascular endothelial growth factor VSMC – vascular smooth muscle cell xviii Acknowledgements I am forever thankful to Professor Phil Ainslie, for having confidence in me, for always pushing and encouraging me (and for having the balls to take on a philosophy student!). Your approach to science has taught me to be ambitious and questioning, to strive, and to compromise only as a last resort. But, your approach to life has taught me more important lessons, more than I can say. Thank you. Dr Ryan Hoiland, thank you for sharing so much of your time and work with me, but most of all for enduring my unintelligible emails on cerebral haemodynamic processes I didn’t understand. Dr Josh Tremblay, likewise, thank you for the insights you provide, and remember: act normal. Hannah Caldwell (soon to be Dr), your grasp of acid-base balance is awe-inspiring and I thank you for your insights also. To my friends, the Ainslie Lab, thank you for agreeing to mentor an idiot in the first place. Thank you for all of your help, encouragement, and understanding; for sharing your ideas and your time, and for all the feedback which contributed to the studies herein. The studies in this dissertation would not have been possible without you, to say the least! I have learned more than just lab techniques and research methods from each of you. I am improved as a person for having known you. Amidst the calm-chaos of the Ainslie Lab, this PhD has been without doubt one of the most fulfilling experiences, one that I will never forget. To all the Ainslie Army, past and present, near and far, I thank each and every one of you. I thank my parents for helping me make the right decision in 2018 when I didn’t know what to do. You’ve always encouraged me to follow my interests and figure it out as I go. Still figuring it out, still going. I thank my loving partner Nique, without whom I’m sure the journey of this degree would have been beyond me! Thank you for cheering me on but also giving me a talking to when I needed it, and for supporting me in all the ways you do. To my supervisory committee members, Professors Greg DuManoir and Chris McNeil, I thank you for always being open to discussion. Additionally, thank you for holding on; my near constant changes in direction and interest resulted in more than a slight change of research question, and I’m sure some confusion on your part. Professor Trevor Day, thank you for your friendship, bonhomie, and invigorating scientific discussion. Cheers! Jay xix Dedication In loving memory of Gabriel Ursus Dix (1995-2021) “People will forget what you said, people will forget what you did, but people will never forget how you made them feel.” Maya Angelou “It’s a marathon not a sprint. But do you know who wins marathons? People who run fast!” “It’s just a teally good time.” INTRODUCTION 1 Chapter 1: Introduction As the seat of consciousness, proper and continued activity of the human brain is essential. Given the near constant energy demand and because it contains little to no essential energy substrate stores, the brain requires a large supply of oxygen- and nutrient-rich blood to remain functional. Even though the brain is ~2% of the total body mass it receives ~13% of cardiac output (CO) at baseline (Table 1-1). The typical adult healthy brain is supplied with 50 mL of blood/ 100 g of brain tissue/ min, compared to the upper limb, for instance, which requires around 3 mL/min at rest. This great relative flow in the brain is because the brain requires ~3 mL of O2/ 100 g/ min and ~5 mg of glucose/ 100g/ min. Per the Fick Equation, the total uptake of a substance by a tissue is equal to the product of blood flow to the area and the arterial to venous difference of the substance. In essence, this equation outlines the importance of blood flow control in the possible uptake of cerebral tissues, cerebral blood flow (CBF) being one of the two main factors determining total possible uptake of a substance. Thus, the primary factors in determining continued cerebral functioning include CBF, the arterial content of a substance, and the cerebral extraction of such (for Fick Equations, see Appendix: Equations:). Therefore, appropriate vascular regulation of CBF is of the utmost consequence; even minor dysfunction, malformation, or hemorrhage of any intra- or extra-cranial vessel can elicit considerable adverse effects, in both the short- and long-term. The various requirements of vessels throughout the body to be able to respond to constantly changing stimuli (pressure, shear stress, blood gases, autonomic innervation, various endocrine factors) mean that experimentally testing various features of vascular function also require targeted and specific assessments. Table 1-1 – Approximate circulation blood flow values in humans at rest Vasculature Blood flow mL/min (% of total CO) Cerebral 750 13 Coronary 250 4 Splanchnic 1400 24 Renal 1100 19 Skeletal muscle 1200 21 Skin 500 9 Other organs 600 10 CO, cardiac output. Data from (Nichols et al., 1998) INTRODUCTION 2 1.1 Aims and hypotheses The aim of this dissertation was to characterize the contribution of arterial CO2 to vascular regulation of CBF. Equivalent responses in peripheral vessels were investigated in tandem to aid in delineation of cerebral vascular control by contrast. The overarching hypotheses were: - Large artery shear-mediated regulation of tone in the cerebral vasculature bears similarity to peripheral responses; nevertheless, these responses represent a smaller percentage of available vascular reserve in the cerebral extra-cranial arteries compared to the peripheral arteries. - Large artery responses to CO2 are more sensitive and of a greater magnitude in the cerebral vasculature than in the peripheral vessels. 1.2 Individual study aims and hypotheses: 1.2.1 Study 1 – Internal carotid artery and brachial artery shear-dependent vasodilator function in young healthy humans This study compared endothelium-dependent vasodilation between the internal carotid artery (ICA) and brachial artery (BA), as well as endothelium-independent vasodilation between the arteries. We hypothesized that endothelium-dependent and -independent vasodilation of the ICA and BA would be greater in the BA than the ICA and that these responses would not be nor correlated between vessels. 1.2.2 Study 2 – Stability of cerebrovascular CO2 reactivity following attainment of physiologic steady-state The purpose of this study was to assess potential time-dependency of the cerebrovascular reactivity to CO2 (CVR) response following the initiation of a CO2 stimulus. We conducted retrospective data analyses such that CVR values were extracted from different time points during a 5-minute CO2 exposure. We hypothesized that: 1) there would be no difference in CVR values when calculated with CBF measures at different time points following the attainment of physiologic steady-state and, 2) once steady state was achieved there would be no difference in values when 60 s and 30 s means were extracted. 1.2.3 Study 3 – Cerebral O2 and CO2 transport in isovolumic haemodilution: compensation of cerebral delivery of O2 and maintenance of cerebrovascular reactivity to CO2 Whether impairments in CBF regulation in pathologic anemia are related to anemia per se or a consequence of underlying pathology remains to be determined. This study investigated the influence INTRODUCTION 3 of acute reductions in CaO2 (anaemia via haemodilution) on global CBF (gCBF) and CVR in the absence of pathology. We hypothesized that acute experimental haemodilution in healthy humans would increase CBF and CVR, while cerebral delivery of O2 (CDO2), CO2 washout, and cerebral metabolism would be preserved. 1.2.4 Study 4A – Brachial artery responses to acute hypercapnia: the roles of shear stress and sympathetic tone This purpose of this study was to determine BA responses during hypercapnia, while accounting for the influences of shear stress and 𝛼-1 adrenergic innervation. We conducted within-subject bilateral BA Duplex ultrasound assessment during an acute steady-state hypercapnic stimulus; with increases in shear stress in one BA attenuated via manual arterial compression (compression), and shear stress in the other BA unhindered (control). We hypothesized that hypercapnia would cause vasodilation in the control but not the compressed BA. We repeated the experiment in a placebo-controlled, double-blind, and randomized design with 𝛼-1 adrenergic receptor blockade (Prazosin). We hypothesized that Prazosin would obviate any sympathetically-mediated constraint of vascular tone and allow for greater presentation of any shear stress- or CO2-mediated vasodilation. We also reasoned that with Prazosin, BA flow and diameter would be elevated in both arms compared to during placebo, and that during hypercapnia with Prazosin, the control arm would dilate to a greater degree than the compression arm. 1.2.4.1 Study 4B – Adrenergic nervous activity in cerebral endothelium-dependent function and reactivity to hypercapnia Despite extensive study of the influence of sympathetic nervous activity (SNA) on the control of CBF, the role of SNA on CVR during changes in the arterial partial pressure of CO2 (PaCO2) remains somewhat controversial (Brassard et al., 2017). In addition to bilateral BA assessment during Study 4A, we also conducted simultaneous and continuous ICA duplex ultrasound during CO2. Here, we also included assessment of ICA shear-mediated endothelium-dependent function - with and without 𝛼1 adrenoreceptor inhibition - in order to determine the role of sympathetically-mediated maintenance of vascular tone on macrovascular function during an acute cerebral hypercapnic challenge. LITERATURE REVIEW 4 Chapter 2: Literature Review In this chapter, topics relevant to the following studies within this dissertation are described. First, before exposition on the relevant physiology, an initial short section summarizes why the gas carbon dioxide (CO2) is a significant environmental consideration (2.1). Second, the fundamental vascular construction will be outlined (2.3) in order to lay the groundwork for a section on the cerebral vasculature and neurovascular unit (2.4). Third, with focus on CO2 (2.5.1), key stimuli that act to regulate cerebral blood flow (CBF) are described (2.5). Fourth, key stimuli that act to regulate peripheral vascular blood flow are described (2.6). Finally, three assessments of vascular function often used in research and clinical settings, and which are crucial in the subsequent dissertation chapters, are described with regard to physiological mechanisms and with some background on common usage and implications (2.7). 2.1 Atmospheric CO2: history and projections Atmospheric CO2 concentrations fluctuate naturally across annual and monthly cycles with the short-term carbon cycle due to global carbon sequestration (e.g., sedimentary soil, seawater, and plant capture) and carbon emissions (e.g., volcanoes, biomass decomposition, fossil fuels), as well as rising and falling with ice and thaw ages (Figure 2-1). However, since the beginning of the industrial revolution, CO2 concentrations have been rising as a direct result of anthropogenic emissions. Subterranean stores of carbon in rocks, sediments, and biomass debris - i.e., fossil fuels - that outweigh the current biomass CO2 some 10000-fold, have been rapidly mined, exposed, and burned rather than remaining buried and slowly releasing CO2 into the atmosphere over millions of years. In the year 1950, the global atmospheric CO2 average surpassed 300 ppm (0.03%) and continues to rise to this day (Figure 2-1). Figure 2-1. Atmospheric CO2 concentrations within the last 800000 years Atmospheric CO2 data measured in air bubbles ice cores. Kya, thousands of years ago; ppm, part per million. Data from (Bereiter et al., 2015). 0200400600800150200250300350400Time (Kya)CO2 (ppm)Present day CO21950, CO2 reaches 300 ppmLITERATURE REVIEW 5 While the average global CO2 concentration is controlled by many factors, the rise in the contemporary era is almost solely the result of anthropogenic fossil emissions (Höök & Tang, 2013) (Figure 2-2). Figure 2-2. Atmospheric CO2 and fossil fuel related CO2 emissions Left y-axis: historical (1959-2020) fossil fuel-related CO2 emissions in megatons per year (Mt CO2/year) data from the Integrated Carbon Observation System (Friedlingstein et al., 2022), total for the world (black line +/-1SD), and by major continental area. Major historical events leading to decreases in CO2 emissions highlighted in red. Right y-axis: historical (1959-2021) Mauna Loa atmospheric CO2 concentration (ppm) data from the National Ocena and Atmospheric Administration [Dr. Pieter Tans, NOAA/GML (gml.noaa.gov/ccgg/trends/) and Dr. Ralph Keeling, Scripps Institution of Oceanography (scrippsco2.ucsd.edu/)]. Inlay: Historical (1950-2020) and growth projection (2020-2100) population data for the world and by major continental area from (Roser et al., 2013). Population growth is also a major driver of CO2 emissions (Höök & Tang, 2013). Figure 2-3. Global CO2 projections Global CO2 projection data from the Intergovernmental Panel on Climate Change Representative Concentration Pathway (RCP) projections for total atmospheric CO2 concentrations with best (RCP2.6) to worst (RCP8.5) scenarios of carbon emissions over the next ~80 years. Data and projections originally published in 2014 (marked at the dotted line) (Prather et al., 2014). 1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 2015 20200500010000150002000025000300003500040000300320340360380400420440YearFossil emissions (Mt CO2/year)AfricaAsiaCentral AmericaEuropeMiddle EastWorld1973 Oil CrisisCollapse of Soviet UnionGreat RecessionCovid-19Global CO2 (ppm)Mauna Loa monthly averageCO2 (ppm)North AmericaOceaniaSouth America1975 2000 2025 2050 2075 210002×1094×1096×1098×1091×1010YearPopulation2000 2020 2040 2060 2080 21004006008001000YearGlobal CO2 (ppm)RCP2.6RCP4.5RCP6.0RCP8.5LITERATURE REVIEW 6 The worst-case scenario predicts that the planet will reach CO2 concentrations of 500 ppm (0.05%) by ~2040, 800 ppm (0.08%) by ~2080, and 1000 ppm (0.1%) by the beginning of the next century (Figure 2-3). These changes may seem small, however, exposures to 1000 ppm (0.1%) and above begin to effect cognition (Du et al., 2020; Zhang et al., 2021), with the physiological effects worsening with greater concentrations. 2.2 CO2 toxicity While CO2 is a normally occurring gaseous product of physiological metabolism, it nevertheless has toxic and lethal effects. Fortunately, the ease with which CO2 diffuses through cell membranes in the body means that humans can usually offload the metabolic waste. However, given the worst-case scenario described above (Figure 2-3), within approximately the next hundred years, atmospheric CO2 concentrations will reach levels that will begin to have measurable physiological effects. The effects of prolonged exposure across the lifetime, even to very low concentrations, lacks investigation in humans; however, there is evidence in animals to indicate that any such effects would likely be detrimental for humans. For example, animal models of chronic exposure to CO2 suggest a causative role for perinatal developmental abnormalities (Haring, 1966; Sprenger & Milsom, 2021; Wyrwoll et al., 2022), bone calcium depletion (Schaefer et al., 1980), deterioration of cognitive abilities akin to hyperactivity and increased anxiousness (Burgraff et al., 2018) associated with increased corticosterone (Wyrwoll et al., 2022), increased neuronal dysfunction (Burgraff et al., 2019), and negative impacts on both lung structure and function in female mice (Larcombe et al., 2021). No human research exists investigating the effects of chronic CO2 in humans during pre-natal development and across the lifetime. What modern research does exist in humans investigating prolonged mild hypercapnia often focuses on the effects in the setting of sedentary behaviour (and therefore is likely confounded) and is usually only of short to mid durations (e.g., >1 hour but <3 hours; e.g., Headid et al., 2020; Park et al., 2022). Those studies that do include longer exposures, varied activity levels, and higher concentrations of CO2 (Consolazio et al., 1947; Faucett & Newman., 1953; Schaefer, 1963, 1979; Cutler et al., 1964; Schaefer & Baker III, 1969; Sinclair et al., 1971) are mostly from Naval submarine environmental studies. These latter pioneering studies have indicated that healthy participants can successfully live and work in environments of 1 to 5.4% (10000-54000 ppm) for anywhere from 3 to 43 days, though the potential effects post-exposure where not examined: “Repetitive psychomotor and psychologic testing was done daily. No decrements were noted in any of these studies and would indicate that the CO2 atmosphere had no adverse effects. It was concluded that a 5-day exposure to 3% CO2 was easily adapted to and failed to adversely affect LITERATURE REVIEW 7 moderate exercise ability, psychomotor performance, or mineral metabolism.” - Glatte et al. (1967) Despite the above extract, the fact of CO2 toxicity cannot be discounted (National Research Council, 1996; Langford, 2005; Guais et al., 2011). For example, in dogs exposed to 100% CO2, electroencephalogram signals became isoelectric within 30 seconds, and death occurred within three minutes; dogs given a gas mixture of 50% CO2 and 50% O2 (to account for the inevitable hypoxia in the 100% CO2 dogs) nevertheless suffered circulatory breakdown – death – within 30 to 90 minutes of exposure (Ikeda et al., 1989). In humans, case reports of immediate loss of consciousness with exposure to liquid CO2 spillage (Halpern et al., 2004), death from dry ice vapour (Hsieh et al., 2005), and deaths attributed to CO2 accumulation in the holds of fishing boats (Dalgaard et al., 1972) denote the noxiousness of CO2. Miners, submariners, welders, construction workers, astronauts - the dangers are great in any situation where CO2 could accumulate in high concentrations, because of the substantial and swift effects on neuronal, respiratory, cardiovascular, and cerebrovascular systems. Fundamentally, the danger of excess molecular concentrations of CO2 in tissues stems from the inter-relationship between CO2 and hydrogen ion concentration ([H+]). Almost all proteins in the body are susceptible to changes in hydrogen bonds due to an altered milieu of H+, which inevitably results in physical-chemical deformation, thereby changing the shape of the protein and impairing function (Broda et al., 1959; Kraig et al., 1987). The cardiovascular and cerebrovascular effects constitute the main subject matter of this dissertation, though the subsequent material is predominantly concerned with the clinical prognostic applications of deliberate short duration hypercapnic exposure. 2.3 Fundamental circulatory anatomy and vascular constitution The cardiovascular system serves to transport essential substrates and signals across the body and remove metabolic waste by-products, as well as aiding in several homeostatic systems such as thermoregulation. The circulatory system accomplishes this with a pump – the heart - and vast complexes of parallel and serial distribution and collection ducts – the arterial and venous circulations. While simple transport could be managed by passive tubes, the structure of vessel walls throughout the circulatory system allows for dynamic and responsive coordination of blood flow. Through the heart, appropriate directional flow is managed by muscular walls and valves. Blood collected from the systemic circulation enters the right atrium, is pumped into the right ventricle and subsequently ejected through the pulmonary arteries to be dispersed through the pulmonary vasculature wherein - via the alveolar microvasculature - it is oxygenated and the metabolic by-product CO2 is off-loaded. Upon return to the heart through the pulmonary veins, the oxygen rich blood is then collected LITERATURE REVIEW 8 into the left atrium – then left ventricle – and finally dispersed across the whole body through the aorta. The arterial system progressively branches, vessels becoming narrower and walls becoming thinner upon each branching. From the largely elastic aorta, peripheral arteries become progressively more muscular until the arterioles, which are the primary means of resistance development throughout the vascular tree. Vascular smooth muscle cells (VMSC) are almost completely absent at the level of the capillaries (Figure 2-4). Ultimately, the circulatory system’s raison d'être - the foundation for its existence - involves the capillary complexes within organs and tissues, wherein the exchange of gases, metabolic substrates, ions and other signalling and messaging factors occurs from intra-luminal to extra- and intra-cellular spaces, allowing for continued metabolism and survival of those tissues. Capillaries then anastomose and reconnect into venules and progressively larger veins which conduct blood towards the heart to be recirculated. Arteries and veins share some common features of structure, however artery walls are much thicker and generally more functionally active due to higher pressures and higher requirement to actively direct blood flow, whereas veins are larger with thinner walls and lower pressures. The venous portion of the circulatory system contains the majority of the blood volume in the body. The following sections will describe the general wall structure shared by most arteries, from the outer to innermost layers. Figure 2-4. Constitutions of vessel walls across the circulatory system. Typical diameter, wall thickness and relative amount of principle components of vessel walls across the circulatory system. Redrawn from (Burton, 1954). 2.3.1 Tunica externa The outermost layer of arteries consists of collagenous, fibrous, and elastic component fibers which provide structural support within which the inner layers exist. In the largest arteries and some large veins, very small blood vessels (vasa vasorum) are also present within the tunica externa, which provide blood to the vessels themselves as the walls of these vessels are thick enough that luminal blood does not diffuse nutrients to all of the cells. Elastin fibers which provide support and promote pressure wave Aorta Artery ArterioleTerminalarterioleCapillary Venule Vein Vena cavaEndotheliumElastic tissuesSmooth muscleFibrous tissuesTypical diameter 25mm 4 mm 30 µm 10 µm 8 µm 20 µm 5 mm 30 mmTypical wall thickness2mm 1 mm 6 µm 2 µm 0.5 µm 1 µm 0.5 mm 1.5 mmLITERATURE REVIEW 9 propagation along artery walls, are abundant in the largest arteries such as the aorta, where elastic wave propagation allows downstream resistance arteries and arterioles to modulate pressure and velocity. 2.3.2 Tunica media The mid-layer of arterial wall structure consists mostly of VSMCs, as well as more elastic and collagenous tissues in the largest arteries. Smooth muscle provides arteries and arterioles with the capacity to actively contract or relax and dilate. 2.3.3 Tunica intima The innermost layer of artery walls consists of endothelial cells (EC) and an internal lamina upon which the endothelial layer resides. The EC layer exists evenly throughout the vascular system and is the thickness of merely a single cell; yet - as in the other tunicae - the proportions of other constituent components of the tunica intima depend on vessel type. The endothelium is ubiquitous throughout the circulation and although the total weight is only ~1kg in an average adult human, it covers a total surface area between 4 to 7 km2 (Aird, 2013). The endothelium has been recognized as more than a passive barrier for decades, as the functional mechanisms through which EC contribute to vessel tone have been uncovered (e.g., Pohl et al., 1986). The endothelium plays a critical and integral role in cardiovascular homeostasis through regulation of vascular tone, immune function, inflammatory responses, and vessel permeability, among many other functions (Aird, 2006, 2007). Dysfunction of ECs is a precursor to - and pivotal aspect of - several pathophysiological conditions (Yamashiro & Yanagisawa, 2020), such as Alzheimer’s dementia (Wang et al., 2018), brain injury following subarachnoid hemorrhage (Lenz et al., 2020), atherosclerosis (Davies, 2009), and many other cerebrovascular diseases (Toda, 2012; Iturria-Medina et al., 2016; Kalaria, 2016; Wolters et al., 2016, 2017). Blood flow through an artery is regulated in large part by the endothelium (Furchgott & Zawadski, 1980) via several mechanisms which mediate vascular tone; predominant amongst these is shear stress (see 2.6.3). Nitric oxide (NO) is produced tonically in ECs, VSMCs, and the blood via a number of mechanisms and is a primary regulator of vascular tone through modulation of VSMC Ca2+ influx, most of which is directly regulated by ECs. The endothelium also regulates the release and scavenging of several other vasoactive paracrine factors such as angiotensin-II, endothelin-1, reactive oxygen species (ROS), vascular endothelial growth factor (VEGF), prothrombotic factors and anti-inflammatory factors, as well as factors influential to leukocyte adherence and platelet aggregation (Yanagisawa et al., 1988; Zieman et al., 2005; Neves et al., 2018). LITERATURE REVIEW 10 Throughout the circulation, embryonic origins determine distinct phenotypes of ECs that create specialized EC layers specific to organs. These differences are evident even within organs between the large arteries and microvasculatures (Aird, 2007; Augustin & Koh, 2017; Minami et al., 2019). The paracellular space where ECs meet can be securely bound such that a tight junction (TJ) is created and ion or ligand transport can only occur transcellularly, as in the brain (see 2.4.4). Several other specialized capillary EC organotypes exist, differentiated during the embryonic stages of development and further configured according to hemodynamics stresses, that contribute to the specific nature of water, small solute, and lipid transport from the blood into surrounding tissues (Augustin & Koh, 2017; Minami et al., 2019). The endothelium can be continuous, as in the brain wherein interstitial transport is tightly regulated (Obermeier et al., 2013; Zhao et al., 2015): fenestrated, as seen in filtration sites such as the glomerular apparatus of the kidneys where intracellular membranous pores of basal lamina break up the contiguous EC layer allowing for the passage of small solutes and peptides. ECs can also be sinusoidal, as seen in organs of extensive exchange between blood and tissue (e.g. liver and several endocrine organs) with gaps between ECs rather than pores and inadequate basal lamina coverage such that there is free exchange of water and large solutes (Strauss et al., 2017). Organotypic EC differentiation is evident throughout the vasculature. For example, while the choroid vessels of the eye are similar to the cerebral vasculature in that there is a great capillary density and the vessels are surrounded by pericytes and glial cells which form a brain-retinal barrier, the EC layer within the retinal barrier form phenotypes unique from the cerebrovasculature. Schlemm’s canal in the anterior chamber of the eye contains a specialized EC type allowing for plentiful aqueous humour outflow by high-rate transcytosis of large vacuoles (transcellular packages) while maintaining the BRB (Jabłońska, 2016; Augustin & Koh, 2017). Even within Schlemm’s canal ECs differ in morphology between the inner and outer luminal walls (Jabłońska, 2016). 2.3.3.1 Historical sidebar - Andreas Vesalius Some 500 years ago, Flemish anatomist Andreas Vesalius eschewed long standing tradition to describe in detail human anatomy from first-hand dissection. Lauded as one of the most influential figures in the history of physiology, Vesalius rejected the commonplace dependence on outdated anatomical texts of the Galenic orthodoxy. Cultural, scientific, and religious norms had produced an environment wherein most anatomists did not themselves dissect bodies, but instead used assistants under direction, and even then, most investigation acted to solidify the paradigm rather than refute the errors of Galen. In 1543, Vesalius published De Humani Corporis Fabrica Libri Septem, (“On the fabric of the human body in LITERATURE REVIEW 11 seven books”) a comprehensive atlas of the human body in seven books which disputed over 300 inaccuracies in Galen’s tradition. There are some mistakes and misrepresentations – see Figure 2-5 – e.g., the internal and external carotid arteries are dickered, and both carotid arteries branch from a common trunk off the aorta rather than separately, characteristic of simian anatomy rather than human. These observations indicate that, although Vesalius did overturn some Galenic canon, he did not completely throw it out, and still looked to simian anatomy. While Vesalius’ account of the body has not stood the test of time, nonetheless, the improvements on previous understanding was prodigious and subsequently transformed medical practices (Barr, 2015). Vesalius’ account was the first to fully describe the whole course of all human vessels through the body and created the earliest comprehensive depiction of the vascular system (Saunders & O’Malley, 1950). However, the dogma of the time regarded the venous system as more important than – and isolated from - the arterial system, fulfilling different functions; and this is represented in Vesalius’ anatomical plates by the detail of the veins and the lack thereof in the arterial diagrams (see Figure 2-5). Figure 2-5. Integra to tius magnae arteriae & Venarum arteriarumque cerebri “Integra to tius magnae arteriae ab omnibus partibus liberae delineation” translated as “complete delineation of the great arterial system”, and “Venarum arteriarumque cerebri”, “cerebral arteries and veins”. The plate illustrating the venous system was far more comprehensive than this one of the arterial system, indicating the paradigm of the era that the venous system was more important in body processes and circulating the vital spirit – e.g., note the emphasis on the cerebral veins in the illustration depicted both systems. The images are in the public domain. LITERATURE REVIEW 12 2.4 Cerebral vascular anatomy The overarching layout of the cerebrovasculature is one of multiple redundancy and collateral supply. In resting states, blood supply to the various cerebral territories (anterior, posterior, etc.) comes from the predominant contributing artery, however, if one delivery path is blocked or otherwise unable to provide the required blood supply, other vessels can supply a portion of the requirement via many anastomoses (see Figure 2-6 for the collateralization of CBF). From the aorta, the right innominate artery branches into the right subclavian and common carotid artery, while the left common carotid artery branches directly from the aorta separately from the left innominate artery. The subclavian arteries from the innominate arteries supply the vertebral arteries (VA) which anastomose into the single intracranial basilar artery following entry into the intracranial space, supplying the posterior portion of the cerebral circulation through the posterior cerebral artery (PCA). The common carotid arteries bifurcate into the internal and external carotid arteries (ICA and ECA, respectively) which supply the cerebral and facial vasculatures, respectively. Approximately 70% of global cerebral blood supply flows bilaterally through the ICAs (Zarrinkoob et al., 2015) and into the middle and anterior cerebral arteries (MCA and ACA, respectively). The left and right ACAs and MCAs are networked together with both PCAs via posterior communicating arteries to create the Circle of Willis, an arrangement allowing for blood to be collaterally supplied from other routes should inflow fail through one or more of the four major suppliers (VAs and ICAs; see Figure 2-6). Figure 2-6. Organization of cerebral blood flow and collateral blood flow CBF is supplied by four extracranial arteries (two ICAs and two VAs). Intracranially, the arteries anastomose to become the Circle of Willis which then branches into the major intracranial vessels perfusing various territories: two anterior cerebral arteries (ACA), two middle cerebral arteries (MCA), and two posterior cerebral arteries (PCA). Normally, blood is supplied to cerebral tissues via the dominant artery supplying the area (e.g., frontal lobes supplied by ACA; occipital lobe supplied by PCA). The extensive interconnectivity of the cerebrovasculature allows for considerable collateral flow in the case of obstruction. From (Hoiland et al., 2019). LITERATURE REVIEW 13 From the major cerebral arteries, many deep perforating arteries occupy and supply the deep parenchymal tissues of the brain, while the leptomeningeal branches (also known as pial vessels) create an anastomotic complex on the surface of the cerebral hemispheres from which cortex perforating vessels descend. Regulation of blood flow via changes in vessel resistance is controlled at every level of the cerebral vasculature, the estimated contributions of the various levels to flow control are indicated in Figure 2-7. Figure 2-7. Segmental vascular resistance of the cerebrovascular tree The component (percentage) of the total vascular resistance each cerebrovascular segment offers to blood flow, which reflects their potential for flow control, is indicated. Therefore, vessels outside of the brain are responsible for ~40% of the resistance and vessels within the substance of the brain for ~60% (Stromberg & Fox, 1972; De Silva & Faraci, 2016). Reproduced with permission from (Iadecola, 2017). These pial vessels knit into the surface and penetrate the cortex through invaginations called perivascular - or Virchow-Robin - spaces. The penetrating arterioles are also collaterally anastomosed through the microvasculature within the parenchymal space, to create a network providing protection against vascular insult or obstruction. These networks of arterioles each radiate a rich net of capillaries into the parenchyma, the reach of which overlays regions from other nets, such that, as with the larger collateral networks, the occlusion (by thrombosis or reduction below critical closing pressure) of one or more capillaries does not completely preclude blood supply to an area. Due to the capillary density of the brain, neurons are typically no further than 50 μm from the nearest capillary (Schlageter et al., 1999; Nicholson, 2001). The capillary density of the parenchyma is somewhat heterogenous, with gray LITERATURE REVIEW 14 matter holding a greater density than white matter (Schlageter et al., 1999), being thereby better supplied and having a greater local blood flow and glucose utilization (Klein et al., 1986). 2.4.1 The blood-brain barrier and neurovascular unit Given the sensitive nature of neural cells to their microenvironment, molecular exchange between the blood and brain tissues or its fluid spaces must be tightly regulated. In order to maintain control of brain tissue concentrations of all relevant molecules, the blood-brain barrier (BBB) isolates the brain from the circulation. Three barriers form the BBB; the vascular endothelium; the choroid plexus epithelium; and the arachnoid epithelium. While all are needed for complete protection, the extensive meshing of the vasculature with the brain tissues means that the BBB formed by the EC layer exerts the greatest control of the environment of the brain (as such, and given the topic of this dissertation, only the vascular BBB will be considered in any depth herein, without great recourse to the BBB at the choroid plexuses). It is now well appreciated that, while the cerebral circulation shares fundamental characteristics with all other vasculatures of the body, the differences of function and structure create a more integrated and active network than elsewhere (Zhao et al., 2015; Iadecola, 2017; Ross et al., 2020). In the brain, the typical components of vessel wall structure present with characteristics such that they form a semi-permeable barrier, the BBB. Permeability across the vessel wall from luminal to interstitial spaces of ligands, substrates, and solutes is controlled by ECs. Just as in the rest of the body, the luminal surface of all vessels within the cerebral circulation consists of the EC layer, however, TJ between individual ECs in the BBB create a complete and continuous physical barrier, ensuring that all inter-compartmental (i.e., intravascular to interstitial) movement requires transcellular transport. See Figure 2-8 for BBB overview. LITERATURE REVIEW 15 Figure 2-8. The neurovascular unit Panel A: general cerebrovascular structure, from pial vessel to capillary network. The neurovascular unit (NVU) is comprised of endothelial cells, vascular smooth muscle (SMC) cells, and mural cells such as pericytes and astrocytes. Endothelial and mural cells of the BBB vary in some aspects of both phenotype and molecular function at different levels of cerebral vasculature, creating heterogeneity between arterial and venous areas. Penetrating arteries (left inset), are formed of endothelial cells which are enveloped by 1 to 3 layers of SMCs. Cerebrospinal fluid (CSF) fills the Virchow-Robin space between pia mater and the basement membrane, while extracellular fluid (ECF) fills the space between astrocytes and neurons. Following the branching of the arterial system into arterioles, SMCs are reduced to a single layer, while endothelial cells remain continuous. At the capillaries (right inset), pericytes and endothelial cells share a basement membrane. Astrocytic endfeet are positioned adjacent to arteriolar and capillary walls. Panel B: cells that constitute the NVU and regulate cerebral blood flow, BBB integrity. Reproduced with permission, from (Sweeney et al., 2019). While O2, CO2, and water can diffuse freely across the lipid membranes of ECs, other molecules can be moved across cell membranes by specific active transport systems (see Figure 2-9). LITERATURE REVIEW 16 Figure 2-9. Transport across the BBB. Summary of molecular transport across the blood brain barrier. Small ions, water, and small lipophilic molecules can cross the blood-brain barrier via ion channels, or are transported passively (i.e., simple diffusion) across cell membranes. Some polar molecules such as glucose, amino acids, organic anions and cations, and nucleosides, can cross the BBB via carrier-mediated transport, and this mode of transport can be uni- or bi-directional. Other modes of transcytosis include receptor-mediated (which requires binding of specific ligand to membrane receptors) or adsorption-mediated transcytosis (which requires binding of non-specific ligands), and efflux pumps. Plasma proteins, e.g., albumin, can be transported by cationization which increases their uptake by adsorptive-mediated endocytosis and transcytosis. MCT-1, monocarboxylic transporter-1; IgG, immunoglobulin G; GLUT-1, glutamate transporter 1; MRP 1,2,4,5, multidrug resistance-associated proteins; BCRP, breast cancer resistance protein. Reproduced with permission from (Wong et al., 2013). In addition to ECs, two other cell types play substantial roles in the BBB – astrocytes and pericytes (along with the basement membrane upon which EC and pericytes are situated). At the level of the microvasculature, astrocytes, pericytes, and ECs are also commonly referred to together as the neurovascular unit (NVU) given their interwoven activity and functions. The NVU is a functional unit responsible for regulation of minute local blood supply in response to neuronal energy demand, and as such plays a great role in regional blood flow responses to metabolic demand. Cerebral EC and the BBB generally are crucial in mediating NVC and CBF regulation mechanisms (Cleary et al., 2020; Longden et al., 2021). Blood–brain barrier breakdown is an early biomarker of human cognitive dysfunction (Zhao et al., 2015; Nation et al., 2019; Sweeney et al., 2019). Given the complexity of mechanisms which contribute to BBB maintenance, and the degree with which ECs, astrocytes, pericytes, interneurons, perivascular macrophages, and neurons are intricately woven, it is LITERATURE REVIEW 17 unsurprising that the upkeep and health of the cerebrovasculature relies on synergistic functions of all of these cell types. In the following, discussion of the three major components of the NVU/BBB will lay the foundation for further discussion of the importance of the endothelium in the cerebral circulation in the context of health, adaptation, and disease. 2.4.2 Pericytes Pericytes - originally named Rouget cells after one of the founders (Eberth, 1871; Rouget, 1873) - are mural contractile cells that, in conjunction with ECs and the basement membrane, form part of the structure of capillaries throughout the body, and regulate capillary diameter and therefore capillary blood flow (Hall et al., 2014; Gonzales et al., 2020). Pericytes lie on the abluminal surface of ECs with protruding nuclei and extensions longitudinally aligned with the vessel lumen, as well as processes the enwrap the capillary perpendicular to the lumen axis (see Figure 2-9). Pericytes are contractile cells that finely regulate CBF through capillary tone (Peppiatt et al., 2006; Hall et al., 2014; Attwell et al., 2016; Longden et al., 2021), although for a time this was unknown, and was subsequently controversial (Hill et al., 2015) before becoming the accepted theory. Per findings in pre-clinical studies, it was thought that while pericytes could constrict (e.g., Tilton et al., 1979) active vasodilation through local relaxation was the sole remit of VSMCs in terminal arterioles (Fernández-Klett et al., 2010). However, it is now known that pericytes mediate vasodilation of cerebral capillaries via numerous Ca2+ and NO related mechanisms, drawing blood flow to areas of higher demand (Hall et al., 2014; Gonzales et al., 2020; Hartmann et al., 2021; Longden et al., 2021) and are therefore major regulators of CBF through mechanisms related to NVC (see 2.5.4) and CVR (see 2.5.1). Pericytes and ECs interface via numerous adhesive proteins, such as fibronectin (Courtoy & Boyles, 1983), as well as membranous interdigitations - pegs and sockets - forming structural connections, promoting pericytic control of EC tone by securely anchored transfer of force (Ornelas et al., 2021). Along with regulation of capillary diameter, other functions of pericytes include; stabilization of newly formed capillaries during angiogenesis (Chan-Ling et al., 2004), maintenance of neuronal health and function of the BBB antecedent to progressive age-mediated vascular-mediated neurodegeneration (Armulik et al., 2010; Bell et al., 2011) and neuroinflammation. Not only are pericytes able to control blood flow within a capillary, the electrochemical sensitivity with which they can do so allows for segmental fine-tuning of blood flow. For example, post-arteriole pre-capillary contractile pericytes lying at capillary bifurcations receive hyperpolarizing signals from downstream neurons and ECs which allows for branch specific preferential dilation and constriction, thereby regulating red blood cell flow to specific areas and increasing regional efficiency (Gonzales et al., 2020). LITERATURE REVIEW 18 2.4.3 Astrocytes Astrocytes are glial cells that contribute to the structural integrity of cerebral tissues and the NVU as well as to communication between all connected cells by ensheathing the cerebral vasculature in processes and end-feet which form the superficial tissue depth connecting blood vessels and neural tissues (Abbott et al., 2006; Zhao et al., 2015; Dalkara, 2017; Herndon et al., 2017; Iadecola, 2017; Lo, 2017; Ross et al., 2020) (see Figure 2-9). A number of BBB features are maintained by astrocytes, including EC TJ tightness, and the expression and localization of EC transcellular transporters. Astrocytes are integral components of blood pressure sensing in the brain (Marina et al., 2020), potentially contributing to cerebral responses to MAP, as well as complexly involved in neurovascular coupling (NVC) (Marina et al., 2018; Hosford et al., 2022) and generation of respiratory rhythms in neuronal circuits controlling ventilation (Marina et al., 2020). Of particular relevance to the current dissertation, astrocytes perform a critical role in the cerebrovascular response to hypercapnia (Howarth et al., 2017; Hosford et al., 2022), as most glial cell types express acid-sensitive receptors (Sofroniew & Vinters, 2010; Storozhuk et al., 2021; Zha et al., 2022) that variously influence neurotransmission between astrocytes and endothelial cells on one side and neuronal cells on the other. Between the cells of the vasculature and cells of brain tissue (i.e., neurons and various phenotypes of glial cells) is the extracellular space wherein lies the extracellular fluid (ECF) / interstitial fluid (ISF). The space between each cell membrane within the brain is small - ~200 Å (20 μm) - and varied across different regions (Villegas & Fernández, 1966), nevertheless, cumulatively the extracellular space makes up ~20% of total brain volume (Nicholson, 2001). ECF creates an essential medium for diffusion of nutrients and energy substrates from capillaries to cells, as well as being a reservoir of various ions for ionic traffic in and out of cells (Lei et al., 2017). 2.4.3.1 Historical sidebar - Rudolph Virchow The concept of neuroglial cells arose before the discovery of the cells themselves. German pathologist Rudolph Virchow – after whom Virchow-Robin spaces are named along with the anatomist Charles-Phillippe Robin – and other physiologists in the early 1800s began characterizing cell theory by the study of plants, animals, and human cadavers that all tissues are built from matrices of constituent cells. In 1846, Virchow published a collection of papers in which he described connective tissues between the brain and epithelium, forming a cement in the brain, spinal cord, and higher sensory nerves which embedded the nervous elements together (Virchow, 1846). Thus began the first accurate characterizations of neuroglia and of what eventually were coined “astrocytes” due to preliminary LITERATURE REVIEW 19 observations of star-shaped cells. Larger glial cells, including oligodendrocytes, astrocytes, and ependymoglial cells can be categorized by shape and cell processes, although there are a number of phenotypic morphologies of all of these cell types. Astrocytes are more or less star-shaped but this arrangement is often changed with contiguous neuronal cell bodies. Categories of astrocytes include protoplasmic, fibrous, interlaminar, and marginal (Reichenbach & Wolburg, 2004). 2.4.4 Endothelial cells Endothelial cells within the cerebral vasculature display a number of distinguishing characteristics. As mentioned above, ECs throughout the vasculature are phenotypically differentiated according to required function, embryonic origin, and hemodynamic milieu. In the cerebral circulation, junctions between ECs are stitched together more closely than elsewhere such that they create TJ and a continuous physical barrier exists as the first-line of defense of the BBB (Zhao et al., 2015). Cerebral TJs are composed of several protein types - e.g. occludin and claudin - which create junctional adhesion by strands which mesh to correspondent protein strands on neighbouring ECs (Sweeney et al., 2019) . These adhesion molecules and proteins have both intra- and extra-cellular strands. Within ECs, protein strands bind with intracellular actin molecules to anchor neighbouring TJ complexes within the cytoskeleton. See Figure 2-9 for molecular BBB transport, which is predominantly regulated by cerebral ECs, and Figure 2-10 for an overview of the BBB and molecular facets of BBB communication and connection.LITERATURE REVIEW 20 Figure 2-10. Overview of the vascular triad of the Blood-Brain Barrier ECs of the BBB are interconnected via tight junction (TJ), adherens junction (AJ), and gap junction (GJ) proteins. TJs, which form a barrier to fluids, consist of occludin, claudins and junctional adhesion molecules (JAMs) and are connected to F-actin filaments by zonula occludens (ZO-1, ZO-2 and ZO-3) scaffolding proteins. GJs are specialized connections for intercellular communication formed by connexin proteins. AJs consist of VE-cadherin, platelet endothelial cell adhesion molecule-1 (PECAM-1) and Nectin. VE-cadherin is linked to F-actin by catenins, while nectin is linked with F-actin by afadin. Pericytes communicate with ECs via growth factor-mediated signaling [platelet-derived growth factor BB (PDGF-BB); platelet-derived growth factor receptor-b (PDGFRb)], adhesion via N-cadherin binding, and GJs. Pericytes can influence BBB permeability through regulation of gene expression in the ECs provoking TJ protein upregulation, inhibition of transcytosis, and upregulation of brain endothelial specific docosahexaenoic acid (DHA) transporter, a major facilitator domain-containing protein 2A (MFSD2a). Both ECs and pericytes are embedded in the basement membrane (BM) and anchored to it via integrins; Astrocytes regulate expression of matrix metallo-proteinase-9 (MMP-9) in pericytes by secreting apolipoprotein E (ApoE). ApoE3, but not ApoE4, binds to the low density lipoprotein receptor-related protein 1 (LRP1) in pericytes, which suppresses the proinflammatory cyclophilin A (CypA) nuclear factor kB (NFkB) MMP-9 pathway and degradation of TJ andBM proteins causing BBB breakdown. Astrocytes signal ECs by Src-suppressed C-kinase substrate (SSeCKS) to increase TJ protein expression. Release of glutamate, dependent on neuronal metabolism, increases [Ca2+] released by astrocytic endfeet, which regulates vascular tone (see 2.5.4). Glucose enters the brain via the EC transporter Glut1, and enters neurons via Glut3. Glucose is taken up by astrocytes mainly by Glut2 and is metabolized to lactate, which is exported to neurons by the monocarboxylic MCT1 and MCT4 transporters. From (Zhao et al., 2015). LITERATURE REVIEW 21 2.5 Regulation of cerebral blood flow As described above, regulation of CBF is crucial for continued neuronal operation and survival. As such, the brain is able to autoregulate blood supply via a number of vascular mechanisms which can work concurrently and additively or dominate independently, depending on the circumstances and in response to several stimuli. These determinants - which are discussed below - include; the arterial blood gases (O2 and CO2) prevailing blood pressures, neuronal metabolism, and, although to a lesser extent, sympathetic and parasympathetic nervous activity. Ancillary to – or underlying – all of these factors are a number of other factors which also influence CBF regulation; namely, hematocrit (Hct) (through the influence on viscosity and shear stress) and hemoglobin (Hb) (through its roles in NO and O2 signalling and scavenging). 2.5.1 Carbon dioxide In order to maintain cerebral cellular pH within a normal physiological range, inflow and outflow of PCO2 and [H+] is regulated by CBF. It is known that CO2 is proportional to [H+] within the CSF (Leusen, 1954; Lambertsen et al., 1961a) to which neuronal cells including astrocytes and pericytes are acutely sensitive. The direct effects of changes in PCO2 and [H+] cause immediate (<10 seconds) changes in terminal arteriole and microvascular cross-sectional area within the brain; as such blood flow increases rapidly, and with it shear stresses on the upstream (proximal) vessels also increase, the mechanisms of which are described in detail below. CBF responses to hypercapnia occur both more rapidly and to a greater magnitude than CBF responses to hypocapnia; see Figure 2-11. Fundamentally, the relationship between, brain tissue PCO2 (PbtCO2), PaCO2, CBF, and the cerebral metabolic rate of CO2 production (CMRCO2) can be succinctly summarized thusly:1 an increase (for example) in PaCO2 that was not subsequently followed by either a proportional increase in CBF or decrease in CMRCO2 would result in elevated PbtCO2 and subsequent acidification (see 2.5.1.1 and 2.5.1.2 for the CO2 vs pH relationship) which, if severe enough, would initiate cell apoptosis (Lagadic- 1 This is a simplification, a great number of interactive phenomena are involved with the determination of each of these variables. That is: 1) alveolar ventilation and whole body metabolic production of CO2 (V̇CO2) contribute to determination of PaCO2, as well as arterial acid-base balance and oxygenation contributing to CO2 transport and therefore to PaCO2 at the capillary level; 2) CaO2, [Hb], hematocrit, and MAP are all relevant to CBF; 3) CMRCO2 is dependent on neuronal activity, e.g. waking status, cognitive engagement, O2 and substrate availability etc; and finally, 4) even intracellular PbtCO2 is influenced by cytosolic bicarbonate ion concentration ([HCO3-]) and water content. LITERATURE REVIEW 22 Gossmann et al., 2004). As cerebral metabolism is indispensable,2 CBF then is readily adjusted to compensate. Figure 2-11. Relationship between cerebral blood flow and PaCO2. Relationship between CBF (relative to baseline; black line) and absolute PaCO2 (while holding arterial partial pressure of O2 [PaO2] constant). Note the asymmetry between hypo- and hypercapnia, and the limits of response representing the limit physical limits of vasoconstriction and dilation, at approximately -15 mm Hg and +15 [ modified from: (Willie et al., 2012)]. The CBF response to changes in PaCO2 is labelled cerebrovascular reactivity (CVR), and is generally presented as ∆CBF/∆PaCO2, though often cerebral blood velocity is taken as a surrogate of CBF (despite problematic methodological considerations) and PETCO2 often taken as a surrogate of PaCO2 (see 2.7.2). The disparity between the slopes of the hyper- and hypo-capnic responses, as well as the difference in total magnitude of the responses, is representative of the priority of CDO2 over acid-base regulation, and the limits of vascular reserve, respectively (Wasserman & Patterson, 1961; Harper & Glass, 1965; Tominga et al., 1976; Ide et al., 2003; Willie et al., 2014). In a teleological sense, the shallower slope 2 Though not fixed. While cerebral metabolism varies regionally and temporally, and global metabolism can change minutely, generally, the total average cerebral metabolic rate of O2 consumption (CMRO2) in the brain in healthy humans at rest is 3.3±0.5 mL100 ml−1 min−1 (Ito et al., 2004). Additionally, a distinction should be made between metabolism quantified by CMRO2 and that defined by CMRCO2, since the ratio of O2 consumed to CO2 produced can change depending on metabolic pathway/fuel substrate utilization. 10 20 30 40 50 60 70-50050100150DCBF (% from baseline)PaCO2 (mm Hg)LITERATURE REVIEW 23 and lesser relative magnitude of the hypocapnic response represent the contra-interaction of the hypocapnic response and the need for maintained CDO2. Hypocapnia causes vasoconstriction, reducing CBF and CO2 washout, thereby maintaining PbtCO2 and pH by balancing PaCO2 and CMRCO2. However, this reduction in CBF also reduces CDO2.3 That CBF does not decline equally with decreasing PaCO2 indicates the prevalence of vasodilator (i.e., CDO2 saving) mechanisms over vasoconstrictor (i.e., CDO2 limiting) mechanisms. There is also a degree of hysteresis of the CBF responses to PaCO2 (Shapiro et al., 1966; Tominga et al., 1976; Ide et al., 2003). The slope of the CBF response to changes in PaCO2 when increasing from baseline to high (hypercapnia) is the steepest, however, if an increase is immediately preceded by a decrease from baseline to low (hypocapnia), then the subsequent response slope is shallower. This hysteresis is, in part, the result of the time delays of changes in PaCO2, PbtCO2, and vascular resistance (Wasserman & Patterson, 1961; Shapiro et al., 1965, 1966). This hysteresis is not apparent when CBF is indexed against the jugular venous PCO2 (PjvCO2), indicating that the cerebral tissue and/or VSMC PCO2, rather than the arterial PCO2, is the main effective stimulus (Shapiro et al., 1965, 1966). If the PaCO2 change is of sufficiently long duration, and time is allowed for CSF PCO2 to equilibrate with PaCO2, then this hysteresis should also be minimized. The subsequent paragraphs describe the putative mechanisms contributing to CVR. The cerebral vasculature is capable of resistance modulation at all levels of the cerebral vascular tree, including large extra-cranial (Willie et al., 2012; Carter et al., 2016; Hoiland et al., 2017; Carr et al., 2020) and intra-cerebral arteries (Giller et al., 1993; Verbree et al., 2014; Coverdale et al., 2015, 2017), pial vessels (Wolff & Lennox, 1930; Lennox & Gibbs, 1932; Kety & Schmidt, 1948b; Shapiro et al., 1971; Faraci et al., 1988; Faraci & Heistad, 1990) through endothelial cells (Iadecola, 2017; Chow et al., 2020) and astrocytes (Marina et al., 2020); and although the contribution of pericytes has been disputed (Hill et al., 2015), mounting evidence suggests they are an integral part of cerebral resistance control (Peppiatt et al., 2006; Hall et al., 2014; Gonzales et al., 2020; Hartmann et al., 2021) (Figure 2-12). This regulation of vascular resistance to control CBF - and therefore CSF and ISF PCO2 and [H+] - is mediated via several mechanisms, some converging some distinct. It is perhaps not surprising, therefore, that differentiating the individual contribution of a single mechanism in vivo in humans is a challenging aspect of study design and should always be a consideration in blockade investigations 3 Although reduction in CDO2 can be compensated for by increases in O2 extraction fraction (OEF) (Yamauchi et al., 1993; Fan et al., 2020), this compensation has limits. For example, McHenry et al. (1961) found that syncope generally seemed to occur when CBF reached approximately 25 to 30 mL100g-1 min-1, around 50% of the resting CBF of ~50 mL100g-1 min-1, indicating a “critical level” of supply below which elevation of extraction cannot suffice to maintain oxygenation. LITERATURE REVIEW 24 (e.g., Smith et al., 2020). Nonetheless, in vitro and in vivo animal and progressive human studies are steadily confirming or refuting candidate mechanisms. Certainly, CVR is the result of the combined effects of changes in PCO2 on intra- and extra-cellular [H+], initiating both direct and secondary effects on vascular smooth muscle cells (e.g., Peng et al., 1998), endothelial cells (e.g., Cleary et al., 2020), astrocytes (e.g., Howarth et al., 2017), and pericytes (e.g., Hartmann et al., 2021) in the cerebral microvasculature. The gaseous signalling molecule nitric oxide (NO) is almost certainly required for maintenance of basal CBF, but, in regards to steady-state changes in CBF by the hypercapnic vasodilator response, NO seems to play a permissive rather than obligatory role (Brian, 1998; Hoiland et al., 2019). This is supported by animal in vitro and in vivo gene knockout and pharmacological blockade, as well as human pharmacological blockade studies (Horvath et al., 1994; McPherson et al., 1995; Takahashi & Sokoloff, 1998; White et al., 1998; Ide et al., 2007; Hoiland et al., 2021) although there is some equivocation (Adachi et al., 1992; Ngai & Winn, 1995; Schmetterer et al., 1997). The majority of pre-clinical studies indicate that the CVR response is likely mediated by several mechanisms including pH-induced neuronal nitric oxide synthase (nNOS) activation (Wang et al., 1995; Iadecola & Zhang, 1996; Okamoto et al., 1997) and acidic disproportionation of nitrites to NO (Zweier et al., 1995; Weitzberg & Lundberg, 1998; Modin et al., 2001). Perhaps also influential is adenosine tri-phosphate (ATP) release from red blood cells due to reduced pH (Bergfeld & Forrester, 1992; Ellsworth et al., 1995) and shear deformation (Wan et al., 2008) causing electrical and chemical gradient changes across the erythrocyte membrane. Increased ATP release causes endothelial P2Y2 receptor stimulation, endothelial nitric oxide synthase (eNOS) activation, and subsequent vasodilatation. These responses all occur within milliseconds-to-seconds with changes in capillary pH and shear stress, and cause the initial pial vessel and pericyte vasodilation, signals for which are then propagated upstream via endothelial cell junctions. LITERATURE REVIEW 25 Figure 2-12. Regulation by CO2 across the cerebral vasculature. Inlay A: relative diameter change of murine cerebral precapillary arterioles and capillaries at the end of hypercapnia (5% CO2 in air for 6 minutes), and following return to normocapnia (Hartmann et al., 2021); showing the vasodilator action of both ensheathing pericytes, which wrap precapillary arterioles (also synonymous with precapillary vascular smooth muscle cells), and capillary pericytes, which control flow to individual capillary branches. Hartmann et al., (2021) showed that both pericyte phenotypes relax with hypercapnia and constrict upon return to normocapnia, and precapillary arteriole pericytes constricted ~3.5 fold faster than capillary pericytes. Inlay B: photographs of feline pial vessel. Left to right: at normoxic normocapnic rest; minor vasoconstriction with hypoxic hypocapnia; extreme dilation with hypoxic hypercapnia (Wolff & Lennox, 1930). Inlay C: high-resolution magnetic resonance images of human MCA. Left to right: white box highlights the area of interest; zoomed images of MCA at ~-7.5, normocapnia, ~+7.5, and ~+15 mm Hg end-tidal partial pressure of CO2. Note only moderate hypercapnia seems to elicit marked dilation (Verbree et al., 2014). Inlay D: Mean (∎) and individual (○) Duplex ultrasound measurements of ICA diameter across the vasomotor response to steady state changes in PaCO2 (Willie et al., 2012). Anatomical diagram from (Willie et al., 2014). Both prostaglandin and cyclooxygenase activity likely also significantly contribute to CVR, though again these may be permissive rather than essential. Cyclooxygenase inhibition substantially reduces pial arteriolar dilator responses (Degiulio et al., 1989; Wagerle & Degiulio, 1994) in a manner specific to extracellular [H+] related vasodilation (Wagerle & Mishra, 1988), the effects of which have been shown in vivo in humans (Eriksson et al., 1983; Kastrup et al., 1999; Xie et al., 2006; Hoiland et al., 2015; Peltonen et al., 2015; Hoiland et al., 2016b; Peltonen et al., 2016). Furthermore, vessel diameter changes contributing to CVR steady-state blood flow changes occur as far upstream as the large extra-cranial arteries (Figure 2-12) such as the ICA (Willie et al., 2012; LITERATURE REVIEW 26 Hoiland et al., 2016b, 2017; Carr et al., 2020) and common carotid arteries (Lavallée et al., 2006; Carter et al., 2016; Iwamoto et al., 2018b, 2018a). Increased blood flow in response to the mechanisms conducted proximally from capillaries upregulates shear stress mediated eNOS activity (described more fully in 2.6.3) in larger vessels; indeed, large cerebral vessels and pial vessels are sensitive to shear stress independent of hypercapnia per se (Heistad et al., 1978; Lee, 1980; Gourley & Heistad, 1984; Rubanyi et al., 1990; Fujii et al., 1991, 1992; Mashour & Boock, 1999; Carter et al., 2016; Hoiland et al., 2017; Carr et al., 2020). In short, and as illustrated in Figure 2-13, as with most integrative physiological responses, numerous mechanisms involving possibly dozens of receptors types and transmitter molecules are involved in mediating CVR. Figure 2-13. Signaling mechanisms underlying the cerebrovascular response to CO2 Individual data showing the percent reduction of the cerebrovascular response to CO2 in studies conducting pharmacological or genetic blockade of hypothesized signaling pathways, in in vivo animal and human studies. Color gradient and size of each symbol denote the statistical power of each study included (total study n). Mean and 95% confidence intervals (black symbols) show the reduction of CVR observed under experimental conditions. From (Hosford et al., 2022), licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/legalcode). 2.5.1.1 Driving Stimuli: Hydrogen or carbon dioxide? Ventilatory (V̇E-CO2) and cerebrovascular (CVR) responses to CO2 are commonly said to be driven by PaCO2. In fact, the respiratory control centre neurons and central chemoreceptors are actually sensitive to intra- and extra-cellular [H+], while the vasculature is sensitive to changes in both PCO2 and [H+] of the vascular tissue determined by luminal and extravascular fluids. The [H+] and pH in the intraluminal, interstitial, and intracellular spaces is constantly varying according to each compartment’s hydration LITERATURE REVIEW 27 equilibrium of CO2 and HCO3-, with by-products H+ and H2O, catalyzed by carbonic anhydrase (CA)4 via carbonic acid (H2CO3): CO2 + H2O H2CO3↔𝐶𝐴 HCO3- + H+ Equation 1 Changes in CO2 and HCO3- both alter the pH of various compartments, expressed via the Henderson-Hasselbalch equation below. The negative logarithm of the acid dissociation constant (pKa) of 6.1 at 37◦C for the equation above, gives the following equation: pH = 6.1 + log [HCO3−]0.03 ∙ PCO2 Equation 2 The driving stimulus of cerebral vascular responses to CO2 is intracellular pH at the abluminal side of vascular walls determined by the equations outlined above. Intracellular and interstitial HCO3- status does also fluctuate; HCO3- is “moved” transcellularly, but via a series of ionic exchanges rather than by direct whole transport, making it slower than CO2. This means that the [HCO3-] and other ions within the cerebrospinal and interstitial fluids are relatively stable over time (Siesjö, 1972; Fencl, 1986; Hladky & Barrand, 2016). Because PaCO2 can change relatively rapidly and CO2 is transported across cellular membranes, both intra- and extra-cellular brain pH are tightly coupled with PaCO2 - dependent on local blood supply – thus, the chemoreceptors are often said (slightly misleadingly) to be directly receptive to PaCO2. While the predominant view is that pH/[H+] alone is the stimulus for CVR, there is also some evidence for the independent effects of PCO2, [H+], and [HCO3-] (Shams, 1985; Nattie & Li, 2012; Meigh et al., 2013; Guyenet & Bayliss, 2015; Gonçalves & Mulkey, 2018). 2.5.1.2 [H+]: Henderson-Hasselbalch vs. Stewart’s physicochemical approach The aforementioned conception of acid-base balance relevant to cerebrovascular control is described in terms of the “standard” approach to acid-base balance. This standard and traditional approach came into being piecemeal over the course of the early decades of the 20th century, and, despite much debate on various nuances (e.g., Bunker, 1965), has nonetheless survived and is the predominant clinical and research approach to acid-base balance (Severinghaus & Astrup, 1985; Rastegar, 2009; Kimura et al., 2018). The traditional approach describes acid-base balance and pH as being the sole remit of [HCO3-] 4 Carbonic anhydrases are ubiquitous zinc enzymes found in most cell types in some form or another in both eukaryotes and prokaryotes. There are some 14 isoforms of CA in mammals alone which exhibit reactive attributes, yet the interconversion of CO2 and HCO3- by CA seems to be its most significant physiological reaction in humans (Maren, 1967; Geers & Gros, 2000; Supuran et al., 2003). LITERATURE REVIEW 28 and PCO2. Using concepts such as the anion gap - the difference between unmeasured plasma anions and unmeasured plasma cations; and base excess - the amount of acid or base needed to titrate 1 L of whole blood to a pH of 7.40 at a constant PCO2 of 40 mm Hg – the traditional approach provides the framework within which most metabolic and respiratory disturbances to acid-base balance can be defined. However, an alternate approach to the fundamental determination of [H+] which does not focus on [HCO3-] and PCO2 alone was proposed in 1983 by Peter Stewart (Stewart, 1983). In this approach, Stewart suggested a re-conceived acid-base approach based on the following issues that he regarded as crucial; the standard model (e.g. [HCO3-] and PCO2) appears to break down at physiologic extremes; there are non-carbonate buffers in physiologic fluids (e.g., albumin, phosphate, and hemoglobin); and, finally, while the standard approach can describe the magnitude of acid-base disturbances it gives no insight into the causal mechanisms (Stewart, 1983; Corey, 2003; Rastegar, 2009; Kimura et al., 2018). So, Stewart suggested that determination of [H+] - and therefore pH – should be based on the physical and chemical properties of strong and weak ions structured by the laws of Mass Action, Conservation of Mass, and Conservation of Charge. Specifically, Stewart’s approach realizes [H+] in regards to three independent variables; the total strong ion difference concentration ([SID]), the total weak acid concentration ([ATOT]), and PCO2. These three independent variables give six dependent variables [HA], [A-], [HCO3-], [CO32-], [OH-], and [H+], from the application of six equilibrium equations; 1), dissociation of water; 2), weak acid dissociation; 3), conservations of mass; 4) bicarbonate ion formation; 5), carbonate ion formation; and 6), electrical neutrality. The [SID] is the sum of all the strong cation concentrations (e.g., [Na+], [K+], [Ca2+] etc.) in the solution minus the sum of all the strong anion concentrations (e.g., [Cl-], [lactate], [albumin]) in the solution. The [ATOT] is a composite concentration representing all non-carbonate (non-volatile) buffers; that is, the sum of [A-] (dissociated non-carbonic weak acids, principally albumin and phosphate) and [HA] (the concentration of plasma weak acids understood for convenience as a single pseudo-monoprotic acid). The [HCO3-], [H+], and hydroxyl ion concentration ([OH-]) are then considered to be dependent variables, unable themselves to cause any acid-base changes per se; while metabolic disorders are the causal result of changes in [ATOT] or [SID]. Physiologically, all tissues, but especially the kidneys, contribute to [SID] regulation through varying and transporting [NA+], [K+], [Ca2+], and [Cl-] etc., while the liver mainly determines [ATOT], and the respiratory arc controls PCO2. The fundamental premise of Stewart’s model that critics appeal against is that which states: [HCO3-] and [H+] are dependent variables which cannot be manipulated directly, therefore, the concentrations LITERATURE REVIEW 29 of these variables are not effected by movement of the ions per se, only by movements of strong ions which changes the [SID]. This is obviously difficult to reconcile with many physiological theories of sub-cellular and molecular processes; for example, various membrane transporters based at least in part on the movement of H+ have been described (DeCoursey & Cherny, 1994; Shimada et al., 2006; Pedersen & Counillon, 2019), and many processes involved in mitochondrial oxidative phosphorylation depend on the understanding that movement of H+ will result in a change in the compartmental concentration (Mitchell, 2011). Though Stewart takes only three values ([ATOT], [SID], and PCO2) as independent variables for acid-base balance, the calculation of the first two requires measurement of several ions. For any model, the inaccuracy of the overall model increases with the number of variables that need to be measured. Given the number of ions that are required to be measured for the Stewart approach to be clinically successful, the degree of inconsistency in interpretations and findings of studies using this approach is not surprising (Kimura et al., 2018). Table 2-1 highlights some of the strengths and weaknesses of the standard and Stewart models. Table 2-1. Standard vs Stewart models of acid-base balance Standard Stewart Measures magnitude Yes Yes Mechanistic insight No a Yes Classifies disorder Yes Yes Independent variables Base excess, [HCO3-], PCO2 [ATOT], [SID], and PCO2 Intracellular buffers Yes No Noncarbonate buffers No Yes Advantages Familiar, relatively simple Comprehensive, indicates causal link Disadvantages Some components somewhat artificially derived and mechanistically uninformative (i.e., base excess) Definition of acids and bases not widely accepted. [ATOT] and [SID] are mathematical constructs and not physicochemical properties or entities. a, unless the anion gap is also used. Adjusted from (Corey, 2003) The Stewart approach is potentially more useful than the standard model in the clinical setting where appropriate treatment of acid-base disorders often requires all-encompassing analyses and diagnoses, provided that analysis and interpretation are not prohibitively time-consuming. In a literature review of comparisons of prognostic performance of the two models; however, Kimura et al., (2018) reported LITERATURE REVIEW 30 that 10 of 17 studies supported the Steward model, while the rest either supported the performance of the traditional model or were unclear. The authors surmised that inconsistency in performance of the models likely arises from differences in specific model and measured ions used (for there are a number of variations of Stewart’s original model), cumulative measurement errors, and errors in interpretation using Stewart’s model, which requires combinatorial interpretation of several parameters simultaneously to give suitable diagnoses (Kimura et al., 2018). Finally, in a rather definitive review of the two models Kurtz et al. (2008) showed that both are quantitatively equivalent even in the presence of non-bicarbonate buffers, that the Henderson-Hasselbalch and Stewart equations are in fact interchangeable, and that there is no evidence to suggest that SID mechanistically causes changes in [H+] as Stewart claimed. The authors suggest that the Henderson-Hasselbalch equation formulation of acid-base balance is a mechanistic formulation that actually reflects the underlying physiology and associated phenomenology, and therefore the Stewart approach adds complexity without reason. For more complete comparisons of the standard and Stewart approaches than the brief precis above, the reader is directed to earlier reviews (Corey, 2003; Kurtz et al., 2008; Morgan, 2009; Rastegar, 2009; Kimura et al., 2018). 2.5.1.3 CO2 bulk transport As described above in 2.5.1, the cerebrovasculature is sensitive to [H+] and PCO2 in the ECF and PCO2 in the blood; and as described in 2.5.1.1, a primary determinant of [H+] (whether by the traditional or Stewart approach) is the CO2 that reaches the area. As such, the bulk delivery and removal of CO2 from a region will contribute to both the PCO2 and the [H+] in that area. CO2 in blood is transported in three main modes; freely dissolved (i.e. that which is measured as PCO2), bound as bicarbonate (HCO3-), and bound as carbamate to Hb (HbCO2; both in free Hb and erythrocytic Hb). It is the latter two (HCO3- and HbCO2) by which the majority of CO2 is carried (van Slyke, 1921; Margaria, 1931, 1957; Roughton, 1935; Geers & Gros, 2000). As such, any scenario in which any haemorheological variable is changed which might affect [HCO3-] or [Hb] - such as isovolumic haemodilution- would cause a reduction of total CO2 carrying capacity. At the level of the capillaries, where most gas exchange occurs, the appropriate measure is surely as a partial pressure; however, since dehydration and rehydration of CO2 is catalytically accelerated by carbonic anhydrase to ~2 milliseconds (Geers & Gros, 2000), at sites beyond the reach of active gas exchange such as in large conduit vessels where blood is conveyed to and away from tissues and diffusional areas, the partial pressure of CO2 represents only a fraction of the total CO2 being transported. Therefore, total CO2 content may be a useful measure in physiological studies in vivo, allowing for calculation of a value for the washout of CO2 from tissues. LITERATURE REVIEW 31 Because CO2 in blood is transported in these three main modes, calculation of a value of total blood content can provide an overall picture of CO2 and acid-base status. Calculating total arterial and cerebral venous CO2 content (CCO2) per Douglas et al. (1988) for comprehensive blood gas interpretation is conducted thusly. First, one calculates the plasma content of CO2 (PCCO2) by Equation 3 : PCCO2 = 2.226 ∙ s ∙ PCO2 ∙ (1 + 10pH – pK`) Equation 3 Where s is the solubility coefficient of CO2 and pK` is the apparent pK [both calculated per Kelman (1967), with the assumption that core temperature was 37.5°C and unchanged with haemodilution], and 2.226 is the conversion factor for mM to mL/100mL. PCCO2 can be calculated for both arterial (PCaCO2) and jugular venous (PCjvCO2) blood using arterial pH, PCO2, and pK` for PCaCO2 and jugular venous pH, PCO2, and pK` for PCjvCO2. Then, by Equation 4, one calculates the total CCO2: CCO2 = PCCO2 ∙ (1 – 0.0289 ∙ [Hb]) (3.352 – 0.456 x SO2) ∙ (8.142 – pH) Equation 4 Where SO2 is O2 saturation, while 0.0289, 3.352, 0.456, and 8.142 are constants determined by Douglas et al. (1988). CCO2 can then be calculated for both arterial (CaCO2) and jugular venous (CjvCO2) blood, using arterial Hb, SO2, and pH for CaCO2, and jugular venous Hb, SO2, and pH for CjvCO2. 2.5.2 Oxygen As depicted in Figure 2-14, the cerebral vasculature responds to reductions in PaO2. It is known that VSMC ATP-mediated K channels contribute to hypoxic vasodilation, as well as ATP acting on endothelial P2Y2 receptors, and release of Hb bound s-nitrosothiols and reduction of nitrites in response to reductions in CaO2 contribute to vasodilation in the cerebral capillaries (reviewed in Hoiland et al., 2016a). The endothelial signals are then propagated upstream via numerous communications through ECs and astrocytes (Longden et al., 2017; Gonzales et al., 2020). Increased CBF during hypoxia maintains CDO2; where CDO2 is not maintained however, the degree of O2 extracted from the arterial blood (oxygen extraction fraction; OEF) is often increased to sustain appropriate brain tissue PO2 and aerobic metabolism. When PaO2 is reduced below around 50 mm Hg, CBF is significantly affected and begins to increase exponentially with further reductions (Figure 2-14). LITERATURE REVIEW 32 Figure 2-14. Relationship between cerebral blood flow and PaO2. Relative CBF (black line) changes by PaO2 (while holding PaCO2 constant) 2.5.2.1 Hypoxic hypoxaemia vs anemic hypoxaemia Although hypoxaemia and hypoxia in tissues can be induced in a number of ways, four categories of hypoxia are commonly used: hypoxic hypoxia, anemic hypoxia, circulatory (ischemic) hypoxia, and cellular hypoxia. Hypoxic hypoxia describes reduced PaO2 resulting from reduced PiO2 or reduced diffusion from atmosphere to blood across alveoli. Figure 2-14 above depicts the typical CBF response to progressive hypoxic hypoxaemia. Anemic hypoxia describes a reduction in the O2 carriage of blood, which can arise from reduced haeme availability, impaired haeme-oxygen binding, or a simple reduction in blood volume (e.g., haemorrhage). Anemic hypoxaemia also causes increases in CBF in order to maintain CDO2, although there is equivocal evidence on this, due to the majority of published findings having been conducted in pathological samples with many disparate methodologies (see Figure 2-15). 0 50 100 150-50050100150DCBF (% from baseline)PaO2 (mm Hg)LITERATURE REVIEW 33 Figure 2-15. Effects of anaemic hypoxaemia via haemodilution on CBF Relative changes in CBF, OEF, and CDO2 in response to reductions in CaO2 in investigations wherein venesection or haemodilution (both iso- and hypovolemia) has been conducted in the setting of CBF assessment. Filled symbols are studies in pathological patients, e.g., cerebrovascular diseases, ICA occlusive disease, brain tumour resection surgery patients etc. Open symbols are studies in “healthy” participants. Dashed lines are studies that report Hb but not CaO2, dotted lines are studies that report Hct or packed cell volume but not Hb or CaO2, and solid lines are studies that report CaO2. CBF as measured via duplex ultrasound, xenon clearance, 1/Ca-vO2, or supposed by MCAv via TCD; OEF as reported in each study; and CDO2 either as reported in each study or calculated as the product of CBF and CaO2 or Hb or Hct. Grey symbols are studies that measured MCAv. PCO2 is either peripheral venous or arterialized blood (dotted lines), or arterial (solid and dashed lines). Data from (Paulson et al., 1973a; Thomas et al., 1977a, 1977b; Humphrey, 1979; Humphrey et al., 1980; York et al., 1980; Henriksen et al., 1981; Wade, 1983; Wood et al., 1984; Hino et al., 1992; Yamauchi et al., 1993; Bruder et al., 1998; Mühling et al., 1999; Ekelund et al., 2002; Daif et al., 2012). Relative to reductions of CaO2 independent of PaO2, anaemia is a hallmark of multiple disease states with well-documented cerebrovascular dysregulation, including sickle cell disease (Nur et al., 2009; Prohovnik et al., 2009) and chronic renal failure (Kuwabara et al., 2002; Zheng et al., 2016; Sprick et al., 2020). For example, sickle cell disease increases the risk of stroke, which may be related to a reduction in cerebrovascular vasodilatory capacity secondary to elevations in resting CBF (Nur et al., 2009; Prohovnik et al., 2009; Kosinski et al., 2017). Similarly, CVR is reduced in chronic kidney disease patients with anaemia concomitant to elevations in resting CBF (Kuwabara et al., 2002). While pre-clinical studies have demonstrated that brain parenchymal O2 tension, and CBF responses to hypercapnia are preserved during subacute anaemia in rats through elevated basal CBF (Mistry et al., 2018), CDO2 is impaired in human chronic kidney disease patients despite similarly increased CBF -30-20-100020406080CaO2 or Hb or Hct (D%)CBF or MCAv (D%)-15-10-505OEF (D%)Hino et al. 1991Mühling et al. 1999Daif et al. 2012Ekelund et al. 2002Yamauchi et al. 1993Thomas et al. 1977aBruder et al. 1998Thomas et al. 1977bWade 1983Humphrey et al. 1979Humphrey et al. 1980Henrikson et al. 1980York et al. 1980Wood et al. 1984Paulson et al. 1973-200204060CDO2 (D%)-15 -10 -5 0 5 1020406080PCO2 (mmHg)CBF or MCAv (D%)LITERATURE REVIEW 34 (Zheng et al., 2016). Whether the differences in CBF regulation observed with anaemia are related to anaemia per se or a consequence of the disease state underlying said anaemia remains to be determined. 2.5.2.2 Hyperoxia Increases of PaO2 above normal physiological pressures (>~100 to 400 mm Hg and up) result in reductions in CBF (Lambertsen et al., 1953; Sicard & Duong, 2005; Willie et al., 2012; Ainslie et al., 2014; Brugniaux et al., 2018), though the magnitude of this response is minimal compared to the effects of hypoxia for instance. Hyperoxia is a stimulant of ventilation which subsequently causes hypocapnia and the associated appropriate CBF response (Fernandes et al., 2021); however, it also leads to minor cerebral vasoconstriction directly (Floyd et al., 2003; Mattos et al., 2019). Elevated PaO2 also causes heightened levels of ROS (Wei et al., 1985; Modun et al., 2012; Ciarlone & Dean, 2016), a complexly influential component of vascular function and health (Faraci, 2006). While prescription of hyperoxic gas is common in many clinical and critical care scenarios as a means to restore arterial oxygen, post-resuscitation arterial hyperoxia following cardiac arrest is associated with increased in-hospital mortality in cardiac arrest patients (Kilgannon et al., 2010). 2.5.3 Cerebral Autoregulation The premise of cerebral autoregulation is that, with increases in MAP - which would increase CBF and CPP without some autoregulatory response - cerebral vascular resistance and vasoconstriction both increase such that blood flow is reduced and CPP is somewhat maintained/re-established. In 1959, Lassen published a review of studies addressing the effects of changes in blood pressure on CBF (Lassen, 1959). Redrawing and presenting data from 11 clinical population cross-sectional studies, Lassen showed an apparent decrease of CBF at a MAP of 50 mm Hg (see Figure 2-16). This data showing a drop off of CBF however came from 2-3 studies which investigated CBF in patients with hypotension, and in which no single CBF value was equivalent to the values presented by Lassen. Nonetheless, Lassen’s curve was propagated and continues to be taught widely, though recent departures (e.g. Brassard et al., 2021), have begun to develop. LITERATURE REVIEW 35 Figure 2-16. Lassen's curve Mean cerebral blood flow and mean arterial pressure values from 11 groups (different studies) of participants with various acute and chronic conditions. Reproduced with permission from (Lassen, 1959). More recently it has been shown that in fact CBF does not plateau over as wide a range of MAP, and is more likely between 80-100 mm Hg, if a plateau exists at all (see Figure 2-17; Lucas et al., 2010; Numan et al., 2014; Claassen et al., 2021; Brassard et al., 2021). Figure 2-17. Modern cerebral autoregulation Relationship between relative changes in MAP from baseline (Δ%MAP) and concomitant relative changes in CBF (Δ%CBF) when MAP decreases and increases including (left panel) and excluding (right panel) the utilization of cardiovascular drugs to manipulate MAP. These data represent an updated analysis initially published by Numan et al. (2014). Note the consistent pressure‐passive nature of CBF in the pharmacological data (left panel) and the small plateau present in the non‐pharmacological data (right panel). Reproduced from (Brassard et al., 2021) under a Creative Commons License. Responses of the cerebral vasculature to changes in blood pressure are strongly affected by the rate at which pressure changes. Indeed, in the field of autoregulation, one factor contributing to the difficulty of agreement on the nature of the response is the influence that the duration and speed of pressure LITERATURE REVIEW 36 changes are as impactful on the response as the magnitude. The faster the MAP change, the smaller the seeming CBF plateau and the sharper the CBF change. To aid in differentiation, autoregulation is often discussed in terms of static or dynamic autoregulation. Static indicating the classic Lassen’s curve describing the typical steady-state relationship between CBF and MAP; while dynamic autoregulation describes the CBF response to rapid or transient changes in MAP. The mechanisms of cerebral autoregulation include myogenic, metabolic, and neurogenic components. That is, VSMC from the carotid bifurcation down to the small arterioles and pial vessels contract or dilate in response to changes in both intraluminal and axial pressure changes. This vasoconstriction occurs mainly through stretch-mediated ion channels in VSMC [(Knot & Nelson, 1998) reviewed in: (Meininger & Davis, 1992; Tzeng & Ainslie, 2014; Claassen et al., 2021)], and potentially with some contribution of neurogenically-mediated sympathetic vasoconstriction, [though the contribution of this is likely minor and is difficult to address experimentally (see Sympathoexcitation)] . In response to decreases in MAP, where CPP would decrease without some autoregulatory response, there is a subsequent reduction in vascular resistance and vasodilation occurs, which increases blood flow to re-establish cerebral perfusion. This occurs predominantly through a combination of neurogenic dilatation and metabolic demand for the maintenance of O2 supply and CO2 washout as described above. Given that, fundamentally, cerebral vascular responses to any stimulus can only entail moderate vasoconstriction or vasodilation, combinations of various stimuli (e.g., sudden MAP change due to postural adjustment, with concomitant CO2 increases due to poor environmental ventilation) must vie for the available cerebrovascular reserve. Therefore, the interrelationship between the effects of MAP and CO2 must be considered and ideally accounted for in experimental design or analyses of CBF studies. Regulation of CBF in response to changes in MAP is also somewhat prioritized over CO2 responses (Harper & Glass, 1965; Aaslid et al., 1989). However, overlapping mechanisms contribute to both responses, and both autoregulatory and CVR can only utilize the available cerebrovascular reserve, and the CBF response to one is often difficult to parse from the other due to the systemic interactions (Przybyłowski et al., 2003; Regan et al., 2014). There is also asymmetry in the CBF response to increases and decreases in MAP, CBF responding more rapidly and with greater efficacy to hypertension that hypotension (Paquette et al., 2017). 2.5.4 Neurovascular coupling CBF is tightly coupled to cerebral tissue metabolic demands due to the paltry substrate storage capacity of the brain itself. The demand for O2 and glucose for oxidative phosphorylation, as well as the need for removal of metabolic by-products - CO2, [H+], inorganic phosphates, etc. - are the drivers underlying LITERATURE REVIEW 37 NVC (Theparambil et al., 2020; Hosford et al., 2022). When neuronal activity is increased, glutamatergic signalling increases nNOS production of NO, as well as arachodonic acid activations of epoxyeicosatrienoic acid and prostaglandins, causing vasodilation in the capillaries, which elicits immediate local increases in blood flow. Subsequently there is upregulation of eNOS – both locally and upstream - due to the flow and shear stress increase, which aids in prolonging and maintaining the increased flow (Longden et al., 2017; Gonzales et al., 2020). These vascular mechanisms are the combinatorial result of both O2 and CO2 reactivity, as described above, occurring on a local area as the result of tissue metabolism demanding O2 and glucose and producing CO2 and [H+]. There is a close relationship between metabolic activity and blood flow; as such, a lack of hyperaemia to local activation can be an indicator of cerebrovascular dysfunction or otherwise dysfunction of some aspect of the NVU (Phillips et al., 2014; De Silva & Faraci, 2016; Iadecola, 2017; Marina et al., 2018; Chow et al., 2020). 2.5.5 Sympathoexcitation While sympathetic and parasympathetic nervous activity are both conceivably involved in CBF regulation given the extensive adrenergic and cholinergic innervation of the cerebral vasculature, extrinsic autonomic influence on CBF is, however, minimal at rest (Gezalian et al., 2021; Koep et al., 2022). Due to the heterogenous density of - and -adrenergic receptor types throughout the cerebral vasculature (see Figure 2-18) the likely main effects of SNA on CBF are mild vasoconstrictive -adrenergic constraint of large extracranial arteries (Hamner et al., 2010; Fernandes et al., 2016), and – though evidence is minimal in humans – some mild vasodilatory  adrenergic activation in the smaller arterioles (Secher, 2000; Ogoh et al., 2007). While cerebral receptor type distribution is mostly based on animal studies (Forbes & Wolff, 1928; Talbott et al., 1929; Chorobski & Penfield, 1932; Cobb & Finesinger, 1932; Pool et al., 1934; Forbes et al., 1937; James et al., 1969), and there are no doubt many differences between species in innervation type and density, this is nonetheless supported by human studies which have investigated SNA control of CBF. For example, Fernandes et al (2016) demonstrated -restraint of larger extra cranial arteries with unilateral handgrip exercise eliciting increased contralateral ICA blood flow with no change in ipsilateral ICA flow. After Prazosin (1 receptor blockade), unilateral handgrip exercise caused increased flow in both contralateral and ipsilateral ICA, indicating the role of 1 adrenergic receptors in cerebral large artery vascular constraint. This study also demonstrated the minor role of SNA in CBF control, as the SNA-mediated constraint was readily superseded by the NCV elicited by unilateral handgrip. LITERATURE REVIEW 38 Figure 2-18. Adrenoceptors and calcium channels in the cerebral vasculature Subtype, distribution, and density of adrenoceptors and calcium channels represented by color and column height. From (Gezalian et al., 2021); gratis reuse, no permission required. While evidence regarding cholinergic influence on CBF regulation is limited (in comparison to the vast uncountable miles of vascular chemosensitivity literature) and was once controversial (e.g., D’Alecy & Rose, 1977 vs. Busija & Heistad, 1984), both animal (Hill, 1896; Forbes & Wolff, 1928; Wolff, 1929; Chorobski & Penfield, 1932; Cobb & Finesinger, 1932; Pool et al., 1934; Forbes et al., 1937; James et al., 1969; D’Alecy & Rose, 1977) and human (Ko et al., 1996; Henry et al., 1998, 1999; Vonck et al., 2000; Conway et al., 2006; Hamner et al., 2012; Hamner & Tan, 2014) studies indicate that parasympathetic innervation of the cerebral vasculature may function similarly to that of the coronary circulation, i.e., parasympathetic stimulation induces vasodilation. Though this, as with adrenergic innervation, occurs in a regionally heterogenous manner, mostly effecting extra-parenchymal vessels The complexly integrated nature of CBF regulation means that discerning the direct role of autonomic innervation from the other regulators described above (i.e. CO2, O2, and MAP) is difficult (Brassard et Figure 2. Schem tic description of subtyp s, distribution, and ensity of adrenoceptorsand calcium channels along the cerebrovascular tree. Distribution and density ofadrenoc ptor subtypes and calcium channels depicted by color and column height alongprincipal cerebral vessels, respectively (Figure Legend and insets). Also shown is the cell-specific localization of adrenoceptor subtypes and calcium channels.Article Copyright © 2020 Authors, Source DOI: 10.1177/0271678X20972869 .See content reuse guidelines at: sagepub.com/journals-permissionsLITERATURE REVIEW 39 al., 2017). For example, while many studies have addressed CBF regulation with and without adrenergic blockade, the changes (or lack of apparent changes) to various measures of CBF are difficult to assign directly to SNA control, due to the co-reflexivity of CBF with MAP (Tzeng & Ainslie, 2014) and CO (Ide et al., 2000; Meng et al., 2015), each of which can be acutely altered with adrenergic blockade (see Table 3 in Brassard et al., 2017; and Table 1 in Koep et al., 2022) or other interventions (e.g., postural effects on PaCO2) (Bjurstedt et al., 1962; Weissman et al., 1982; Gelinas et al., 2012; Claassen et al., 2021). However, a method which circumvents some of these issues is the measurement of the cerebral arterio-venous norepinephrine difference, which provides an index of sympathetic nervous activation of the extrinsic innervation to the cerebral vasculature (Mitchell et al., 2009). Other obstacles to study comparison and interpretation include: the uncertainty of the permeability of the BBB to adrenergic receptor blockade drugs; the use of some measurement techniques, e.g., TCD blood velocity rather than volumetric flow; the fact that with some stimuli there is a tapering off of the increases in SNA, meaning that there is time dependency of the responses. Though conducted in felines, Table 2-2 concisely summarizes the effects of sympathetic and parasympathetic nerve stimulation on various sized pial vessels. Table 2-2. Feline pial artery responses to sympathetic and parasympathetic stimulation Feline large artery and pial artery responses to sympathetic and parasympathetic nerve stimulation. Reproduced with permission from (Forbes & Wolff, 1928). LITERATURE REVIEW 40 2.6 Regulation of peripheral blood flow Blood flow through all arteries in the body is the result of the end-organ perfusion requirements. Accordingly, blood flow through the large conduit peripheral arteries (e.g., the brachial artery) is determined by the needs of distal skeletal muscle and cutaneous microvascular beds where the main determinants are metabolism and thermoregulation, respectively, which are both heavily regulated by SNA due to the systemic requirement for MAP regulation. The following sections describe the regulation of peripheral blood flow and vascular tone, with focus on skeletal muscle metabolic demand and the overarching sympathetic nervous activity. 2.6.1 Metabolism During increased metabolic demand (e.g., skeletal muscle contraction) blood flow is increased and matched to the degree of metabolic demand; that is to say, the greater the contraction intensity, the greater the coupled blood flow increase. Additionally, the muscle mass involved also determines the blood flow responses proportionally, that is to say, activation of smaller muscle masses elicits relatively lesser responses, and larger masses greater responses. The majority of blood flow demand for skeletal muscle metabolism is the result of the requirement for O2, as there are some stores of carbohydrates and fat for use as fuels in skeletal muscle, while O2 is stored in myoglobin in muscles in small amounts (Hoppeler & Weibel, 2000). At rest, healthy human adults consume ~3 to 4 mLof O2 /kg/min, the majority of which is directed to the vital organs; however, during exercise, O2 consumption can reach values 10 to 15-fold higher as a result of skeletal muscle metabolic demand (Joyner & Casey, 2015). This results in a major redistribution of cardiac output to the active muscle beds (see Figure 2-19). LITERATURE REVIEW 41 Figure 2-19. Idealized schematic of the destination of cardiac output during rest and exercise Schematic shows idealized distributions of blood flow at rest (left), during maximum exercise in a healthy untrained young male subject (middle), and in a male elite endurance athlete (right). Reproduced with permission from (Joyner & Casey, 2015). This increase in cardiac output distributed to metabolically active muscle represents the considerable vascular reserve of the peripheral circulation. Blood flow to working muscles, for instance through the femoral artery, can increase by as much as 3000% (Saltin et al., 1998) during dynamic knee-extensor exercise. The local mechanisms of vasodilation caused by exercise are many, but include increased pCO2, lactate, potassium, [H+], and adenosine from ATP breakdown (Brechue & Stainsby, 1994; Marshall, 2007; Sarelius & Pohl, 2010). Additionally, complexly integrated roles have also been postulated for other factors such as NO (Grange et al., 2001). During exercise, blood flow to non-exercising limbs is muted by an increase in sympathetically-mediated vasoconstrictor tone (Blair et al., 1960), until greater exercise intensities or prolonged duration causes thermoregulatory cutaneous vasodilation. Exercise also acutely leads to an increase in sympathetic activity and MAP (Alam & Smirk, 1937; Mark et al., 1985; Thomas & Segal, 2004) via central command, as well as afferent mechano- and metabo-reflexes arises directly from the working muscle. The local release or escape of exercising skeletal muscle blood flow from sympathetic nervous constraint, despite the increase in systematic SNA signalling is commonly referred to as functional sympatholysis. LITERATURE REVIEW 42 2.6.2 Sympathetic mediation of vascular tone Although to differing magnitudes, sympathetic innervation can exert influence on peripheral (Barcroft & Edholm, 1946; Richardson et al., 1960; Ferret et al., 1989; Boutouyrie et al., 1994), cerebral (Van Mil et al., 2017, 2018; van Mil et al., 2019), coronary (Zeiher et al., 1989, 1991; Schächinger et al., 2000), and splanchnic (Gollwitzer-Meier & Bohn, 1930; Mohamed & Bean, 1951; Epstein et al., 1961, 1966; Schaefer & Baker III, 1969; Elam et al., 1981; Davidson et al., 1986) circulations. In the peripheral circulation, SNA can impose marked vasoconstrictor tone and is seemingly dominant in maintaining resting vascular tone (Barcroft et al., 1943; Barcroft & Edholm, 1946; Hijmering et al., 2002). The main neurotransmitters involved in sympathetic signalling on VSMCs are norepinephrine (NE) and epinephrine (both adrenergic), and acetylcholine (cholinergic), though others are involved such as ATP, NO, and neuropeptide-Y. As indicated in 2.5.5 and Figure 2-18, adrenoreceptor type (e.g., 𝛼-1, 𝛼-2) distribution, density, and sensitivity are heterogenous within the brain, and this is true across the circulatory system (Thomas & Segal, 2004; Shoemaker et al., 2016; Brassard et al., 2017; Koep et al., 2022). Figure 2-20. Adrenoreceptor densities, and sensitivity to sympathetic stimulation in the peripheral vasculature α1; alpha 1 adrenergic receptor; α2; alpha 2 adrenergic receptor; Y1, neuropeptide Y Y1 receptor; P2X, purinergic vasoconstrictor receptor. 1A-5A, first through fifth-order arterioles. Number of asterisks reflects magnitude of effect. Blank boxes indicate lack of information. Reproduced with permission from (Shoemaker et al., 2016). LITERATURE REVIEW 43 2.6.3 Shear stress The various factors described above are foremost in determining vascular blood flow regulation in the cerebral and peripheral vasculatures respectively, however, throughout the circulatory system, another predominant factor determining vessel tone and therefore diameter is shear stress. Shear stress is the co-planar dragging force exerted on the intraluminal surface of ECs by the movement of blood. Vascular regulation of blood flow through changes in conductance/resistance occur according to changes in shear stress, as well as change sympathetic nervous activity and circulating vasodilators and constrictors. The regulation of blood flow and maintenance of vascular health is dependent on functional endothelium responses; increases in shear stress (through increased blood velocity; Figure 2-21 inlays 1 and 2) on the endothelial wall are normalised through vasodilation, so that blood flow is maintained at the level required for the demand of the supplied tissues while also maintaining the stress on the vessel walls (see Figure 2-21 inlays 3 and 4). There is a positive linear relationship between acute changes in shear stress and vessel diameter in peripheral arteries which has been well-established (Anderson & Mark, 1989), and in cerebral arteries to a lesser degree (Fujii et al., 1992; Carter et al., 2016; Hoiland et al., 2017; Carr et al., 2020). LITERATURE REVIEW 44 Figure 2-21. Regulation of vessel diameter by shear stress Exposure of the endothelium to an increase in shear stress (step 2) causes vasodilation (step 3), a homeostatic functional change that moderates the stress on the vascular wall. When exposed to prolonged periods (chronic or repeated exposure over weeks) of change in flow and shear, vessel remodeling can occur, whereby drag forces are “structurally” normalized (step 4). Reproduced with permission from (Green et al., 2017). Primarily, arterial blood flow is regulated at the level of muscular resistance arteries and arterioles by endothelial mechanotransduction of shear stress through the glycocalyx, caveolae, cilia, g proteins, tyrosine kinases, and torsion of intracellular matrices [reviewed in: (Chistiakov et al., 2017; Fels & Kusche-Vihrog, 2020; Yamashiro & Yanagisawa, 2020)]. These shear stress-sensitive mechano-transducers up/downregulate a number of homeostatic vascular tone mechanisms (He et al., 2020), including eNOS phosphorylation (on Ser1177 sites, among others) through eNOS mediation of l-arginine and O2, a major regulator of NO within the endothelium (Dimmeler et al., 1998; Fisslthaler et al., 2000). From the endothelium, NO is transduced across the abluminal EC surface into VSMCs whereby it LITERATURE REVIEW 45 upregulates soluble guanylate cyclase (sGC) and cyclic guanosine monophosphate (cGMP), which phosphorylates myosin light chain kinase and desensitizes VSMCs to Ca2+ – causing VSMC relaxation (see Figure 2-21). Cyclooxygenase, via arachidonic acids, and endothelial-derived hyperpolarizing factors also contribute to shear-mediated endothelial-dependent vasodilation (see Figure 2-21). The endothelial production and concentrations of NO and other vasodilators is tightly coupled with the contiguous shear stress on the luminal endothelial surface; therefore, VSMC tone is also tightly coupled with endothelially-sensed shear stress. Given the vast number of pathways mediated in the vasculature through ECs, in part by shear stress – e.g., inflammation and anti-inflammation signalling, apoptosis, NO bioavailability, cell alignment and proliferation (Chistiakov et al., 2017; Fels & Kusche-Vihrog, 2020; Yamashiro & Yanagisawa, 2020) – the importance of endothelial function in vascular blood flow regulation in both health and disease cannot be overstated. Despite substantial regional heterogeneity (Aird, 2007; Leloup et al., 2015; Augustin & Koh, 2017; Minami et al., 2019), this is true of the endothelium throughout the body – dysfunction of the endothelium playing a critical role in the early development of several major diseases, including, for instance, atherosclerosis (Bonetti et al., 2003; Aird, 2006; Nixon et al., 2010; Conway & Schwartz, 2013; Vlachopoulos et al., 2015; Fernandes et al., 2018) and vascular and Alzheimer’s dementias (Kalaria, 2016; Wolters et al., 2016, 2017; Wang et al., 2018; Nation et al., 2019; Pitchaya et al., 2019). 2.7 Assessments of vascular function in research and clinical settings 2.7.1 Peripheral flow mediated dilatation Non- invasive tests of endothelium-dependent function include Duplex ultrasound assessment of peripheral artery velocity and diameter (from which flow can be calculated) during flow-mediated dilation (FMD), in response to both reactive hyperaemia (RH) and steady state increases in flow. The typical (RH) FMD test requires arterial occlusion using an inflatable cuff to elicit distal microvascular NO and metabolite mediated dilation, which upon cuff release, causes a rapid increase in blood flow and shear stress to far above baseline values [shear rate increases of ~1000% (Carr et al., 2020)]. Shear stress increases sharply, typically peaking at <10 seconds, and in response, endothelium-dependent mechanisms (e.g., eNOS) are upregulated and NO production is increased, causing subsequent vasodilation (Pohl et al., 1986; Rubanyi et al., 1986; Melkumyants et al., 1989; Fisslthaler et al., 2000). This dilation peaks ~45-90 seconds following peak shear stress. Peripheral artery FMD is predictive of cardiovascular risk and outcomes (Inaba et al., 2010; Green et al., 2011; Ras et al., 2013); this FMD has also been correlated with coronary artery endothelium-dependent vasodilation assessed via LITERATURE REVIEW 46 acetylcholine infusion (Anderson et al., 1995; Broxterman et al., 2019) and increases in shear stress (Takase et al., 1998). A 1% increase in FMD has been associated with a 13% decrease in risk of adverse cardiovascular events, while an increase by one standard deviation was associated with a 41% decrease in cardiovascular event risk (Inaba et al., 2010). However, while shear mediated dilation assessments in peripheral arteries are practiced widely in research to assess vascular endothelium-dependent function (Thijssen et al., 2011a, 2019), FMD has not yet been endorsed as a clinical surrogate endpoint, or a method of refining risk stratification, or monitoring treatment efficacy (Vlachopoulos et al., 2015). Nonetheless, the utility of this technique in physiological research for evaluating the effects of interventions on vascular function means that it remains a useful research tool. Peripheral artery FMD tests examine endothelial function specifically, rather than overall vascular function since they provoke endothelium-dependent mechanisms without a great influence of VSMCs. In comparison, coronary (Zeiher et al., 1989, 1991; Schächinger et al., 2000) and carotid (Van Mil et al., 2017, 2018; van Mil et al., 2019) artery responses to stimulation of the skin by a cold water stimulus (“cold pressor test”) indicate the functionality of the vasculature generally as responses in these arteries combine both endothelium-dependent and -independent mechanisms. In the these arteries, cold pressor-mediated increases in SNA promote both vasoconstriction (through 1 adrenergic receptor activation) and vasodilation [through increased endothelially-transduced epinephrine activation of -adrenergic receptors as well as increased heart rate which raises myocardial demand and increases blood flow promoting shear-mediated vasodilation in the coronary arteries (Zeiher et al., 1989)]. With a healthy, functioning endothelium, the net result is vasodilation, while the presence of endothelial dysfunction results in vasoconstriction. As such, the cold pressor test assesses the endothelium but the net result of the response is representative of the overall health of the vessel. The cold pressor test then is an assessment that is able to indicate vascular health, but conducted without other tests is unable to further illuminate the mechanisms that are changed or effected by an intervention or disease state. Rather, assessments that can aid in the discernment of various mechanisms are of great utility, particularly in research settings. 2.7.2 Cerebrovascular reactivity to carbon dioxide Assessment of CVR (the physiology of which is described in 2.5.1 Carbon dioxide) has been determined using a group of methods that generally use controlled respiratory hypercapnia/acidosis to assess the efficacy of cerebral global and/or regional vascular regulation of CBF to changes in PaCO2. Methods of eliciting hypercapnic challenges include breath-holding, increases in the fraction of inspired CO2 (FICO2), and computerized targeting and forcing systems (see Table 2-3) (Robbins et al., 1982; Duffin, LITERATURE REVIEW 47 2011; Fierstra et al., 2013; Fisher, 2016; Fisher & Mikulis, 2021).5 The primary feature common to these methods is the elevation - either ramped, stepped, or steady-state - of end-tidal PCO2 (PETCO2) above individual baseline in order to elevate Pa CO2. Crucially, several of these methods generate incidental physiological responses which can present substantial confounds to interpretation (Regan et al., 2014), discussed further below. 5 Other methods which can be used to determine CBF CO2 reactivity - though which are meant for use in the determination of ventilatory rather than cerebrovascular sensitives to CO2 – included rebreathing techniques. Though of immense utility for ventilation assessment, alas, rebreathing is beyond the scope of this literature review, given these techniques focus on ventilatory sensitivity, while the focus of this thesis is vascular reactivity, despite the interconnectedness of these physiological reflex arcs (Ainslie & Duffin, 2009; Carr et al., 2021a). Additionally, CA inhibitors e.g., acetazolamide (which can be used to manipulate/increase arterial pH by impeding the interconversion of CO2 and HCO3-) have also been used extensively to assess CBF reactivity to acidosis. However, this too has been excluded here for the sake of brevity. LITERATURE REVIEW 48 Table 2-3. Attributes of the different experimental approaches used for the assessment of CVR Attribute Breath hold Fixed inspired CO2 Dynamic end-tidal forcing Prospective targeting Target PetCO2 No No Yes Yes Target PetO2 No No Yes Yes Target VA No No No Yes Breath-to-breath variability Yes Yes No (operator dependent) No Time to steady state Never 1-3 min 10 sec to 2 min 3-15 sec Ramp PETCO2 protocol achievable Depends on participant breathhold ability No Yes Yes End-tidal-arterial gradient during test NA Yes Variable No Automated targeting N/A No Yes Yes Equipment NA Douglas bag; at least one specific mix (e.g., 6% CO2, 21% O2, balance N2 or similar) gas tanks; breathing circuits 100% O2, 100 CO2, and 100% N2 medical grade gas tanks; gas analyzer; appropriate computer hardware and software, breathing circuit 100% O2, 90% CO2 10% O2, and 10% O2 90% N2 specific mix gas tanks; appropriate computer hardware and software, breathing circuit Quality assurance No No No RespirActTM: ISO13485 Compatibility TCD TCD, MRI, PET, duplex ultrasound TCD, MRI, PET, duplex ultrasound TCD, MRI, PET, duplex ultrasound Costs Zero Minimal costs $10000 to $50000 $60000 Key attributes that contribute to the strengths and limitations of each technique are summarized. PetCO2, partial pressure of end-tidal carbon dioxide; PetCO2, partial pressure of end-tidal oxygen; VA, alveolar ventilation; MRI, magnetic resonance imaging; PET, positron emission tomography; TCD, transcranial Doppler ultrasound; US, ultrasound. Redrawn and altered based on (Hoiland et al., 2019) The methods by which CVR is assessed can contribute considerably to differences in the stimulus efficacy and response isolation. A fundamental consideration is that PaCO2 is determined by both V̇CO2 and V̇A Although in healthy individuals at rest, metabolically-produced CO2 remains relatively constant, tidal volume and V̇A fluctuate within individuals breath-by-breath and, as such, PetCO2 also changes to a small degree breath-by-breath. This makes the accurate targeting of a specific PaCO2 difficult when using changes in fixed FICO2 (Peebles et al., 2007; Duffin, 2011; Fisher, 2016). LITERATURE REVIEW 49 Additionally, the PetCO2-PaCO2 gradient can vary with increasing hypercapnia [likely due to higher ventilation (Tymko et al., 2016)], affecting determination of CVR according to the variable indexed (i.e. PetCO2 or PaCO2) 6. Given that the PaCO2 changes the CSF and ISF pH most rapidly, indexing CVR against PaCO2 is superior to PetCO2 and is recommended (Robbins et al., 1990). Nonetheless, PetCO2 is commonly used and sufficient provided that the limitation of arterial-end tidal gradient is acknowledged and considered in the experimental interpretation. The efficacy of a CO2 stimulus in causing targetable alterations in PaCO2 that can be standardized depends on the technique’s capability to prospectively and reflexively manage PetCO2 each breath while accounting for variations in minute ventilation and allowing for tight “clamping” or maintenance of desired values. While PaCO2 is integral to the determination of PbtCO2 and [H+], and is the independent variable in many CO2-related CBF studies in humans, CVR is likely better indexed against PjvCO2 than PaCO2. As PbtCO2 may be more representative of the PCO2 on the abluminal surface of the vasculature (Bradley & Semple, 1962; Bradley et al., 1965; Shapiro et al., 1965, 1966; Pontén & Siesjö, 1966; Fencl, 1986; Peebles et al., 2007; Fan et al., 2010; Hoiland et al., 2015), and experimental and ethical limitations often forbid the direct measurement of PbtCO2 in healthy humans. So, because PbtCO2 varies inversely with CBF, and the PbtCO2-PaCO2 is reduced with increasing flow, CO2 washout and therefore PjvCO2, or the PaCO2-PjvCO2 gradient (or the arithmetic mean of PaCO2-PjvCO2 +1 mmHg) may be more representative of the CSF PCO2. For comprehensive discussion on the nuances of CVR testing and the need for standardized research and clinical guidelines for these assessments see Fierstra et al., (2013), Hoiland et al. (2015, 2019), and Fisher (2016). 2.7.2.1 Influence of MAP: During typical CVR tests, it is well known that MAP is markedly increased via sympathoexcitation (Ainslie & Duffin, 2009; Willie et al., 2012). One important consideration is that MAP contributes to increased cerebrovascular resistance (Przybyłowski et al., 2003; Ainslie et al., 2012). While the myogenic mechanisms of these responses may be distinct or may be overlapping (e.g., White et al., 2000; Tzeng et al., 2011), changes in resistance for the sake of CVR and cerebral autoregulation are nevertheless enacted through changes in the very same vasodilator reserve. That is to say, vasodilation is vasodilation, regardless of whether for the purposes of acid-base or pressure regulation. As such, changes in MAP during CVR tests, or changes of PaCO2 during cerebral autoregulation assessment, 6 Incidentally, although PetCO2-PaCO2 gradients can be a confounding factor in research studies, assessment of CO2 and O2 alveolar-arterial gradients can be of use in pulmonary function assessment should they be present, to non-invasively determine the degree of ventilation/perfusion mismatch by the ratio of deadspace (alveolar volume not contributing to gas exchange) to shunt (blood anastomoses bypassing gas exchange) (Wagner et al., 2022). LITERATURE REVIEW 50 confounds interpretation if not accounted for. Therefore, it is essential to report MAP, CO2 (whether PaCO2 or PjvCO2, or both), and CBF at baseline and with any intervention as descriptive clarity is crucial for interpretation. However, further clarity can be achieved with though additional approaches. First, calculation of cerebrovascular conductance (CVC; mL/min blood flow / mm Hg MAP) is a common method of analytically accounting for MAP changes during CVR. Vascular conductance represents blood flow at a given blood pressure. Assessing CVC and the CVC change with changes in PaCO2 (conductance reactivity, CVC CVR; mL/min blood flow / mm Hg MAP / mm Hg PaCO2) provides further indication as to the blood flow changes with hyper/hypocapnia while accounting for the change occurring with MAP. It should be noted that the CVC index is limited when used alone; it presumes that pressure and flow are linearly related, and that the remaining response after accounting for CVC CVR is fully explained by CVR. Second, statistically accounting for MAP with multiple linear regression analyses or as a covariate in linear mixed model analyses can aid in discerning MAP from PaCO2 related CBF changes, though this is also prone to the similar issues as the calculation of CVC. Third, simple correlational analyses can reveal the relationship between changes in MAP with changes in CBF/CBV during CVR testing. This relationship can indicate the degree to which MAP is influential on CBF and provide insight into this pressure-passive relationship if the relationship is altered following an intervention (e.g., Przybyłowski et al., 2003; Ainslie et al., 2012). The relationship between elevations in MAP and CBF/CBV during hypercapnia is up to R2=0.9 (Willie et al., 2012). Finally, appropriate study design is perhaps the best option for removing the influence of MAP changes in CVR, whereby changes in MAP are offset during CVR testing, for example, by the use of lower body negative pressure to simultaneously reduce MAP by the same degree as PaCO2 elevates it, or to separately elevate MAP and assess the degree of CBF change then account for that mathematically from the CVR response within-individuals. Although this route is difficult to achieve, has limited utility clinically, and adds further complexity to study interpretation. Lastly, while responses elicited by CO2 at the microvasculature are near-instantaneous, peak dilation of the larger upstream arteries [e.g., middle cerebral artery and internal carotid arteries (ICA)] is delayed from the onset of CO2 by up to 3 minutes (Coverdale et al., 2015; Carter et al., 2016; Hoiland et al., 2017; Iwamoto et al., 2018a; Carr et al., 2020). The mechanisms described above increase shear stress in larger cerebral vessels, which elevates eNOS mediated NO production and causes dilation even in the absence of hypercapnia (Heistad et al., 1978; Lee, 1980; Rubanyi et al., 1990; Fujii et al., 1991, 1992; Mashour & Boock, 1999; Carter et al., 2016; Hoiland et al., 2017; Carr et al., 2020). The changes in large artery diameter due to increased shear stress during steady-state hypercapnia represent only part of the response, other mechanisms – likely those described above – also contribute to the diameter change (Carr & Ainslie, 2020; Hoiland et al., 2021). LITERATURE REVIEW 51 2.7.3 Cerebral shear-mediated dilatation Following animal data indicating FMD of cerebral vessels (Fujii et al., 1991, 1992; Paravicini et al., 2006), an assay of extra-cranial cerebral artery shear-mediated dilation (cSMD) was developed (Hoiland et al., 2017). In brief, a 30 second bolus of hypercapnia (+9 mm Hg above baseline PETCO2) briefly elevates CBF - and thereby shear stress - through the cerebral arteries causing shear mediated vasodilation measured in the ICA (Hoiland et al., 2017; Carr et al., 2020). Upregulated shear stress-mediated eNOS activity in larger vessels causes dilation even absent hypercapnia (Heistad et al., 1978; Lee, 1980; Rubanyi et al., 1990; Fujii et al., 1991, 1992; Mashour & Boock, 1999; Carter et al., 2016; Hoiland et al., 2017; Carr et al., 2020). Thus, measurement of cerebral artery diameter during controlled and transient elevations of cerebral shear stress provides an experimental paradigm for the specific assessment of cerebral endothelial function (Hoiland et al., 2017; Carr et al., 2020). Using non-selective NOS inhibition by intravenous NG-monomethyl-l-arginine (L-NMMA) cSMD has been shown to be NOS dependent, while CVR was unchanged (Hoiland et al., 2021) (see Figure 2-22). Figure 2-22. CVR, cSMD, and BA FMD during saline and nitric oxide synthase inhibition Global cerebral blood flow (gCBF) cerebrovascular flow reactivity to CO2, cerebral shear-mediated vasodilation (cSMD), and brachial artery flow-mediated dilation (BA FMD) conducted during infusion of saline (orange) and NG-monomethyl-l-arginine (L-NMMA; green). CVR and cSMD figures reproduced with permission from (Hoiland et al., 2021) and data for BA FMD redrawn from (Doshi et al., 2001). Doshi et al. (2001) used upper arm cuff occlusion in the data presented here, rather than the standard upper forearm (Thijssen et al., 2019). As mentioned above (2.5.1 Carbon dioxide), while the literature is equivocal on the NO-dependency of CVR, the endothelium-dependent nature of cSMD is unlikely to be similarly challenged given the extensive and historical literature describing eNOS pathway involvement in analogous vascular responses in many other regions (Pohl et al., 1986; Rubanyi et al., 1986; Melkumyants et al., 1989; Fisslthaler et al., 2000). The very short stimulus duration perhaps underlies the difference between CVR LITERATURE REVIEW 52 and cSMD, with longer durations allowing for full presentation of many of the mechanisms described above in 2.7.2. While CVR (ml/min/mmHg) assesses mostly the downstream arteriolar and microvascular response where ICA dilation is secondary, cSMD (%∆mm) is a large artery (ICA) response. Although cSMD is predicated on the CVR response (requiring elevated blood flow in response to transiently elevated PaCO2) it is representative of a specific part of the cerebral vascular tree, and can occur in the absence of a CO2 stimulus (being a shear-mediated response). It just so happens that the hypercapnic stimulus is the most effective method (not to mention one of the only few) to rapidly and transiently yet safely elevate CBF in humans in vivo. Thus far, cSMD is limited to a small number of laboratories around the world, in part due to the complex and expensive equipment as well as prodigious operator knowledge and experience required. There have been several studies which have utilized CVR methods (longer duration hypercapnic exposures) as a means to assess cerebral endothelium-dependent and/or shear-mediated function (Lavallée et al., 2006; Iwamoto et al., 2018a, 2018b, 2020; Suzuki et al., 2020); however, given the number of mechanisms contributing to the CVR response, as well as the number of complexly intertwined factors (e.g., MAP) which could also play a role in the response, treating CVR style tests as “endothelium-dependent” and interpreting them using shear stress focused analyses may be a misrepresentation (Carr & Ainslie, 2020). 2.8 Summary: The cerebral and peripheral vasculatures are both sensitive to a number of stimuli, and likely hundreds of mechanisms exist for effective homeostasis of both local vascular areas and end-organ perfusion. Discerning vascular responses into local regulation (e.g., FMD) versus distal regulation (e.g., CVR) requires multifaceted understanding of the integrative physiology of both the supplier (arteries) and the supplied (end-organ). In the following chapters, studies are presented wherein discernment of this kind has been the overarching theme, while the focus of each study has variously been the cerebral and peripheral vasculatures during hypercapnic challenges. SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 53 Chapter 3: Study 1 – Internal carotid artery and brachial artery shear-dependent vasodilator function in young healthy humans Study 1 - Key points • Brachial artery (BA) shear-mediated dilation is a widely used assessment of vascular function with links to coronary artery health and cardiovascular risk. • Cerebral vascular health is often interrogated using cerebrovascular (middle cerebral artery velocity) reactivity to carbon dioxide. • We show that endothelium-dependent diameter (dilator) responses are not significantly related between the internal carotid artery (ICA) and BA; nor are endothelium-independent responses. • Additionally, ICA endothelium-dependent responses are not related to MCA velocity or ICA blood flow reactivity responses to carbon dioxide. • Therefore, assessment of large extracranial cerebral artery vascular health should be quantified via methods specific to the vessel, not via peripheral endothelial function or cerebrovascular reactivity to carbon dioxide. Abstract This study compared internal carotid artery (ICA) and brachial artery (BA) endothelium-dependent and -independent vasodilation. We hypothesized that endothelium-dependent and -independent vasodilation of the ICA and BA would be neither similar in magnitude nor correlated between vessels. In 19 healthy adults (23±6 years, 24±3 kg/m2, 6 female), endothelium-dependent dilation in the ICA was determined via Duplex ultrasound during transiently elevated shear stress caused by increased partial pressure of end-tidal carbon dioxide using dynamic end-tidal forcing (+9mmHg; cerebral flow-mediated dilation, cFMD). BA endothelium-dependent dilation was assessed via standard flow-mediated dilation (FMD). Endothelium-independent dilation in the ICA and BA was assessed concurrently for 10-minutes following administration of 400 µg sublingual glyceryl trinitrate (GTN). Endothelium-dependent vasodilation of the ICA (3.4±2.4%) was lower than (P=0.013) and not correlated to that of the BA (7.9±3.3%; r2=0.00, P=0.93). Including baseline diameter and shear-rate area under the curve as covariates maintained the difference between cFMD and FMD (3.3±4.2 % vs 7.8±3.8 %, P=0.03), while including baseline diameter and baseline shear rate-adjusted area under the curve as covariates abolished it (5.9±3.7 % vs 5.9.8±3.5 %, P=0.99). GTN-mediated vasodilation of the ICA (14.3±2.9%) was lower than (P=0.002) and not correlated to that of the BA (25.5±8.8%; r2=0.12, P=0.19). Adjusting for baseline diameter eliminated the differences in GTN-induced vasodilation (ICA: SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 54 20.1±5.8 % vs BA: 20.4±5.5 %; P=0.93). Differences in endothelium-dependent responses, and the lack of correlations between arteries, indicates that endothelium-dependent function cannot be assumed to be related across cerebral and peripheral vasculatures in young, healthy humans. SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 55 3.1 Background: Cardiovascular and cerebrovascular risk and disease are due, in part, to antecedent structural and functional vascular changes, including nitric oxide (NO) mediated endothelial dysfunction. Assessment of endothelial function has become synonymous with assessment of endothelial health. In conduit arteries, shear stress causes vasodilation through eNOS activation (Pohl et al., 1986; Rubanyi et al., 1986; Melkumyants et al., 1989; Fisslthaler et al., 2000); this includes the cerebrovasculature (Rubanyi et al., 1990; Fujii et al., 1991, 1992; Mashour & Boock, 1999). Shear-mediated pathways contribute to vascular function and health in the peripheral arteries (Naylor et al., 2011; Green et al., 2017) and peripheral artery conduit function is predictive of cardiovascular risk and outcomes (Inaba et al., 2010; Green et al., 2011; Ras et al., 2013). As such, flow (shear) mediated dilation (FMD) assessments are widely practiced in peripheral arteries to assess endothelial function (Thijssen et al., 2011a, 2019). However, while brachial artery (BA) FMD is correlated with coronary artery endothelium-dependent vasodilation assessed via acetylcholine infusion (Broxterman et al., 2019) and increases in shear stress (Takase et al., 1998) it is not a reflection of, nor correlated to, FMD in other conduit arteries (e.g., superficial femoral artery, popliteal artery, radial artery) (Nishiyama et al., 2007; Thijssen et al., 2008, 2011b; Jazuli & Pyke, 2011). Endothelial dysfunction in the cerebral vasculature is a major risk factor for cerebrovascular diseases such as ischemic and hemorrhagic stroke, cognitive decline, dementia, and Alzheimer’s disease (reviewed in: (Toda, 2012; Iturria-Medina et al., 2016; Kalaria, 2016; Wolters et al., 2016, 2017). Indeed, reduced cerebral NO bioavailability may precede amyloid angiopathy (Merlini et al., 2016) as well as histological and angiographic evidence of Alzheimer’s disease (Vita et al., 1990; Yasue et al., 1990; Mano et al., 1996). It remains unknown if BA FMD reflects cerebrovascular endothelial function. Our group has extended early animal studies (Heistad et al., 1978; Lee, 1980; Fujii et al., 1991) to develop a cerebral flow-mediated dilation (cFMD) endothelial function test specific to extra-cerebral arteries in humans (Carter et al., 2016; Hoiland et al., 2017). Specifically, cFMD assesses vasodilation in the ICA measured via Duplex ultrasound imaging of blood velocity and vessel diameter during transiently elevated shear stress. In theory, cFMD assessed endothelium-dependent function of the ICA analogously to FMD in the BA, although the specific endothelium-dependency has yet to be demonstrated. While middle cerebral artery (MCA) insonation using transcranial Doppler ultrasound (TCD) is often utilized for investigations requiring temporal definition of cerebrovascular responses, Duplex ultrasound has an advantage over TCD because continuous assessment of arterial diameter is possible in addition to contemporaneous velocity measurement, enabling quantification of shear stress and blood flow. Duplex assessment of the ICA is also preferred over TCD assessment of MCA because of the greater reactivity of the ICA compared to the MCA, for both physiological and technical reasons SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 56 (Willie et al., 2012). Whether ICA endothelial function assessed using cFMD is correlated to endothelial function examined by BA FMD remains to be determined. The development of techniques analogous to BA FMD for early detection of cerebrovascular endothelial dysfunction in humans may be clinically significant, particularly given that typical measures of cerebrovascular function (e.g., CO2 reactivity; CVR) using MCA velocity are not predictive of cerebrovascular disease specific risk (Portegies et al., 2014). While previous work has linked BA FMD to CVR (Lavi et al., 2006; Ainslie et al., 2007), CVR does not seem to hold predictive value (Portegies et al., 2014) in populations other than those with steno-occlusive disease (Markus & Cullinane, 2001). This may, in part, be due to the fact that CVR responses only partially reflect endothelium-dependent cerebral vasodilation. Furthermore, the stimuli of BA FMD (transient peak shear stress) and CVR (steady state mean shear) are dissimilar, so it remains unclear whether exposing the ICA and BA to similar shear stress stimuli (i.e. transient increases in shear stress) provokes correlated responses between these vessels, such as those which exist between brachial and coronary FMD (Takase et al., 1998). The purpose of the current study was to compare endothelium-dependent shear-mediated dilation in conduit vessels of the peripheral and cerebral vasculatures, specifically the brachial and internal carotid arteries. Based on a previous study demonstrating a cFMD magnitude of ~3-4% (Hoiland et al., 2017), and given that BA FMD is typically ~4-10% in healthy young adults (Witte et al., 2005; Holder et al., 2019), we hypothesized that cFMD of the ICA would be of a lesser magnitude than BA FMD. Given the common failure to find correlations between shear-mediated function in the peripheral arteries (Nishiyama et al., 2007; Thijssen et al., 2008, 2011b; Jazuli & Pyke, 2011), and the likelihood of distinct endothelial cell phenotypes between the ICA and BA (Gerritsen, 1987; Jow et al., 1999; Nilius & Droogmans, 2001), in addition to other characteristics which differentiate the cerebrovasculature from other vascular beds (Kety & Schmidt, 1948b; Shapiro et al., 1971; Baumbach & Heistad, 1983; Faraci et al., 1988; Vallance et al., 1989; Faraci & Heistad, 1990; Ainslie et al., 2005a; Thijssen et al., 2008; Hall et al., 2014; Brassard et al., 2017; Carter et al., 2017), we also hypothesized that endothelium-dependent responses in the ICA and BA would not be correlated. To account for potential differences in smooth muscle cell function between the BA and ICA, we also assessed vasodilator responses to sublingual glyceryl trinitrate (GTN) in both arteries; we hypothesized that GTN responses would be dissimilar in magnitude (ICA GTN dilation would be lesser than BA), and not correlated between arteries. As steady-state CO2 reactivity of the cerebral vasculature is often considered analogous to cerebral vascular endothelial function (Lavi et al., 2006; Ainslie et al., 2007) we also investigated whether a common test of CVR (indexed by both MCA velocity and ICA blood flow) was related to ICA endothelial function. SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 57 3.2 Methods: Participant characteristics: Nineteen participants (6 female; 23±6 years, 73±12 kg, 175±12 cm, 24±3 kg/m2) volunteered for and participated in this study. Exclusion criteria included the presence or history of cardiovascular, musculoskeletal, or metabolic disease, present smokers or a history of smoking, or use of medication (except oral contraceptives). Whether phase of menstrual cycle affects endothelium-dependent function is unclear (Stanhewicz & Wong, 2020; Wenner & Stachenfeld, 2020), with studies having reported both variation (Hashimoto et al., 1995; Williams et al., 2001) and no variation (Shenouda et al., 2018) across the cycle. Therefore, in order to include females, we attempted to standardize for menstrual phase (Thijssen et al., 2019) by only assessing women during phases when estradiol is lowest i.e. in the first seven days of the follicular phase in normally menstruating women (MacNutt et al., 2012) (n=1), or during the placebo week in those taking oral contraceptives (Minson et al., 2000) (n=1), or during menses in those with an intra-uterine device (Faundes et al., 1980) (n=4). The study was approved by the University of British Columbia’s Clinical Research Ethics Board (H17-01810) and conformed to the Declaration of Helsinki (except registry in a database). All participants were informed of any potential associated risks and familiarized with all experimental procedures, after which all provided written informed consent prior to any experimental sessions. Experimental measures: Cardiorespiratory variables: Cardiorespiratory variables included mean arterial pressure (MAP), systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), minute ventilation (VE), electrocardiogram (ECG), inspired and expired volume, respiratory flow, partial pressure of end-tidal CO2 and O2 (PETCO2 and PETO2 respectively). Beat-by-beat blood pressure was assessed via non-invasive finger photoplethysmography (Finometer PRO, Finapres Medical Systems, Amsterdam, Netherlands). Absolute correction of Finometer values was based on duplicate brachial artery blood pressure measurements from the right arm using an automated sphygmomanometer (EM-759-E; Omron, Kyoto, Japan). The HR was assessed by a lead II configuration ECG. Breath-by-breath CO2 and O2 were continuously sampled at the mouth by a calibrated gas analyzer (model ML206, ADInstruments). The PETCO2 and PETO2 were calculated in LabChart using peak detection analysis with daily correction for barometric pressure. Respiratory flow and inspired and expired volume were measured with a SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 58 pneumotachometer (model HR 800 L, Hans Rudolph, Shawnee, KS). All variables were continuously sampled at 1000 Hz via analogue-to-digital converter (PowerLab 16/30, AD Instruments, Colorado Springs, CO) and recorded in LabChart 7 software and saved for subsequent offline analysis. Dynamic end-tidal forcing: We controlled PETCO2 and PETO2 using a dynamic end-tidal forcing system described previously (Tymko et al., 2015, 2016). This approach allows for computerized breath-by-breath regulation of end-tidal gases, and therefore arterial gases, independent of ventilation. Notably, this method of arterial blood gas manipulation provides lesser arterial-to-end-tidal gradients of PETCO2 than other methods of blood gas manipulation (e.g. CO2 inhalation from a reservoir/Douglas bag), precludes any influence of V̇E on CVR (Howe et al., 2020), and therefore provides an accurate quantification of stimulus response relationships (Fisher, 2016; Fisher et al., 2018). This gas delivery system was used for both the cFMD and CVR tests described below. Transcranial Doppler (TCD): Blood velocity of the right middle cerebral artery (MCAv) was measured using transcranial ultrasound (2 MHz, TCD, Spencer Technologies, Seattle, WA). Optimal M-mode imaging was achieved using a headframe (M600, Spencer Technologies) allowing precise alignment of insonation angle and minimal movement artifact. Raw MCAv data was exported via analogue-to-digital converter (PowerLab) to LabChart 7 for offline analysis. The MCA was insonated through the trans-temporal window using techniques described in detail elsewhere (Willie et al., 2011). ICA and BA ultrasonography: Right ICA and BA blood velocity and vessel diameter were measured using Duplex ultrasound (10-MHz multi-frequency linear array, Terason uSmart 3300, Teratech, Burlington, MA). Diameter and velocity of the ICA and BA were imaged at least 1.5 cm distal to the common carotid bifurcation and 2 cm proximal to the brachial-radial bifurcation, respectively, to avoid capturing the effects of turbulent flow and nonuniform shear. An insonation angle of 60° was maintained throughout each test. After initial image acquisition, B-mode gain was not changed, to avoid inconsistency in apparent arterial wall characteristics between tests. All recordings were made in accordance with published technical recommendations (Thomas et al., 2015). Continuous images were screen captured and stored as video files for offline analysis (see data analysis section). SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 59 Experimental protocol: Participants arrived to the laboratory after a ≥6-hour fast, having avoided alcohol and vigorous physical activity for 24 hours prior and caffeine for 12 hours prior (Thijssen et al., 2019). Upon arrival participants rested supine for 20 minutes during instrumentation to promote hemodynamic stabilization. Briefly, a BA FMD was performed, after which, five minutes of baseline respiratory data was collected before the cFMD. Following the cFMD test, participants completed a CVR test, and finally, administration of GTN. Adequate recovery (10 minutes) was provided between the cFMD, CVR, and GTN tests to allow for CO2 wash-out (see protocol schematic Figure 3-1). A return to normal blood pressure and MCAv were verified by an investigator prior to commencing a subsequent test. Figure 3-1. Study 1 - Protocol schematic. Brachial flow-mediated dilation (FMD): Flow mediated dilation is a routine test of brachial artery endothelial function (Thijssen et al., 2011, 2019). Briefly, following a one minute baseline Duplex ultrasound scan of BA diameter and blood velocity, arterial inflow was occluded using a manual inflation cuff for five minutes at 220 mm Hg. After five minutes the cuff was rapidly (<1 second) deflated, causing reactive hyperaemia. Duplex imaging was continuously maintained throughout the five minutes of occlusion and three minutes following cuff deflation (Thijssen & Green, 2020). Cerebral (ICA) flow-mediated dilation (cFMD): This test follows the protocols established by Hoiland et al., (2017). Once baseline end-tidal gas concentrations have been established, participants breathed through the dynamic end-tidal forcing system, and were provided appropriate tidal volumes of O2 and CO2 concentrations on a breath-by-breath basis to maintain baseline PETCO2 and PETO2. Duplex ultrasound of the right ICA captured one minute of end-tidal forced baseline breathing, followed by two minutes of coached ventilation at 20 breaths per minute. The purpose of hyperventilation clamped at baseline end-tidal values is to ensure a SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 60 rapid increase in PETCO2 at the onset of hypercapnia as well as to aid sonographer imaging during hypercapnia (detailed in: Hoiland et al., 2017). The PETCO2 was then swiftly elevated to 9 mm Hg above baseline for 30 seconds to drive a transient increase in ICA blood flow and arterial shear stress, after which PETCO2 was returned to and maintained at baseline values for a further three minutes. Duplex imaging of the right ICA was maintained continuously throughout. Cerebrovascular reactivity to carbon dioxide (CVR): End-tidal gas pressures were controlled via dynamic end-tidal forcing. Baseline PETCO2 and PETO2 were re-established until steady and maintained for one minute of baseline Duplex imaging of the right ICA, after which, PETCO2 was elevated to 9 mm Hg above baseline and maintained for five minutes. Duplex imaging was maintained throughout. Endothelium-independent dilation (GTN): Administration of glyceryl trinitrate (GTN) was used to assess endothelium-independent dilation in the BA and ICA. Right BA and ICA were simultaneously scanned for one minute, at which point 400 µg of GTN was sublingually administered; thereafter, Duplex imaging continued throughout and for the following 10 minutes. Before and immediately following GTN administration, PETCO2 and PETO2 were continuously recorded. This test concluded the study. GTN dose-responsiveness sub-study: To determine and compare BA and ICA smooth muscle sensitivity to direct NO donation, we compared the dose-response curves to 400, 800, 1200, and 1600 µg GTN doses in a subset of volunteers (n=5 males). We carried out three follow up visits during which we administered 800, 1200, and 1600 µg GTN doses. No difference in the time course or peak dilation between 400 – 1600 µg in the BA, nor in the ICA were identified (see results); therefore, for the main investigation, only 400 µg GTN doses were employed. Data analysis: Reconstructed brachial arterial blood pressure waveforms (from which MAP was calculated), MCAv, PETCO2, and PETO2, were exported from LabChart in one-second averaged bins into Microsoft Excel. BA and ICA diameter, blood velocity (v), flow (Q), and shear rate were analysed using customized wall SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 61 tracking/edge detection software (BloodFlow Analysis, version 5.1) (Woodman et al., 2001). The software calculates shear rate: Shear rate = 4 × peak envelope blood velocity / arterial diameter, And flow: Q = peak envelope blood velocity/2 × [π(0.5 × diameter)2] × 60. This software was designed to mitigate investigator bias; it has been validated and is described in detail elsewhere (Woodman et al., 2001; Black et al., 2008). Brachial FMD: Using BloodFlow Analysis 5.1, an area of interest encompassing an appropriate section of the Duplex image (vessel walls and velocity spectral trace) allowed for beat-by-beat detection of reactive hyperaemia and blood flow responses during the FMD test (Woodman et al., 2001). One minute of baseline data was analysed against the three minutes of response data to give baseline, peak, and time to peak diameter, and Q and shear rate characteristics. This approach followed standardized guidelines (Thijssen et al., 2011, 2019). Cerebral (ICA) FMD: Full details of cFMD analysis are described in detail in Carter et al., (2016) and Hoiland et al., (2017). In brief, blood flow and shear rate were automatically calculated from diameter and velocity data at 30Hz (Bloodflow Analysis 5.1). General Purpose Data Graphing Software (version 1) then smoothed the data with a low frequency filter and calculated median baseline diameter and shear rate during the last minute of hyperventilation prior to elevation of PETCO2. Automatically detected increase thresholds and peaks were then assessed visually by two independent investigators to ensure that: the detected threshold preceded the detected peak; the variable did not decrease below the threshold point before reaching the detected peak. From a total of 19 cFMD tests, 12 met the above criteria, the detected values were accepted, used in all analysis and are reported herein. Of the remaining seven files, four were of insufficient quality to provide determinable values, either due to overt angle changes (n=3) or unclear wall detection (n=1). Thresholds in the remaining three cases were manually adjusted by the two investigators to where each independently judged there to be a considerable deviation from baseline that met the algorithmic criteria above (as per previous work: Carter et al., 2016; Hoiland et al., 2017). SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 62 The mean of the manually assessed thresholds were used for all statistical analysis and are reported herein. For all data, shear rate area under the curve (SRAUC) was then calculated from the onset of hypercapnia to peak diameter (Black et al., 2008; Thijssen et al., 2011a). Cerebrovascular reactivity to carbon dioxide: Averages of one minute of baseline data and the final two minutes of elevated PETCO2 during the steady state test were used to determine flow, velocity, and PETCO2 values. Reactivity of QICA to elevations in PETCO2 was determined by: Absolute CVR = QICA/PETCO2, Relative CVR= %QICA/PETCO2, GTN: Baseline data was averaged for one minute prior to GTN administration and compared to 10 minutes of response immediately post GTN using peak detection software (Bloodflow Analysis 5.1). Data exclusion criteria: For all Duplex ultrasound files, any video images where image quality was insufficient to provide continuous and consistent data were excluded from analysis. This included, four files for cFMD [lost due to either overt angle changes (three) or unclear wall detection (one)], one for CVR (lost due to participant movement), and four for the ICA GTN scans (lost due to overt insonation angle changes). Any statistical outliers (determined as lying beyond the mean ± [3 × SD]) were excluded from any analysis involving that variable or variables related to the pertinent outcome. This included, one participant for all CVR calculations due to extreme PETCO2 values. Other data that were excluded include, one participant for all MCAv related outcomes due to TCD error, and one for BP related outcomes during cFMD due to Finometer finger-cuff malfunction. Additionally, per previous work (Hoiland et al., 2017) bivariate outliers were detected using bagplot analysis (https://cran.r-project.org/web/packages/aplpack/aplpack.pdf), which functions as a bivariate box plot (Rousseeuw et al., 1999), this included one for cFMD × SRAUC. As such, statistical analyses were run on: BA FMD n=19, ICA cFMD n=14; BA GTN n=19, ICA GTN n=15; ICA CVR n=17. SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 63 3.3 Statistical analysis: Percent dilation and absolute diameter change in BA versus ICA for FMD versus GTN was compared using linear mixed model (LMM) analysis with compound symmetry covariance (factors: artery & test). Subsequently, all responses were allometrically scaled to account for differences in baseline artery diameter as previously described (Thijssen et al., 2008). For administration of GTN at 400, 800, 1200, and 1600 μg the percent and absolute dilations were compared by linear mixed model analyses (factors: artery & dose), also, the arteries responses to the different doses were individually compared by linear mixed model analyses (factor: dose). Linear mixed model analyses compared allometrically scaled FMD and GTN between the BA and ICA. Furthermore, to control for SRAUC differences between the cFMD and BA FMD tests, SRAUC was included as a covariate in linear mixed model analysis assessing FMD (Atkinson & Batterham, 2013a); likewise for allometrically scaled FMD. Additionally, in order to account for differences in baseline shear rate between the arteries and because the methods of eliciting shear increases differs between the arteries (i.e. cuff release vs CO2 bolus), we calculated the total area under the curve for 3 second bins of the delta between mean baseline shear and response shear from stimulus onset to 180 seconds post (delta SRAUC, DSRAUC). Separate analysis of BA and ICA FMD was conducted using both DSRAUC and the natural logarithm of baseline diameter (DLogBase) as covariates. Fisher’s least significant difference test was used for pairwise post hoc comparisons in all linear mixed model analyses. For each comparison, linear mixed model estimated means (EM) were back calculated per Shenouda et al. (2017). To determine whether vasodilatory function is related between vessels, each ICA response was compared to the equivalent BA response using Pearson’s r correlations (i.e. BA FMD versus ICA cFMD, and BA GTN versus ICA GTN). Pearson’s r correlations were also run testing back-transformed allometrically-scaled data, calculated as per two methods from previous studies (Atkinson et al., 2013; Shenouda et al., 2017). All statistical analyses were performed with the Statistical Package for the Social Sciences (SPSS, Version 24). Absolute, relative, allometrically-scaled, and scaled-SRAUC-adjusted means, standard variations, and p-values are all reported herein. P<0.05 was taken to indicate statistical significance. Data are mean ± standard deviation unless otherwise stated. 3.4 Results: Endothelium-dependent tests: By design, during the cFMD test PETCO2 was rapidly (4-5 breaths, 14.6±3.4 s) elevated by ~9 mmHg (41.6±2.8 to 51.1±2.8 mmHg, respectively, P<0.001). Shear rate increased from 381.7±121.6 at SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 64 baseline to a peak of 507.4±165.1 1/s (P<0.001), with an overall SRAUC of 45095±13677 1/s. ICA diameter increased (+3.4±2.5 %) from 5.01±0.44mm to a peak of 5.17±0.39 mm (P<0.001) following the transient CO2 stimulus (Table 3-1). Group averaged diameter and shear rate data for the cFMD test is depicted in Figure 3-2A. There was no change in MAP during transient hypercapnia (87±10 vs 89±10 mmHg, P=0.10) and variability in MAP was unrelated to ICA dilation (r2=0.07; P=0.38). Other cardiorespiratory data for the cFMD test are reported in Table 3-2. During BA FMD (Table 3-1), diameter increased (+7.9±3.3 %) from 3.84±0.61 mm at baseline to a peak of 4.13±0.62 mm (P<0.001). Group averaged data for the BA FMD test is depicted in Figure 3-2B. SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 65 Figure 3-2. Study 1 - Diameter profiles for each test Diameter and shear rate response profiles by percent change from baseline during ICA cFMD (A), and BA FMD (B), note the difference in right y-axis scales between A and B. (C) Diameter response profile by percent change from baseline during GTN in BA and ICA. (D) Diameter change for each artery for each test compared with linear mixed model analysis (factors: artery, test). Main effects for both artery (P<0.001), and test (P<0.001). (E) Allometrically scaled BA FMD and ICA cFMD diameter changes compared with BA ICA BA ICA01020304050Diameter change (%)FMD GTNArtery, P<0.001Test, P<0.001Interaction, P=0.58BA ICA04812Diameter change (%)Scaled for baseline diameter and SRAUCFMD0 200 400 6000102030-5Time (seconds)Diameter change (%)GTN 400ugICABABA ICA01020304050Diameter change (%)Scaled for baseline diameterGTNA B CD E FBA ICA051015Diameter change (%)Scaled for baseline diameterFMD0 60 360 420 480 540-2024604008001200Time (seconds)Diameter change (%)Cuff releaseDiameterShear RateShear rate change (%)0 100 200-202460102030Time (seconds)Diameter change (%)+9mmHgShear rate change (%)DiameterShear RateGBA ICA04812Diameter change (%)Scaled for baseline diameter and DSRAUCFMDH*SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 66 linear mixed model analysis (factor: test. Covariate: DLogBase). (F) Allometrically scaled BA FMD and ICA cFMD diameter changes compared with linear mixed model analysis while controlling for SRAUC (factor: test. Covariates: SRAUC, DLogBase). (G) Allometrically scaled BA FMD and ICA cFMD diameter changes compared with linear mixed model analysis while controlling for DSRAUC (factor: test. Covariates: DSRAUC, DLogBase). (H) Allometrically scaled BA and ICA GTN diameter changes compared with linear mixed model analysis (factor: test. Covariate: DLogBase). A, B, and C, are mean (solid lines) ± 95% confidence intervals (dashed lines), D is mean (bars) ± standard deviation (lines) with individual data (circles). E and H are back-transformed individual data (circles) (Shenouda et al. 2017) with means (bars) ± standard deviations (lines). F and G are back-transformed estimated means (bars) ± back-transformed estimated standard deviations (lines). Back-transformed individual values for F and G cannot be given as the calculation to back-transform individual values only includes natural logged diameter values and the common slope of the log transformed DLogBase vs peak diameter relationship, but not shear rate area under the curve, as such, for F and G, only back-transformed estimated means and standard deviations have been presented (back calculated per Shenouda et al. 2017). * P<0.05. SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 67 Table 3-1. Peripheral and cerebral vascular characteristics for endothelium-dependent and -independent tests TEST FMD GTN ARTERY BA ICA BA ICA N= 19 14 19 15 Baseline Diameter (mm) 3.84±0.61 5.01±0.44* 3.74±0.55 5.15±0.48* Peak Diameter (mm) 4.13±0.62* 5.17±0.39* 4.65±0.49* 5.89±0.56* Delta Diameter (mm) 0.29±0.11† 0.16±0.11‡ 0.92±0.22‡ 0.74±0.17† Delta Diameter (%) 7.9±3.3† 3.4±2.5‡ 25.5±8.8‡ 14.3±2.9† Time to Peak Diameter (sec) 69.4±45.1 102.0±29.9 422.0±137.9 440.4±144.3 Baseline shear rate (1/s) 90.5±63.2 381.7±121.6* - - Response SRAUC (1/s) 25769±11760 45095±13677* - - DSRAUC (1/s) 39185.9±17.121.1 3110.1±3633.3* Time to Peak Shear (sec) 8.5±2.7 38.48.6* - - Time from Peak Shear to Peak Diameter (sec) 60.9±45.6 63.0±30.3 - - BA, brachial artery; DSRAUC, total area under the curve of the difference between baseline shear rate and response shear rate until peak diameter; FMD, flow-mediated dilation; GTN, glyceryl tri-nitrate; ICA, internal carotid artery. Baseline diameter compared within artery with paired two-tailed student’s T-tests. Peak diameter compared to baseline diameter within each artery and test with paired two-tailed student’s T-tests. Baseline shear rate, Response SRAUC, DSRAUC, Time to peak shear, and Time from peak shear to peak diameter compared between BA and ICA with paired two-tailed student’s T-tests.; * P<0.05. Delta diameter compared between artery and test with linear mixed model analysis (factors: artery, test); † P<0.05 between cFMD and GTN within artery, ‡ P<0.05 between BA and ICA within test. Data are mean ± standard deviation.SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 68 Table 3-2. Mean vascular and cardiorespiratory variables one minute before, and during administration of CO2 for cFMD (30 second), and CVR cFMD CVR Baseline During Change (%) Baseline During Change (%) PETO2 (mmHg) 88.1±4.0 88.1±3.5 - 88.9±3.5 87.6±3.3 - PETCO2 (mmHg) 41.6±2.8 (Peak)51.1±2.8* 23.1±6.8 41.9±2.5 50.8±2.5* 21.2±1.9 MAP (mmHg) 87±10 89±10 - 89±11 95±13* 7±5 HR (bpm) 76±17 76±20 - 70±14 77±16* 10±16 MCAv (cm/s) 67.3±17.9 74.8±19.8* 11.6±6.1 63.0±14.7 86.6±21.3* 39.1±8.9 MCACVR (% ∙ mmHg-1) - 1.3±0.9 - - 4.4±1.0 - ICAv (cm/s) 49.8±9.3 55.6±10.5* 11.6±7.5 46.0±8.3 66.5±12.7* 45.2±15.0 QICA (ml/min) 211.9±47.2 239.2±56.7* 12.9±7.6 277.9±61.8 437.5±108.1* 57.4±17.2 ICACVR (% ∙ mmHg-1) - 1.5±1.1 - - 6.5±1.9 PETO2, partial pressure of end-tidal oxygen. PETCO2, partial pressure of end-tidal carbon dioxide. MAP, mean arterial blood pressure. HR, heart rate. MCAv, middle cerebral artery blood velocity. MCACVR, middle cerebral artery cerebrovascular reactivity to carbon dioxide. ICAv, internal carotid artery blood velocity. QICA, internal carotid artery blood flow. ICACVR, internal carotid artery cerebrovascular reactivity to carbon dioxide. Data were compared pre vs during for cFMD and CVR using paired two-tailed students t-tests. cFMD n=14, CVR n=17 *P<0.05. Data are mean ± standard deviation.SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 69 Endothelium-independent test: Sublingual administration of 400 μg GTN lead to a 14.3±2.9 % vasodilation of the ICA and 25.5±8.8 % vasodilation of the BA (Figure 3-2C). The ratio of ICA cFMD to GTN dilation was 0.22±0.16, and the ratio of BA FMD to GTN dilation was 0.33±0.15. During the GTN test, there was a slight (<1 mmHg) decrease in PETCO2 from baseline (39.8±3.2 vs 39.1±3.0 mmHg; P=0.02) and a modest reduction in MAP from 91±10 to 89±9 mmHg (P=0.02). For the dose-response subset, there was a main effect of artery (P=0.006) but no effect of dose (P=0.92) (Figure 3-3A and B). Investigated separately, there was no effect of dose for ICA (P=0.11) and no effect of dose for BA (P=0.56). Figure 3-3. Study 1 - Response profiles to different GTN doses. (A) BA response profiles to 400, 800, 1200, and 1600 µg GTN doses (n=5). (B) ICA response profiles to 400 µg (n=4), 800, 1200, and 1600 µg doses (n=5). Mean (solid lines) ± standard deviation (dashed lines). 0 200 400 6000102030-5Time (seconds)Diameter change (%)GTN160012008004000 200 400 6000102030-5Time (seconds)Diameter change (%)16001200800400GTNABSHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 70 Between-artery comparisons: Between-artery and between-test comparisons: Linear mixed model analysis of BA and ICA FMD and GTN absolute responses demonstrated significant main effects of artery (P<0.001), and test (P<0.001, Figure 3-2D). Dilation of the BA was greater than that of the ICA for each test (FMD & GTN), while dilation in response to GTN was greater than that of the FMD of each artery (ICA & BA). ICA vs BA endothelium-dependent function: Linear mixed model analysis of allometrically scaled (covariate: DLogBase) ICA and BA FMDs revealed that responses were not significantly different between arteries (Figure 3-2E). However, when SRAUC was also included in analysis, the difference between ICA and BA remained ([scaled estimated mean ± scaled SD] 3.3±4.2 vs 7.8±3.8 %, respectively; P=0.025, Figure 3-2F). When DSRAUC was included in the analysis in place of SRAUC, ICA and BA were not significantly different ([scaled estimated mean ± scaled SD] 5.9±3.7 % vs 5.9±3.5 %, respectively; P=0.99 Figure 3-2G). ICA vs BA endothelium-independent function: Linear mixed model analysis of allometrically scaled GTN tests (covariate: DLogBase) showed that the vasodilatory responses were not different between arteries ([scaled estimated mean ± scaled SD] ICA: 20.14±5.78 % vs BA: 20.40±5.50 %, respectively; P=0.93; Figure 3-2H). Select Correlations: Neither FMD, nor GTN absolute or percent responses were correlated between arteries (Figure 3-4A & B), nor were the back-calculated allometrically scaled values (Figure 3-5A & B). SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 71 Figure 3-4. Study 1 - Correlational analysis. (A) cFMD and FMD percent change responses compared (n=14). (B) GTN percent change responses (n=15). (C) Relative cerebrovascular reactivity to CO2 (CVR) compared with cFMD ICA dilation (n=13). Figure 3-5. Study 1 - Correlational analysis of allometrically scaled ICA and BA endothelium-dependent responses. Data back transformed with formula according to (A) Shenouda et al. (2017) and (B) Atkinson et al. (2013). BA FMD/GTNICA FMD/GTN0.0 0.2 0.4 0.6 0.80.00.20.40.6 r2 = 0.16p=0.240 5 10 15 20051015BA FMD Diameter change (%)ICA cFMD Diameter change (%)r2 = 0.00p=0.930 20 40 6005101520ICA GTN Diameter change (%)BA GTN Diameter change (%)r2 = 0.12p=0.192.5 3.0 3.5 4.0 4.5 5.03.54.04.55.05.56.0BA baseline diameter (cm)ICA baseline diameter (cm)r2 = 0.03p=0.450 50 100 150 200050100150BAPeak shear to Peak Diameter (sec)ICA Peak shear to Peak Diameter (sec)r2 = 0.08p=0.290 5 10051015cFMD diameter change (%)Relative CVR (%Δ |ml/min ICA| / Δ mmHg PETCO2Δ) r2 = 0.06p=0.46A B CD E F6 8 10 12 14051015BA FMD Diameter change ((DLogPeak/(DLogBaseβ))-1)*100ICA cFMD Diameter change ((DLogPeak/(DLogBaseβ))-1)*100r2 = 0.02p=0.61.22 1.24 1.26 1.28 1.30 1.32 1.341.201.251.301.35BA FMD Diameter change (DPeak/(DBaseβ))ICA cFMD Diameter change (DPeak/(DBaseβ))r2 = 0.03p=0.48ABSHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 72 CVR: During the final two minutes of steady state hypercapnia, PETCO2 was elevated from 41.9±2.5 at baseline to 50.8±2.5 mmHg (P<0.001) and PETO2 was maintained at baseline. MAP increased from 89±11 mmHg to 95±13 mmHg (P<0.001), as did HR (67±10 to 74±14 bpm; P=0.02). During hypercapnia, relative reactivity in the ICA was 6.49±1.94 % per mmHg, and in the MCA was 4.44±0.98 % per mmHg. QICA increased from 277.9±61.8 ml/min at baseline to 437.5±108.1 ml/min (P<0.001) and ICA diameter increased from 5.1±0.5 mm at baseline to 5.3±0.5 mm. cFMD versus CVR: When matched for the change in PETCO2 (cFMD Δ9.5±2.4 vs CVR Δ8.9±0.6 mmHg; P=0.52), ICA diameter change during CVR was not different to cFMD diameter change (4.2±3.0 vs 3.4±2.5 %, respectively; P=0.42). Relative cerebrovascular reactivity and cFMD diameter change were not related (r2=0.06; P=0.46, Figure 3-4C), nor were ICA dilation induced by CVR and that induced by cFMD (r2=0.26; P=0.068). 3.5 Discussion: We assessed the differential magnitudes and potential relationships between BA and ICA endothelial-dependent and -independent vasodilation. We hypothesized that endothelium-dependent responses would be dissimilar in magnitude (smaller in the ICA [cFMD]) and not correlated between BA and ICA; and that endothelium-independent (GTN) responses would be dissimilar in magnitude (smaller in the ICA) and not correlated between BA and ICA. Our main findings are the absence of correlations between endothelium-dependent or -independent vasodilation in the ICA and BA regardless of whether the comparison was between absolute or percent values (Figure 3-4A & B), or allometrically-scaled (Figure 3-5A & B). Furthermore, we determined that while ICA and BA endothelium-dependent diameter changes are not similar in magnitude (Figure 3-2D), this is the result of differences in baseline diameter (Figure 3-2E) and shear rate (Figure 3-2F & G). Collectively, these results indicate that BA measures of endothelium-dependent and -independent function are not indicative of ICA function. Further insight can be garnered from interpretation of covariate analyses. Allometrically scaling to account for different baseline diameters abolished the difference in endothelium-dependent responses between arteries (Figure 3-2E). Controlling for baseline diameter and response SRAUC re-established the difference in endothelium-dependent responses (Figure 3-2F); while accounting for baseline diameter and baseline shear rate-adjusted area under the curve (that is, DSRAUC; Figure 3-2G), once SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 73 again eliminated the difference. These observations indicate that ICA endothelium-dependent vasodilation is relatively smaller in absolute terms than BA endothelium-dependent vasodilation (Figure 3-2D) as a consequence primarily of its larger baseline diameter and elevated baseline shear rate, rather than due to any apparent differences in stimulus shear rate. Furthermore, while ICA and BA endothelium-independent responses were significantly different in absolute terms (Figure 3-2D), allometric scaling abolished the difference (Figure 3-2H). This method of statistical control indicates that differences in the magnitude of endothelium-independent vasodilation between ICA and BA are explained by differences in baseline diameter. The lack of correlations in corresponding responses between arteries (Figure 3-4A and B, Figure 3-5A and B) implies that endothelium-dependent or -independent function in the BA does not inherently imply a similar functional response in the ICA, and vice versa. In the following, we briefly suggest two notable points, that is, shear rate sensitivity and endothelium-independent control, followed by the main body of discussion concerning regional endothelial cell heterogeneity. Finally, we examine the evidence, experimental limitations, and implications underlying the findings of this study. Shear rate sensitivity: The magnitude of hyperemia following BA FMD cuff release (shear rate increases of up to 972 % [95% CI 744-1201] from baseline mean Figure 3-2B) is much greater than that elicited in the ICA by cFMD CO2 bolus (shear rate increases of up to 21 % [95% CI 13-29] from baseline mean, Figure 3-2A), and this appears to account for the smaller endothelium-dependent vasodilation in the ICA (Figure 3-2G). The dissimilarity in the shear rate stimulus between the arteries is likely a consequence of differences in both experimental interrogation of the vessels and vessel baseline haemodynamics, as indicated by BA and ICA baseline shear and DSRAUC (Table 3-1). Baseline shear is significantly greater in the ICA, yet DSRAUC is significantly greater in the BA. Given these differences in baseline shear, controlling for SRAUC does not accurately characterize the FMD stimulus when comparing ICA and BA. As such the difference in magnitude of the endothelium-dependent response between the two arteries appears to be due, at least in part, to a lesser increase in shear rate in the ICA caused by the cFMD test than in the BA caused by cuff release (see experimental considerations section). Differences in baseline shear are paramount in the determination of endothelial cell phenotype expression (Gerritsen, 1987; Jow et al., 1999; Nilius & Droogmans, 2001) and therefore also in the determination of shear stress responses (see regional endothelial cell heterogeneity section below). SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 74 Endothelium-independent control: Although the percent diameter change caused by 400 µg GTN was different between the two arteries (Figure 3-2D), we found after allometrically scaling for baseline diameter that responses were comparable (Figure 3-2H). When considered in conjunction with the findings of the endothelium-dependent tests, these results are interpreted to imply that vasoactive control of cerebral and peripheral vessels, at least in regards to the ICA and BA, is unrelated between vessels of differing magnitudes as a consequence of distinctions in endothelium-dependent function rather than endothelium-independent function, since maximal vascular smooth muscle mediated responsiveness is similar in both arteries once the inherent difference in size is taken into account (Figure 3-2H). Regional endothelial cell heterogeneity: Factors involved in the determination of resting tone and vasomotor activity in all arteries include; degree and activity of innervation (Baumbach & Heistad, 1983; Ainslie et al., 2005a; Brassard et al., 2017), vessel architecture (Thijssen et al., 2008), downstream resistance (Kety & Schmidt, 1948b; Shapiro et al., 1971; Faraci et al., 1988; Faraci & Heistad, 1990; Hall et al., 2014), haematological and humoral factors, endothelial function (Vallance et al., 1989) and endothelial cell phenotype expression (Gerritsen, 1987; Jow et al., 1999; Nilius & Droogmans, 2001). These factors also determine heterogeneity and variability in endothelium-dependent and -independent function between vasculatures, as indicated by the lack of correlations of responses between arteries in young, healthy adults. Differences between the vasculatures are likely the result of one or more of the above factors. In particular, endothelial cell phenotype expression is sensitive to the contiguous haemodynamic milieu (Gerritsen, 1987; Jow et al., 1999; Nilius & Droogmans, 2001) and, since ICA and BA baseline and response hemodynamics are significantly different (Table 3-1), the sensitivity of endothelial mechanotransduction is also different between the arteries. Indeed, differences in the function of mechanoreceptive channels have been demonstrated to underpin regional differences in vascular function in animal (Drummond et al., 2004; Althaus et al., 2007; Ashley et al., 2018) and human in vitro (Wang et al., 2009) models. Haemodynamic conditions at rest in the ICA and BA differ due to the substantially different end-organ perfusion requirements. The brain needs near constant perfusion, ~50-60 ml/min/100g, for which the ICA bilaterally supplies ~70% (Sabayan et al., 2013; Zarrinkoob et al., 2015), whereas resting forearm skeletal muscle requires substantially less, ~3 ml/min/100g (Hartling et al., 1989; Thompson et al., 2015), for which the BA is the primary provider. These baseline conditions also contribute to the different magnitudes of responses between arteries, at baseline the ICA supplies the brain abundantly and so only a small change is needed, whereas the BA supplies minimally at baseline so with stimulation a much larger response ensues and is accounted for by a greater functional SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 75 reserve. We suggest that the malleability and adaptability of endothelial cells to their environmental conditions (dynamic forces and humoral factors) underlie the differences and lack of correlations between endothelium-dependent function between the two sites; although by no means is endothelial heterogeneity the sole determinant of differences between the investigated vessels, given the myriad other differences between cerebral and peripheral artery structure and function noted above. 3.6 Experimental considerations: Baseline diameter: ICA baseline diameter was significantly larger prior to the GTN test than prior to the cFMD test (5.15±0.48 vs 5.01±0.44 mm, respectively, P=0.017). This highlights the prolonged effect that hypercapnia may have on the cerebrovasculature, and indicates that randomization may have improved the study design by possibly negating this order effect. However, analysis with allometric scaling for baseline diameter nevertheless revealed that GTN and FMD diameter change were similar between arteries (Figure 3-2E and H). Additionally, since the different dose subset tests were carried out either on different days or >30 minutes after completion of the previous test, if there had been a physiologically meaningful difference of baseline diameter due to prior dilation, then there would be a significant effect of dose on diameter change, since the 800, 1200, and 1600 µg doses would begin from “true” baseline and the 400 µg dose would represent an interfered with baseline. However, there was no effect of dose on diameter change during GTN when comparing the 400, 800, 1200, and 1600 µg GTN doses. Dissimilarities between stimulus methods: Since endothelium-dependent vasodilation is both rate and magnitude sensitive (Frangos et al., 1996; Butler et al., 2000), the consideration of these differences is paramount to comparing endothelium-dependent responses between arteries; we utilized several post-hoc statistical analyses in order to do so. FMD stimuli in both arteries differed as a result of methodological necessity. In some peripheral vessels it is feasible to compare responses to matched stimuli between arteries (e.g.: Carter et al., 2013) or to various stimuli within arteries (e.g.: Carter et al., 2014; Tremblay et al., 2019). However, in the cerebrovasculature, where uninterrupted blood flow is crucial, a post-occlusion reactive hyperemia experimental model is clearly not feasible (Rossen et al., 1943). Since a ~10 mmHg increase in PETCO2 is close to the physiological limits of tolerance in humans (Willie et al., 2012), it is unlikely that more sudden elevations in shear rate that are greater in magnitude can be induced in vivo in the ICA. The observed shear increase in the ICA is probably representative of the near maximal shear stress SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 76 achievable through transient perturbations in healthy cerebral conduit arteries (Hoiland et al., 2017). In contrast, during BA FMD, shear rate typically peaks less than 10 seconds after cuff release after rapidly increasing as a consequence of the downstream microvascular vasodilation. While methodological differences between the endothelium-dependent tests in the ICA and BA are apparent, we endeavored to rectify this via reasonable analyses (i.e., SRAUC and DSRAUC covariate analyses). Correlational analyses: The difference between correlation and causation should be acknowledged. By investigating the possibility of a correlation between BA and ICA endothelium-dependent or -independent function, we do not intend the implication that there may have been a causal link therein. Rather, our findings would seem to confirm and extend previous findings concerning the lack of relation between various arterial beds (Thijssen et al., 2011b) and the differences in endothelium-dependent responses to various vessel specific tests in the absence of pathology. We conducted post-hoc power analysis (G*Power, https://www.psychologie.hhu.de/) and determined from our FMD and cFMD means and standard deviations (7.86±3.33 % and 3.4±2.5 %, respectively), a Cohen’s d of 1.07, with = 0.05 and a sample size of n=14, that we achieved a power of 0.96 to detect a difference between endothelium-dependent responses of the BA and ICA. From our BA and ICA GTN means and standard deviations (25.49±8.76 % and 14.33±2.93 %, respectively), giving a Cohen’s d of 1.25, and a sample size of n=15, achieved a power of 0.99 to detect a difference between endothelium-independent responses of the BA and ICA. Given that linear mixed model analysis allows and accounts for differing group sizes, we are confident that our direct comparisons between arteries are powered to see differences. Given that our correlational analyses resulted in no relationships where P surpassed our critical significance level P=0.05 we detected no significant relationships. In order to achieve power >0.8, a priori power analyses for correlations with the r2 value we observed for our primary outcome (i.e., the relationship between endothelium-dependent responses), that is, r2=0.0005, we would require a sample size >15000 participants to observe this relationship (if it exists). As such, from our data we infer that there is no meaningful relationship between the endothelium-dependent responses of the BA and ICA. Vascular function sample homogeneity: We studied vascular responses in a cohort with no indications of cardiovascular disease or atherosclerosis, it is possible that inclusion of diseased or aged subjects presenting with a greater range of vascular (dys)function may have increased our chances of detecting a correlation between vascular SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 77 beds, if one exists. For example, Lavallée et al. (2006) found an inverse correlation in their elderly (60±25 years) control group between BA FMD (4 minutes occlusion) and common carotid artery vasodilation after 10 minutes of 5% CO2. Their healthy control group (n=20) included both males and females (n=10), some with hypercholesterolemia (n=4) and some who were current smokers (n=4). Furthermore, Lavallée et al. (2006) found no correlation between BA and common carotid artery responses in the lacunar stroke group (n=17); although this group included hypercholesterolemia (n=3), hypertension (n=16), diabetes (n=1), and smokers (n=5), further confounding interpretation of the relationships between arteries. Lavallée et al.'s (2006) findings are clearly not consonant with ours, and comparison is tenuous given several differences in methods (e.g., non-standard BA FMD and steady-state vs transient CO2 tests) and the complex influences on vascular function of the various pathologies present in both of the study groups (Niedermaier et al., 1993; Dzau et al., 1994; Costa et al., 2020; Suzuki et al., 2020). Nonetheless, the inclusion of a larger sample with a greater range of vascular function may advance the current findings. Dose-response subset: We employed multiple doses of the minimum clinical acute (sublingual spray) dose (400 µg) to assess dose-response curves in both arteries; however, this did not allow the determination of any such dose-response curve since 400 µg promotes maximal vasodilation in both arteries assessed. We found no difference in the time course or peak dilation between 400 – 1600 µg in the BA, nor in the ICA. In order to make a comparison between the ICA and BA smooth muscle sensitivity to exogenous NO donation, lower doses are likely required, e.g. 100, 200, 300 µg. Although sublingual administration provides ecological validity for clinical treatment, titration via intravenous GTN may be a useful experimental approach to explore in future studies. Sex differences: The current study includes too few females (n=6) to conclusively conduct sex-difference investigations; however, preliminary assessment would indicate that endothelium-dependent function is unrelated between BA and ICA in both males and females (Figure 3-6A), likewise for endothelium-independent function (Figure 3-6B). Iwamoto et al. (2018) showed that there were no sex differences in ICA vasodilation response to steady-state hypercapnia, although differences between methods in that study and the current study make comparison difficult. Given that the effect of menstrual cycle on vascular function in general remains controversial (e.g., Hashimoto et al., 1995; Shenouda et al., 2018; Stanhewicz and Wong, 2020; Wenner and Stachenfeld, 2020; Williams et al., 2001), further SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 78 investigation into endothelial function within the cerebral vasculature is essential to determine any sex-related characteristics thereof. Figure 3-6. Study 1 - Correlations by sex ICA and BA endothelium-dependent (A) and -independent (B) percent dilation responses. Implications: It is known that impaired vascular structure and function, cerebral hypoperfusion, and atherosclerosis pose risk factors for all forms of dementia (Kalaria, 2016; Wolters et al., 2016, 2017; Pitchaya et al., 2019). However, as yet there are no bioassays of intra- or extra-cerebral endothelial function; therefore, development of a means to assess impairments to the cerebrovascular (ICA) endothelium in vivo is crucial. The lack of relationship between BA and ICA endothelial function indicates that extra-cranial (ICA) cerebral endothelium-dependent function cannot be inferentially evaluated via peripheral (BA) tests of FMD. Although CVR tests are often interpreted as assays of cerebrovascular endothelial function (e.g., Ainslie et al., 2007; Lavi et al., 2006) and measurement of ICA diameter change during steady-state CVR tests taken to be representative of shear-mediated dilation (Iwamoto et al., 2018a, 2018b, 2020; Suzuki et al., 2020), the current study found no relationship between CVR of QICA and cFMD diameter change (r2=0.06; P=0.46, Figure 3-4C). And while CVR mediated dilation and cFMD mediated dilation was not statistically correlated (r2=0.26; P=0.068), a significant correlation may have been observed with a greater sample size. This would not be unfeasible given the degree of overlap of 0 5 10 15 20051015BA FMD Diameter change (%)ICA cFMD Diameter change (%)r2 = 0.01p=0.71r2 = 0.06p=0.750 20 40 6005101520ICA GTN Diameter change (%)BA GTN Diameter change (%)r2 = 0.06p=0.46r2 = 0.23p=0.61ABSHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 79 vasodilatory mechanisms between the tests (i.e., increased shear stress; central to cFMD, and certainly instrumental to the dilation subsequent to the initial response during CVR). However, CVR is most likely not representative of strictly shear-mediated endothelium-dependent cerebrovascular function since it is additionally influenced by endothelium-dependent mechanisms that are not related to shear stress, such as acidic disproportionation of nitrite to NO (Zweier et al., 1995; Weitzberg & Lundberg, 1998; Modin et al., 2001) and acidic release of adenosine tri-phosphate from red blood cells (Bergfeld & Forrester, 1992; Ellsworth et al., 1995, 2009) as well as shear-related mechanisms besides eNOS upregulation, i.e. shear-deformation of red blood cells causing adenosine tri-phosphate release (Price et al., 2004, 2006). The diameter achieved during steady-state CVR will also be influenced by the responses of cerebrovascular tone to increased MAP in addition to CO2. So, it is plausible that, within-subjects CVR and cFMD mediated dilation may only present a weak relationship. While CVR continues to have prognostic value for general vascular dysfunction, it is our contention that; CVR is in fact less indicative of cerebral endothelium-dependent function than is commonly interpreted due to the several confounding influences mentioned above; and that, cFMD is more closely related to cerebral endothelium-dependent responses (Hoiland et al., 2017; Carr & Ainslie, 2020). Investigation of the relationship between extracranial artery endothelium-dependent function (cFMD) and cerebral microvascular health (e.g., via cerebral capillary dysfunction and transit time, pericyte contractility, neurovascular uncoupling) would aid in determining the prognostic value of cFMD in regards to brain specific endothelial dysfunction; analogously to the cardiac-oriented assessment of associations between other vascular bioassay and coronary artery health [e.g., BA FMD vs coronary artery acetylcholine reactivity (Broxterman et al., 2019); common carotid artery cold pressor test reactivity vs coronary artery cold pressor test reactivity (Van Mil et al., 2017, 2018)]. Studies conducted in diseased populations would extend our findings to include changes in endothelial and smooth muscle responses to shear stress and nitric oxide donation with vascular dysfunction, as well as the association with cerebral specific vascular risk and disease progression. Further investigation of endothelium-dependent and -independent responses in the peripheral and cerebral vasculatures, and the relations, or lack thereof, during various combinations of pharmacological and non-pharmacological arterial pressure changes with and without adrenergic blockade would also undoubtedly extend the current findings. 3.7 Conclusion: Cerebrovascular (ICA) and peripheral (BA) conduit artery endothelium-dependant and -independent vasodilatory function are not correlated. In short, differences in baseline and stimulus shear characteristics explain and determine the unadjusted differences in FMD response magnitude between SHEAR-MEDIATED INTERNAL CAROTID AND BRACHIAL ARTERY FUNCTION 80 ICA and BA. In future studies on the cerebral vasculature in healthy populations, inferences should not be made from peripheral (BA) endothelium-dependent function to cerebral endothelium-dependent function (ICA), given the lack of relation. Translational Perspective: Brachial artery shear-mediated dilation is a widely used assessment of vascular function with links to cardiovascular risk; and assessment of cerebrovascular (middle cerebral artery velocity) reactivity to carbon dioxide is often used to index cerebral vascular health. However, neither test specifically examines cerebral endothelial function. We hypothesized that endothelial function in the internal carotid artery (main supplier of cerebral perfusion) and the brachial arterial (main supplier of forearm perfusion) would be dissimilar in magnitude and not correlated due to a number of differences (e.g., haemodynamics) between vascular beds. Using transiently elevated shear rate to assess both arteries, we found that shear-mediated function differed between vessels due, in part, to differences in baseline shear rate. Recent in vitro animal and human cerebral endothelial cell data indicate a direct link from prolonged elevations in shear stress to mitigated cerebrovascular dysfunction in Alzheimer’s dementia. In humans however, whilst mid-life physical activity levels and cerebral hypoperfusion are associated with the development of vascular dementia, the relationship between Alzheimer’s dementia and vascular shear stress is less clear. Whilst shear stress elevation can improve peripheral vascular function in healthy adults, the differences between vascular beds mean that uniform challenges imposed on the vasculatures may not cause equivalent stress or elicit comparable adaptation. The present findings lend further support to the need for development of preventative measures in the context of cerebrovascular dysfunction that are not only applicable across pathologies but are also specific to cerebral endothelial health. PHYSIOLOGICAL STABILITY OF REACTIVITY 81 Chapter 4: Study 2 – Stability of cerebrovascular CO2 reactivity following attainment of physiologic steady-state New findings: What is the central question of this study? During a steady-state cerebrovascular CO2 reactivity test, do different data extraction time points change the outcome for cerebrovascular CO2 reactivity? What is the main finding and its importance? Once steady-state end-tidal pressure of CO2 and hemodynamics were achieved, cerebral blood flow was stable, and so cerebrovascular CO2 reactivity values remained unchanged regardless of data extraction length (30s vs 60s) and time point (at 2, 3, 4, and 5-min). Abstract: This study assessed cerebrovascular CO2 reactivity (CVR) and examined data extraction time points and durations with the hypotheses that: 1) there would be no difference in CVR values when calculated with cerebral blood flow (CBF) measures at different time points following the attainment of physiologic steady-state, 2) once steady-state was achieved there would be no difference in CVR values derived from 60s and 30s extracted means, and 3) that changes in V̇E would not be associated with any changes in CVR. We conducted a single step iso-oxic hypercapnic CVR test using dynamic end-tidal forcing (end-tidal PCO2, +9.4±0.7mmHg), and transcranial Doppler and Duplex ultrasound of middle cerebral artery (MCA) and internal carotid artery (ICA), respectively. From the second minute of hypercapnia onwards, physiologic steady-state was apparent, with no subsequent changes in end-tidal PCO2, PO2, or mean arterial pressure. Therefore, CVR measured in the ICA and MCA was stable following the second minute of hypercapnia onwards. Data extraction durations of 30s or 60s did not give statistically different CVR values. No differences in CVR were detected following the second minute of hypercapnia after accounting for mean arterial pressure via calculated conductance or covariation of mean arterial pressure. These findings demonstrate that, provided the PCO2 stimulus remains in a steady-state, data extracted from any minute of a CVR test during physiologic steady-state conditions produce equivalent CVR values; any change in the CVR value would represent either a failure of CVR mechanisms, a change in the magnitude of the stimulus, or measurement error. PHYSIOLOGICAL STABILITY OF REACTIVITY 82 4.1 Background: Cerebrovascular reactivity to CO2 (CVR) is a common assessment of integrative cerebral vascular function, representative of several convergent mechanisms in the cerebral vasculature that act in response to changes in the partial pressure of arterial CO2 (PaCO2). Experimental tests of CVR are considered representative of cerebrovascular health as CVR appears indicative of some pathologies by way of impairment (Willie et al., 2014; Fisher, 2016; Fisher et al., 2018; Hoiland et al., 2019). As with all in vivo assays, the outcome value of a CVR test is heavily dependent on the techniques and measures employed [ reviewed in: (Ainslie & Duffin, 2009; Fierstra et al., 2013; Fisher, 2016; Fisher et al., 2018; Hoiland et al., 2019; Carr et al., 2021a)]. For example, applying fixed step changes in the fraction of inspired CO2 (FICO2) with a Douglas bag or reservoir – a technique commonly used due to its accessibility and simplicity – is known to yield high inter- and intra-individual and inter-session variability such that comparison between studies can be challenging (Fisher, 2016; Fisher et al., 2018; Burley et al., 2020). In contrast, computerized gas controlling systems [dynamic end-tidal forcing (DEF) (Tymko et al., 2016) and sequential gas delivery (Slessarev et al., 2007)] allow for accurate and precise end-tidal PCO2 (PETCO2) control (Mark et al., 2010), which reduces within and between individual variability. However, there are several caveats of DEF (and sequential gas delivery), which include but are not limited to the high cost of equipment to purchase and the requirement for highly skilled operators. Gas delivery with DEF results in smaller PETCO2-PaCO2 gradients during hypercapnia compared to other techniques [i.e. CO2 inhalation from a Douglas bag (Peebles et al., 2007)], precludes any influence of V̇E on PETCO2 and PETO2 thereby obviating the influence of V̇E on cerebral blood flow (CBF) or CVR (Smith et al., 2016; Howe et al., 2020). These distinct methodological advantages with DEF systems allow for a more accurate quantification of stimulus response relationships within the cerebrovasculature (Fisher, 2016; Fisher et al., 2018). Minute ventilation (V̇E) is often concurrently measured during CO2 challenges to assess total ventilatory sensitivity to CO2 (V̇E-CO2), representative mostly of the central chemoreflex response (Forster et al., 2008; Forster & Smith, 2010; Smith et al., 2010; Guyenet, 2014; Guyenet & Bayliss, 2015). While the driving stimulus for CVR and V̇E-CO2 is in both cases a change in PaCO2, the two systems are not mutually and equally influential upon each other. CVR influences the exposure of central chemoreceptors to PaCO2 through changes in CBF, thereby regulating V̇E in light of changes to PaCO2, but the reverse is not the case. Indeed, it is changes in PaCO2, PaO2, and MAP subsequent to V̇E that influence CBF (but not CVR mechanisms) (Carr et al., 2021a), and with the use of methods which remove the ability of changes in V̇E to affect PaCO2 or PaO2, V̇E has no influence on CVR (Howe et al., PHYSIOLOGICAL STABILITY OF REACTIVITY 83 2020). In order to further support this, we hypothesized in this study, that variation over time of V̇E-CO2 would not be associated with variation of CVR. The importance of CVR methodology has been extensively discussed given its influence on outcome measures and impact on physiologic interpretation (Ainslie & Duffin, 2009; Fierstra et al., 2013; Fisher, 2016; Hoiland et al., 2019; Burley et al., 2020; Howe et al., 2020; Carr et al., 2021a). Indeed, physiological factors such as the influence of blood pressure on CVR (Harper & Glass, 1965; Willie et al., 2012; Regan et al., 2014), the influence of ventilation on CVR (Ogoh et al., 2019; Howe et al., 2020), as well as methodological factors such as the technique used to measure CBF [e.g. ultrasound or magnetic resonance imaging (Burley et al., 2021), transcranial Doppler ultrasound or Duplex ultrasound (Brothers & Zhang, 2016; Hoiland & Ainslie, 2016)], and the timing and duration of data analysis post initiation of a CO2 stimulus (Burley et al., 2020) have received a large focus in recent years (Samora & Vianna, 2020; Tymko & Steinback, 2020). Indeed, recent work using transcranial Doppler to measure cerebral blood velocity (Burley et al., 2020) has shown that CVR values extracted from different minutes of an ostensibly steady-state CVR test give significantly different values (Burley et al., 2020), notwithstanding the inherent variability in the methods employed (i.e., fixed changes in FICO2). In order to build on this literature regarding the measurement of CVR and promote a consensus regarding data collection, extraction, and analyses that allows for data comparisons across research laboratories and stimulus methods, we conducted retrospective analyses of steady-state CVR data collected using DEF and volumetric assessment of CBF through the internal carotid artery (ICA). We conducted data analyses such that CVR values were extracted from different time points during a targeted and steady CO2 exposure. Given that in healthy individuals, mechanisms regulating CBF in response to changes in CO2 should be relatively consistent across time unless there are added perturbations (Willie et al., 2012, 2014), we hypothesized that: 1) there would be no difference in CVR values when calculated with CBF measures at different time points following the attainment of physiologic steady-state, 2) once steady state was achieved there would be no difference in values when 60 s and 30 s means were extracted, and 3) since DEF controls PETCO2 independent of V̇E (Smith et al., 2016; Howe et al., 2020), changes in V̇E-CO2 sensitivity would not be associated with any changes in CVR. PHYSIOLOGICAL STABILITY OF REACTIVITY 84 4.2 Methods: Ethical approval: We retrospectively analyzed data from a previously published study (Hoiland et al., 2017), which was approved by the University of British Columbia Clinical Research Ethics Board (H17-00929), all procedures conformed with standards set by the Declaration of Helsinki except for registration in a database. Participants completed written informed consent before participation. The original investigation was conducted to provide high fidelity beat-by-beat cerebrovascular data during a 5-minute CO2 reactivity test. While the primary outcomes from this study have been published, the current study’s hypotheses, data extraction, data analysis, and statistical analysis were all newly developed in light of recent publications (Burley et al., 2020), and independent of our previously published analyses. Eleven healthy young volunteers (8M, 3F) that were 22±3 years of age with a body mass index of 22±2 kg/m2 were included in the present analysis. All participants were free from overt cardiovascular, neurological, and respiratory disease, were non-smokers with no history of smoking, and not taking any over the counter or prescription medication at the time of experimentation (apart from oral contraceptives). All females were tested in days 1–3 of the follicular phase. Protocol: We conducted a CVR protocol that included a two-minute baseline period followed by a five-minute iso-oxic hypercapnic stimulus where PETCO2 was rapidly elevated to a target value of +9 mm Hg above baseline and thereafter held steady. This duration was to allow ample time for cerebrovascular responses to CO2 to occur and reach a steady-state. During data collection, steady-state stimulus was determined as ≥ 3 breaths at target PETCO2. Participants laid quietly in supine position and breathed simulated room air for five-minutes, and then were instructed to breathe normally on our DEF system for another two minutes of simulated room air (PETO2 97.7±5.4 mm Hg & PETCO2 40.7±1.7 mm Hg). Thereafter, PETCO2 was elevated as quickly as possible to a target value of +9 mm Hg above baseline and maintained for five minutes. Cardiorespiratory variables: Cardiorespiratory variables included mean arterial pressure (MAP), systolic blood pressure (SBP), diastolic blood pressure (DBP), V̇E, inspired and expired volume, respiratory flow, PETCO2, and PETO2. Beat-by-beat blood pressure was assessed via non-invasive finger photoplethysmography (Finometer PHYSIOLOGICAL STABILITY OF REACTIVITY 85 PRO, Finapres Medical Systems, Amsterdam, Netherlands). Absolute correction of Finometer values was based on duplicate brachial artery blood pressure measurements from the right arm using an automated sphygmomanometer (EM-759-E; Omron, Kyoto, Japan). Breath-by-breath CO2 and O2 were continuously sampled at the mouth by a calibrated gas analyzer (model ML206, ADInstruments). The PETCO2 and PETO2 were calculated in LabChart using minimum and maximum waveform detection analysis with daily correction for barometric pressure. Respiratory flow and inspired and expired volume were measured with a pneumotachometer (model HR 800 L, Hans Rudolph, Shawnee, KS). All variables were continuously sampled at 1000 Hz via analogue-to-digital converter (PowerLab 16/30, AD Instruments, Colorado Springs, CO), recorded in LabChart 7 software, and saved for subsequent offline analysis. Dynamic end-tidal forcing: We controlled PETCO2 and PETO2 using a DEF system described previously (Tymko et al., 2015, 2016). This approach allows for computerized breath-by-breath regulation of end-tidal gases, and therefore arterial gases, independent of V̇E. Notably, this method of arterial blood gas manipulation precludes any influence of V̇E on CVR (Howe et al., 2020), and therefore provides a more accurate quantification of stimulus response relationships (Fisher, 2016; Fisher et al., 2018). CVR assessment: Baseline PETCO2 and PETO2 were re-established used DEF until steady and maintained for two minutes, at the beginning of the second minute ultrasound scanning of ICA commenced and was maintained throughout. After one minute of baseline scanning, PETCO2 was elevated to +9 mm Hg above baseline and maintained for five minutes. PETO2 was maintained at baseline levels throughout (iso-oxia). Duplex imaging was maintained continuously throughout baseline and hypercapnia. Vascular imaging: Duplex ultrasound: Right ICA blood velocity and vessel diameter were assessed with Duplex ultrasound (10 MHz multi-frequency linear array, Terason uSmart 3300, Teratech, Burlington, MA). The ICA was imaged at least 1.5 cm distal to the common carotid bifurcation to avoid turbulent flow and non-uniform shear. An insonation angle of 60° was maintained throughout each test. All recordings were made in accordance PHYSIOLOGICAL STABILITY OF REACTIVITY 86 with published technical recommendations (Thomas et al., 2015). Screen capture software allowed recording and storage of continuous video files for future offline analysis. Transcranial doppler ultrasound: Blood velocity of the right middle cerebral artery (MCAv) was measured using TCD (2 MHz, TCD, Spencer Technologies, Seattle, WA, USA). Optimal M-mode imaging was achieved using a headframe (M600,SpencerTechnologies) allowing precise positioning, control of insonation angle and minimal movement artefact. Raw MCAv data were exported via an analogue-to-digital converter (PowerLab) to LabChart 7 for offline analysis. The MCA was insonated through the trans-temporal window using techniques described in detail elsewhere (Willie et al., 2011) Data analyses: While previous studies have conducted multiple tests of varying duration to assess the time dependency of CVR (Burley et al., 2020), this introduces unavoidable between-test variability as a potential confound in the comparison of CVR from differing durations of CO2 exposure. Therefore, we opted to conduct only one CVR test of five minutes duration (n=7; n= 2 completed only four minutes ~ 45 seconds of hypercapnia; n=2 completed only four minutes of hypercapnia) to compare the influence of data extraction at different times to assess the potential time-dependency of CVR independent of any test-re-test variability in CVR. For all variables, one-minute means were calculated for the minute preceding onset of hypercapnia for baseline values (0 – 60 s), and for each minute from the onset of hypercapnia during the CVR test (60-120 s = minute 1; 120-180 s = minute 2; 180-240 s = minute 3; 240-300 s = minute 4; 300-360 s = minute 5). Ventilatory data (PETCO2, PETO2, V̇E) were extracted breath-by-breath, cerebrovascular [ICA blood flow (Q) and velocity (v), and MCAv] and cardiovascular (MAP) data were extracted beat-by-beat. For QICA, MCAv and CVR values, both 60 s (every minute) and 30 s (last half of each minute) means were extracted. ICA diameter, Q, v, and shear rate were analyzed using customized wall tracking/edge detection software (BloodFlow Analysis, version 5.1) (Woodman et al., 2001). The software calculates Q thus: Q (mL/min) = peak envelope velocity (cms)2 × 𝜋(0.5 × diameter (cm))2 × 60 (s) PHYSIOLOGICAL STABILITY OF REACTIVITY 87 This software was designed to mitigate investigator bias; it has been validated and described in detail (Woodman et al., 2001; Black et al., 2008). Our within-day coefficient of variation for the assessment of ICA diameter using duplex ultrasound is 1.5% (Hoiland et al., 2017). CVR of QICA and MCAv was calculated thus: Absolute CVR = 𝑄ICA or MCAvPETCO2 Relative CVR= %𝑄ICA or %MCAvPETCO2 Finally, to account for the influence of MAP we included it as a covariate in statistical analyses (see below), and additionally calculated cerebrovascular conductance (CVC) of QICA and MCAv as well as CVR of those CVC values (CVC-CVR). CVC = 𝑄ICA or MCAvMAP CVC-CVR = 𝑄ICA CVC or MCAv CVCPETCO2 Data exclusion: Of the participants tested, blood pressure data from two participants was not available due to loss of the Finometer signal. Two CVR tests were ended at minute 4 rather than minute 5 at the request of the research participants. Finally, two tests were ended within the last ~15 s at the request of the research participants meaning that the period of time where the 30-second data would typically be extracted was reduced to only 15-seconds. As outlined in the discussion, the shorter the length of the data extraction period the greater the risk for error, and as such these two 15-second means were not included in the 30 s vs 60 s comparisons. However, these participants remained in all other analyses. Therefore, overall MAP n=9; and for minute 5 comparisons of 60 s vs 30 s n=7. 4.3 Statistical analyses: To assess hypercapnia-induced changes in cardiorespiratory variables (PETCO2, PETO2, MAP, V̇E, MCAv, QICA) all minute means were compared using linear mixed model analyses (Factor: minute. Levels: minutes baseline, 1, 2, 3, 4, and 5). To assess CVR (MCAv-CVR, QICA-CVR, both absolute PHYSIOLOGICAL STABILITY OF REACTIVITY 88 and relative) and V̇E-CO2 values, all minute means were compared using linear mixed model analyses (factor: minute. Levels: minutes 1, 2, 3, 4, and 5). To assess the utility of 60 s vs 30 s means, 60 s and 30 s means from each minute of CVR were compared using linear mixed model analyses (factors: minute and extraction duration. Levels: minutes 1, 2, 3, 4, and 5; 60 s and 30 s), additionally Bland-Altman plots were created to compare the mean and difference between 60 s vs 30 s means of MCAv, QICA, and CVR of MCAv and QICA. Furthermore, relative CVR of MCAv and QICA (factors: minute and artery. Levels: minutes 1, 2, 3, 4, and 5, ICA and MCA) were compared to determine consonance between QICA and MCAv values during CVR. Finally, MAP was also included as a covariate in linear mixed model analysis of CVR as MAP improved the -Log likelihood indicating its acceptability in the model, and separately, CVR of CVC was also examined via linear mixed model analysis. For all linear mixed model analyses, Bonferroni post-hoc corrections were examined in the event of main effects when comparing single variables (e.g., factor: minute), or in the event of interaction events when comparing multiple variables (e.g., factors: minute and extraction duration). 4.4 Results: By design, PETCO2 was rapidly elevated from baseline in minute 1 (+7.0±1.7 mm Hg, P<0.001), higher still in the minute 2 (+10.3±1.7 mm Hg vs baseline P<0.001) and thereafter remained constant for minutes 3, 4, and 5 (all P<0.001 vs baseline). The PETCO2 was not different between minutes 2, 3, 4, and 5 (i.e. steady-state was achieved; Figure 4-1A; combined minutes 2-5 average +9.4±0.7 mm Hg vs baseline), and PETO2 was unchanged throughout. There was no change in V̇E at minute 1, however, V̇E was elevated at minute 2 and remained steady thereafter (all P<0.001 vs baseline and minute 1; Figure 4-1B). MAP was unchanged from baseline in minute 1, but was elevated in minute 2 and remained stable thereafter (all P<0.001 vs baseline and minute 1; Figure 4-1C). MCAv, QICA, CVCMCAv, CVCQICA were significantly elevated from baseline in minute 1 (P<0.001) and were further elevated at minutes 2, 3, 4, and 5 (all P<0.001 vs baseline and minute 1; Figure 4-2). Therefore, minute 2 onwards was representative of physiological steady state, and is denoted where relevant as the shaded (green) area in each figure. PHYSIOLOGICAL STABILITY OF REACTIVITY 89 Figure 4-1. Study 2 - Cardiorespiratory response to hypercapnia. For each panel, individual data are presented as grey line and dot plots, with the group mean and standard deviation overlaid in black. In panels A and B, the two participants who completed only a four minute test are highlighted in red, and means and standard deviations excluding those participants (red) are also highlighted. Time on the y-axis is presented for baseline and each subsequent minute following initiation of the CO2 stimulus. Panel A depicts end-tidal gases (PETO2 & PETCO2) during baseline and hypercapnia with the consequent ventilatory response (V̇E) depicted in Panel B. The mean arterial pressure (MAP) response to hypercapnia is presented in panel C, with the individual changes in MAP from baseline (ΔMAP, mm Hg) presented in Panel D. No differences were seen in any variable from minute 2 onwards. Therefore, the shaded area (green) indicates physiologic steady-state. * P<0.001 vs baseline, # P<0.001 vs minute 1. Baseline 1 2 3 4 5708090100110120MinuteMAP (mm Hg) #Baseline 1 2 3 4 54045505590100110MinuteEnd-tidal partial pressure (mm Hg) #PETO2PETCO2Baseline 1 2 3 4 50204060MinuteVE (l/min) #Baseline 1 2 3 4 5-505101520MinuteDMAP (mm Hg) #A BC DPHYSIOLOGICAL STABILITY OF REACTIVITY 90 Figure 4-2. Study 2 - Cerebrovascular response to hypercapnia. For each panel, individual data are presented as grey line and dot plots, with the group mean and standard deviation overlaid in black. Time on the y-axis is presented for baseline and each subsequent minute following initiation of the CO2 stimulus. Panel A depicts internal carotid artery blood flow (QICA) during hypercapnia with the associated internal carotid conductance (QICA-CVC) response depicted in Panel B. The middle cerebral artery blood velocity (MCAv) response to hypercapnia is presented in Panel C, with the associated middle cerebral artery conductance (MCAv-CVC) response depicted in Panel D. No differences were seen between any variable from minute 2 onwards. Therefore, the shaded area (green) indicates physiologic steady-state. * P<0.001 vs baseline, # P<0.001 vs minute 1. Steady state time point (exposure duration): Absolute MCAv and QICA CVR were both higher at minutes 2, 3, 4, and 5 than minute 1 (all P<0.001 vs minute 1). Indeed, once physiologic steady state was reached (i.e. no change in MAP, PETCO2 and PETO2), there were no differences in either MCAv or QICA CVR (i.e. minutes 2, 3, 4, and 5; Figure 4-3A&B). Analyses of MCAv CVR with covariation for MAP showed steady-state from minute 2 onwards (all P≤0.001 vs minute 1), likewise for QICA CVR (minute 1 vs 2 P=0.025, minute 1 vs 3, 4, and 5 P≤0.001). The absolute CVR of CVCMCAv was elevated in minute 2 (0.029±0.009 ∆(cm/s/mm Hg)∙ ∆mm Hg-1) compared to minute 1 (0.019±0.09 ∆(cm/s/mm Hg)∙ ∆mm Hg-1: P<0.001), CVR of CVCQICA was elevated in minute 3 (0.13±0.024 ∆(mL/min/mm Hg)∙ ∆mm Hg-1) compared to minute 1 (0.066±0.046 ∆(mL/min/mm Hg)∙ ∆mm Hg-1: P<0.001), and both remained steady thereafter (all Baseline 1 2 3 4 5200300400500MinuteQICA (mL/min) #Baseline 1 2 3 4 5255075100125MinuteMCAv (cm/s) #Baseline 1 2 3 4 50246MinuteCVCQICA (mL/min/mm Hg) #Baseline 1 2 3 4 50.00.51.01.5MinuteCVCMCAv(cm/s/mm Hg) #A BC DPHYSIOLOGICAL STABILITY OF REACTIVITY 91 P<0.001 vs minute 1). V̇E-CO2 was elevated in minutes 2, 3, and 4 from minute 1 (all p<0.001), and was higher again in minute 5 vs minute 2 (P<0.01; Figure 4-4). Figure 4-3. Study 2 - Absolute CVR of MCAv (top), QICA (middle), and relative CVR comparing both (bottom). # P<0.001 vs minute 1. 1 2 3 4 50246MinuteMCAv CVR (Dcm/s/Dmmhg)#1 2 3 4 50102030MinuteQICA CVR (DmL/min/Dmmhg)#1 2 3 4 50246810MinuteCVR (%DmL/min or %Dcm/s /DmmHg)Minute, P<0.001Artery, P=0.027Interaction, P=0.150MCAv CVRQICA CVRABCPHYSIOLOGICAL STABILITY OF REACTIVITY 92 Figure 4-4. Study 2 - ?̇?E-CO2 sensitivity during 5-minutes of CO2 exposure. Individual data (circles) with the two participants who completed only a four minute test highlighted in red, and means and standard deviations (squares and error bars) for all (black) and excluding the four minute CVR participants (red). Given that two participants ended the test following four minutes, and are thus not included in the fifth minute, there appears ostensible variation across time in V̇E-CO2 responses from minute 4 to minute 5 in the original analysis (black squares). However, analysis of the data excluding these participants (red squares) shows that while V̇E-CO2 was indeed elevated at minute 4 and minute 5 compared to minutes 1 and 2, the difference is less severe in the n=9 analysis than in the original analysis. This indicates that while V̇E-CO2 never quite reaches true steady-state (no further change given a steady stimulus), there is only minor variation during a 5 minute test while the stimulus is steady. # P<0.001 vs minute 1, 𝛾 P<0.01 vs minute 2. Data extraction length: Linear mixed model analysis of QICA showed a significant main effect of duration of CO2 exposure (p<0.001), no main effects for data extraction length (P=0.170), and no interaction effect (P=0.198); likewise for MCAv, main effect of duration of CO2 exposure (P<0.001), no main effect of data extraction mean length (P=0.177), and no interaction effect (P=0.088) (Figure 4-5A&C). Analysis of absolute QICA CVR demonstrated a significant main effect of duration of CO2 exposure (p<0.001), no main effect of extraction length (P=0.265), nor an interaction effect (P=0.989); likewise for MCAv CVR, main effect of duration of CO2 exposure (P<0.001), no main effect of data extraction mean length (P=0.272), and no interaction effect (P=0.894) (Figure 4-5B&D). 1 2 3 4 5-101234MinuteVE-CO2(DL/min/Dmm Hg)##ggPHYSIOLOGICAL STABILITY OF REACTIVITY 93 Figure 4-5. Study 2 - 60 s means vs 30 s means for QICA, QICA CVR, MCAv, and MCAv CVR. Mean and standard deviation for 60 s mean data and 30 s mean data. Panel A; QICA, main effect of duration of CO2 exposure (P<0.001), no main effect of data extraction mean length (P=0.17), and no interaction effect (P=0.20). Panel B; QICA CVR, main effect of duration of CO2 exposure (P<0.001), no main effect of data extraction mean length (P=0.27), and no interaction effect (P=0.99). Panel C; MCAv, main effect of duration of CO2 exposure (P<0.001), no main effect of data extraction mean length (P=0.18), and no interaction effect (P=0.09). Panel D; MCAv CVR, main effect of duration of CO2 exposure (P<0.001), no main effect of data extraction mean length (P=0.27), and no interaction effect (P=0.89). For all panels, n=11 for baseline (0 to 60 s), minute 1 (60 to 120 s), minute 2 (120 to 180s), minute 3 (180 to 240 s) and minute 4 (240 to 300 s), whereas n=7 for minute 5 (300 to 360 s) since two participants completed only 240 s of hypercapnia (300 s total) and a further two completed >330 s but <360 s. Thus, for comparison between 60 s and 30 s, four participants were excluded from the minute 5 comparison. Additionally, Bland-Altman plots of 30 s and 60 s extraction length means for both QICA and MCAv for minutes baseline, 1, 2, 3, 4, and 5 demonstrated good agreement and minimal bias between the two extraction durations (Figure 4-6). As expected, minute 1 showed strong bias for underestimation of both QICA and MCAv using a 60 s mean, this is to be anticipated given the rising CBF over the first minute of hypercapnia, indicative that CBF has not reached steady state (and is higher when averaged over 60 secs vs the first 30 secs). For minutes 2, 3, 4, and 5, relative to absolute CBF, the limits of agreement were minimal - that is, the limits of agreement were representative of minimal ~3-5 % changes from the mean CBF values (e.g. ±~20 mL/min when average QICA was ~400 mL/min). Similarly, Bland-Altman plots of 60 s vs 30 s extraction length means for CVR of both QICA and MCAv for minutes, 1, 60 s mean 30 s mean0 60 120 180 240 300 3600123Time (s)QICA CVR (DmL/min/Dmmhg) 30 smean60 smean0 60 120 180 240 300 360024Time (s)MCAv CVR (Dcm/s/Dmmhg)30 smean60 smean0 60 120 180 240 300 360200300400500Time (s)QICA (mL/min)30 smean60 smean0 60 120 180 240 300 360406080100120Time (s)MCAv (cm/s)30 smean60 smeanA BC DPHYSIOLOGICAL STABILITY OF REACTIVITY 94 2, 3, 4, and 5, also showed good agreement and minimal bias between the two methods (Figure 4-7). Again, the limits of agreement were minimal compared to the mean CVR values (e.g. ±~3 mL/min/mm Hg when average CVR-QICA was ~14 mL/min/mm Hg).PHYSIOLOGICAL STABILITY OF REACTIVITY 95 Figure 4-6. Study 2 - Bland-Altman plots comparing 60 s and 30 s data means of QICA (top) and MCAv (bottom), at baseline and each minute. Note, minimal bias and physiologically negligible variation of 95% limit of agreement in both MCAv and QICA in all minutes (except minute 1 of CVR where this will be influenced by the dynamic changes in CO2 and CBF that occur). N=11 for baseline to minute 4, n=7 for minute 5. 40 60 80 100 120 140 40 60 80 100 120 140 40 60 80 100 120 140 40 60 80 100 120 140 40 60 80 100 120 140100 200 300 400 500 600 100 200 300 400 500 600 100 200 300 400 500 600 100 200 300 400 500 600100 200 300 400 500 600MCAv (cm/s)QICA (mL/min)40 60 80 100 120 140-15-10-50560 s mean - 30 s mean100 200 300 400 500 600-60-40-200204060 s mean - 30 s meanBASELINE MINUTE 1 MINUTE 2 MINUTE 3 MINUTE 4 MINUTE 5Mean of 30 s mean and 60 s meanPHYSIOLOGICAL STABILITY OF REACTIVITY 96 Figure 4-7. Study 2 - Bland-Altman plots comparing 60 s and 30 s data means of QICA CVR (top) and MCAv CVR (bottom) at each minute. Note, minimal bias and physiologically negligible variation of 95% limit of agreement in both MCAv CVR and QICA CVR in all minutes (except minute 1 of CVR where this will be influenced by the dynamic changes in CO2 and CBF that occur). N=11 for baseline to minute 4, n=7 for minute 5. 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 50 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30MCAv CVR(cm/s/mm Hg)QICA CVR(mL/min/mm Hg)MINUTE 2 MINUTE 3 MINUTE 4 MINUTE 50 1 2 3 4 5-1.0-0.50.00.560 s mean - 30 s mean0 5 10 15 20 25 30-6-4-202460 s mean - 30 s meanMINUTE 1Mean of 30 s mean and 60 s meanPHYSIOLOGICAL STABILITY OF REACTIVITY 97 CVR by artery: When comparing relative CVR of MCAv and QICA, as expected there was a main effect of minute (P<0.001) and main effect of artery (P=0.027), but no interaction effect (P=0.150) (See Figure 4-3C). Comparing relative CVR of MCAv and QICA and including MAP as a covariate resulted in a main effect of minute (P<0.001) but not artery (P=0.475) and no interaction effect (P=0.406). Furthermore, when comparing relative CVR of CVCMCAv [minutes 1-5 respectively: 2.8±1.3, 4.1±1.7, 4.1±1.3, 4.1±1.6, and 4.4±1.9 %∆(cm/s/mm Hg)∙ ∆mm Hg-1)] and CVCQICA [minutes 1-5 respectively: 2.3±1.5, 3.9±1.3, 4.5±0.6, 4.6±0.8, and 4.8±1.4 %∆(mL/min/mm Hg)∙ ∆mm Hg-1)] again there was a main effect of minute (P<0.001) but not of artery (P=0.552), and no interaction effect (P=0.442). 4.5 Discussion: The main findings herein are that following the second minute of hypercapnia administered via DEF and attainment of physiologic steady-state: 1) there were no significant differences between CVR values taken from different time points of CO2 exposure for both the MCA and ICA, even after accounting for MAP, 2) whether data was extracted in durations of 30 s or 60 s did not impact CVR values; and finally, 3) while V̇E was steady from minute 2 of CO2 exposure onwards, calculated V̇E-CO2 sensitivity increased. Despite this elevation in V̇E-CO2, CVR was stable. Combined, these findings indicate that a steady CO2 stimulus will produce a steady physiological response and any change or variation in the CVR value (representative of a change in CBF or PETCO2) during a steady-state test represents either a failure of CVR mechanisms (unlikely in healthy individuals), and/or a change in the magnitude of the stimulus, and/or some other perturbation such as a change in MAP. In the following, we briefly discuss our findings in the context of the physiology underlying CVR, then discuss why our findings differ from those of Burley et al. (2020) in regards to the contributing methodological and physiological factors. Our goal was to follow on from Burley et al. (2020) and characterize steady-state CVR data extraction methods using extracranial volumetric flow as well as intracranial velocity indices, using DEF. In the following discussion we employ thorough explanation of the factors contributing to differences between the outcome of this study and others in the hope that our results will aid in the undertaking (hypothesis building, design, and implementation) and interpretation of future CVR investigations. PHYSIOLOGICAL STABILITY OF REACTIVITY 98 Time point of data extraction: We found no differences between CVR, CVC, MAP, V̇E or any other variable following minute 2 of hypercapnia (Figure 4-1-Figure 4-4). In the first minute of hypercapnia, there was either no change in the mean value (MAP, V̇E), or a significant but small change (MCAv, QICA, etc.), which subsequently rose to a stable value at the second minute onwards. This response dynamic is related to two primary factors: 1) the time taken for the completion of a step change in PETCO2, and 2) the physiologic time dependency of the consequent vascular response to changes in PaCO2. As reviewed in detail elsewhere (Ainslie & Duffin, 2009; Fierstra et al., 2013; Fisher, 2016; Fisher et al., 2018; Hoiland et al., 2019), the ability to control PETCO2 varies widely across gas manipualtion techniques. In the present study, using DEF, we were able to quickly - in approximately 5-10 breaths - elevate PETCO2 to our target (i.e. ~+9 mm Hg from baseline) (Hoiland et al., 2017). This rapid hypercapnic transition allows for the desired change in PETCO2 to occur in roughly one minute and a relatively short transition period between baseline conditions and steady-state hypercapnic conditions. Following hypercapnia onset, the physiologic response of the cerebral vasculature begins rapidly at the level of the capillaries and pial vessels (Wolff & Lennox, 1930; Lennox & Gibbs, 1932; Kety & Schmidt, 1948b; Shapiro et al., 1971; Faraci et al., 1988; Faraci & Heistad, 1990; Hall et al., 2014; Gonzales et al., 2020; Hartmann et al., 2021), mediating an increased blood flow via the large upstream (intra- and extra-cranial) arteries (Giller et al., 1993; Willie et al., 2012; Verbree et al., 2014; Coverdale et al., 2015; Carter et al., 2016; Hoiland et al., 2017). Once a steady PETCO2 is attained, and thus, PaCO2, PCO2 must still reach near or pseudo-equilibration with the cerebrospinal and interstitial fluids (CSF and ISF, respectively) - which bathe the pial vessels and therefore hold a large influence over CBF (Wolff & Lennox, 1930) - before the full extent of the cerebrovascular response can be elicited. True equilibration between blood and brain tissue CO2 will never occur due to constant cerebral metabolic production of CO2. However, for the purposes of time constant estimation and with several assumptions (described below), upon a change in PaCO2 in the intraluminal compartment, equilibration of PaCO2 and PCO2 in the CSF will require at least three time constants [𝜏; 𝜏 is the duration required for fluid equal in volume to that of one compartment to flow across the membrane of the adjacent compartment (Mithoefer & Kazemi, 1963)]. Taking normative values: resting CBF ~750mL/min (Kety & Schmidt, 1948a), CVR to PaCO2 ~7%/mm Hg (Hoiland et al., 2019), and total CSF 140mL (Johanson, 2008); and applying a stimulus of +9 mm Hg PaCO2 leads to a steady state CBF of ~1200mL/min. A 𝜏 of ~7 s would be required for a volume of blood equal to that of the CSF to traverse the cerebral circulation during hypercapnia. If three time constants are required for 95% equilibration, PHYSIOLOGICAL STABILITY OF REACTIVITY 99 this would occur in a total of ~22 s. Given that CSF and ISF freely communicate across the permeable membranes separating them (i.e., the ependyma in the ventricles and pia-glial membrane in subarachnoid space), taking ~420 mLas the total CSF+ISF volume (Johanson, 2008) results in a 𝜏 of ~20 s and near-equilibration around 60 s. These values will fundamentally determine arterial to CSF equilibration; however, this straightforward conception overlooks some crucial factors that should be taken into consideration in reality including: 1), continuous metabolic CO2 production – which may change with hypercapnia (Kogure et al., 1975; Kliefoth et al., 1979; Horvath et al., 1994; Yang & Krasney, 1995; Xu et al., 2011), 2), blood flow and volume surrounding the brainstem specifically, 3), CSF volume changes (Grant et al., 1989), 4), the contributions to CVR and V̇E-CO2 of intra- versus extracellular pH sensing, and 5), the duration of systemic perfusion of PaCO2 changes. As such, time to steady-state is dependent on many factors and changes accordingly; however, even with no increase in CBF with elevated PaCO2 𝜏 would still not exceed ~20 s with the normative values given above. Duration of data extraction: We found no statistical differences between 30 s and 60 s means for any variable beyond minute 2 of hypercapnia. This is, again, indicative of the attainment of steady state in the second minute of hypercapnia. Excessively short data duration means could foreseeably impact CVR values given normal physiological MAP and heart rate variability. In the setting of physiological resting and supine steady-state, CVR does not change over a 30 s period and, as such, 30 s means should be similar enough to 60 s means, as observed herein. This indicates that, if constrained by some other factor (e.g. aspects of experimental design), future studies could utilize a 30 s mean data extraction. That said, and in agreement with Burley et al., (2020) we nevertheless recommend data extraction of a 60 s mean, especially with the use of Duplex ultrasound. At the very least, we contend that the duration of data extraction not be less than 30 s as this introduces the potential for cyclic physiologic changes related to the cardiac cycle and respiratory cycle to influence averaged data. Considerations for the method of CO2 delivery: Given the cyclic or oscillatory nature of the assessed variables, the CVR outcome value is dependent on both the stimulus exposure duration (e.g. in extremis, 30 s stimulus exposure ≠ physiologic steady-state) and the duration of data extraction (e.g. in extremis, 1 s mean ≠ 60 s mean). We have demonstrated that following the attainment of physiologic steady-state CVR is not time dependent, and is not statistically different when extracting 30 s or 60 s for data averaging. Further to these considerations is the arguably more foundational role of the method by which CO2 is manipulated in the discernment of CVR. This will fundamentally determine the variability seen within and between PHYSIOLOGICAL STABILITY OF REACTIVITY 100 subjects related to V̇E, PETCO2, and the consequent cerebrovascular and cardiovascular responses to hypercapnia (Fisher, 2016). Therefore, the method of CO2 delivery impacts the level to which physiological steady-state can be achieved, and so determines the final CVR value. Here, two key factors require consideration: 1) the ability to precisely control and manipulate PETCO2, and 2) the level to which PETCO2 is an accurate surrogate for PaCO2, the true physiologic stimulus. In accordance with the alveolar gas equation, PaCO2 is proportional to the rate of metabolically produced CO2 (V̇CO2) and inversely proportional to alveolar ventilation (V̇A). As V̇CO2 remains relatively constant in healthy individuals at rest, but ventilatory sensitivity to CO2 (i.e. changes in V̇A) may be widely variable between individuals (Hirshman et al., 1975), differing ventilatory responses can markedly impact the prevailing PaCO2 stimulus between research participants. Further, V̇A fluctuates within individuals breath-by-breath and, as such, PETCO2 also changes breath-by-breath (Mark et al., 2010). This makes targeting a specific PaCO2 difficult when using fixed changes in FICO2 (Peebles et al., 2007; Mark et al., 2010; Duffin, 2011; Fisher, 2016). Indeed, changes in V̇E-CO2 may influence CVR when employing fixed changes in FICO2 as changes in V̇E will be reflected in changes in the prevailing PaCO2 stimulus (Burley et al., 2020). This influence of V̇E is obviated with the use of DEF, as precise breath-by-breath alterations in FICO2 allow for the maintenance of a steady PETCO2 in the setting of alterations in V̇E-CO2 , resulting in no impact on CVR (Howe et al., 2020). In regards to data extraction length, since the FICO2 technique may not achieve steady-state - because PETCO2, and therefore, PaCO2 fluctuate - it is plausible that means of different lengths may capture peaks, troughs, or transitions of the constantly changing variables, and as such, we recommend the use of 60 s means especially with FICO2 The majority of work assessing CVR uses PETCO2 in place of PaCO2 as a surrogate for the CO2 stimulus due to ease of measurement and relatively accurate representation of PaCO2. Therefore, the relationship between PETCO2 and PaCO2 (the PETCO2-PaCO2 gradient) is a key factor to consider for the standardization of CVR tests. The PETCO2-PaCO2 gradient can vary (and often increases) with increasing hypercapnia (Peebles et al., 2007; Tymko et al., 2016), affecting the determination of CVR (Tymko et al., 2016). The magnitude by which the PETCO2-PaCO2 gradient increases with hypercapnia appears much larger when employing fixed changes in FICO2 versus DEF (or sequential gas delivery) (Peebles et al., 2007; Tymko et al., 2016). The use of fixed changes in FICO2 as a hypercapnic stimulus is inherently variable and as such, physiological steady-state, if achieved, is likely not as stable as with more sophisticated gas control systems (Mark et al., 2010). This is ultimately the outcome of the findings by Burley et al., (2020), as PHYSIOLOGICAL STABILITY OF REACTIVITY 101 the stimulus durations they tested produced significantly different CVR values in minute 2 vs the last minute of the exposure. However, ascribing this variability in the CVR value to data extraction and analysis methods without recourse to the inherent variability of the stimulus used could be potentially misleading regarding the true physiological steady-state of CVR. The findings of the current study employing DEF show that data extracted from any minute of the CVR test during true physiologic steady-state conditions produces equivalent CVR values (per the definition of steady-state). Considerations in regards to stimulus delivery methods based on the present findings and previous work (Burley et al., 2020) include: during steady state tests, CVR data means can be as short as 30 s with DEF, but should be 60 s with Douglas bag methods; CVR values can be taken from any minute following steady state when using DEF, but it should be noted that when Douglas bag methods are used CVR is variable because of the influence of changes in PaCO2 with V̇E. 4.6 Methodological Considerations: The relevance of regional CVR heterogeneity and it’s bearing on V̇E-CO2 assessment has been discussed elsewhere in detail (Hoiland et al., 2015; Howe et al., 2020; Carr et al., 2021a), and as such here it suffices to say that assessing the anterior (ICA and MCA) rather than posterior circulation feeding the brainstem and central chemoreceptive areas may be a limitation in studies assessing V̇E-CO2 conjointly with CVR. Two participants completed only a 4-minute CVR test, in which particularly low V̇E responses were evident as shown in Figure 4-1 & Figure 4-4. These data were nevertheless included in all analyses since V̇E-CO2 for none were statistical outliers [all minutes were within the range determined by mean±(3×standard deviation)] and because V̇E-CO2 does not directly affect CVR values when using DEF (Howe et al., 2020). However, inclusion of this data may have impacted interpretation if V̇E-CO2 were influential on CVR, as in the use of fixed changes in FICO2. Furthermore, given the rationale of this study (i.e., once steady-state is achieved, the data extraction time point makes no difference to the CVR values), inclusion of this data certainly could have impacted the reported V̇E due to the consolidative and lowering effect on V̇E during minutes 1-4. For example, while minutes 3, 4, and 5 are not different in either the original or n=9 analyses, a shallower V̇E response in the 2 participants who received 4-minutes gives the appearance in the original analysis (Figure 4-4, black line) that V̇E-CO2 is potentially biologically variable even at the 5th minute. Whereas, in the n=9 analysis, total exclusion of the 4-minute data changes the group means of each minute and shows that while V̇E-CO2 is indeed elevated at both minute 4 and 5 from minutes 1 and 2, it is much less variable during minutes 3-5. This PHYSIOLOGICAL STABILITY OF REACTIVITY 102 second interpretation/analysis is physiologically appropriate, since the first interpretation is the result of methodological and technical variability, i.e. a shorter test, and inclusion of these data having a consolidative and lowering effect on the mean values. Crucially, the removal or inclusion of these participants’ data from the CVR analyses does not change the outcome of the findings of this study; however, we highlight this point to show that even sophisticated approaches (e.g., DEF and sequential gas delivery) can produce the appearance of physiological variation if data extraction or other methods differ - serving as an example that not all methods are appropriate for all research questions. Since DEF eliminates the ability of V̇E-CO2 responses to alter PaCO2, these differences (minutes 2 versus 4 and 5) do not influence the CVR values, which is yet more evidence for V̇E alone not impacting CVR (Howe et al., 2020), yet, if the primary outcome herein had been V̇E-CO2 this technical issue would add confounding factors to our interpretation. Additionally, as is the case with all outcomes, the above considerations should also be appreciated in the context of sample size per se. Individuals may well display slight variation from the group mean, as the magnitude and time course of each person’s responses are indeed uniquely determined by the inherent variability among subjects, however, these differences will have a greater effect on the group mean the smaller the overall sample size. We did not address between-day reliability or variability of the steady-state CVR. The inclusion of such data and comparisons would strengthen the possible inferences regarding application of CVR methods. While this is a concern regarding the between day variability of CVR, this is particularly of note in regards again to the choice of methods and techniques. Specifically, the use of FICO2 has been shown to produce large within-day between-test intra-individual coefficients of variation, of between 7-46 % for CVR, and 13-77 % for V̇E-CO2 (Burley et al., 2020). Since between-test and between-day variability is likely to be further augmented with the use of methods that cannot separate confounding factors from the outcome measure, it is essential that interpretation of findings account for methodological contributions to variability. In order to avoid large influences in variability - both physiological and technical - we conducted only one CVR test, thereby narrowing the focus and avoiding test-retest physiological and technical variability. Inclusion of male and female participants is a requisite aspect of modern physiological research generally (Lindsey et al., 2021). While there are both male and females participants analyzed herein, and although we controlled for phase of menstrual cycle (i.e., early follicular), the small sample of females precludes even preliminary investigation into - or inference concerning - sex-related differences of CVR or V̇E-CO2. PHYSIOLOGICAL STABILITY OF REACTIVITY 103 4.7 Conclusions: Once physiologic steady state was attained, as indicated in the present study by plateau of the PETCO2 stimulus and cardiorespiratory responses (e.g., MAP & V̇E), there were no subsequent changes in these variables until the cessation of hypercapnia. As long as PETCO2 is consistently elevated from normocapnia and held stable, CVR mechanisms will maintain cerebral vasodilation such that CBF is elevated and thereby maintained. During this time, any change in the CVR value (representative of a change in CBF or PETCO2) would represent either a failure of vascular mechanisms, and/or a change in the magnitude of the stimulus. In the present study, PETCO2 was elevated in minute 1 and steady at minute 2 onwards, we can therefore presume that PaCO2 was also stable and elevated during those minutes (since V̇CO2 likely did not change). Since it seems unlikely that the responsiveness of CVR mechanisms in healthy individuals would be variable minute to minute, we conclude that any variation in the CVR outcome of other studies is likely the result of stimulus variability.ANAEMIA AND CEREBROVASCULAR REACTIVITY 104 Chapter 5: Study 3 – Cerebral O2 and CO2 transport in isovolumic haemodilution: compensation of cerebral delivery of O2 and maintenance of cerebrovascular reactivity to CO2 Abstract: This study investigated the influence of acute reductions in arterial O2 content (CaO2) via isovolumic haemodilution on global cerebral blood flow (gCBF) and cerebrovascular CO2 reactivity (CVR) in 11 healthy males (age; 28±7 years: body mass index; 23±2 kg/m2). Radial artery and internal jugular vein catheters provided measurement of blood pressure and gases, quantification of cerebral metabolism, cerebral CO2 washout, and trans-cerebral nitrite exchange (ozone based chemiluminescence). Prior and following haemodilution, the partial pressure of arterial CO2 (PaCO2) was elevated with dynamic end-tidal forcing and gCBF measured with duplex ultrasound. CVR was determined as the slope of the gCBF response and PaCO2. Replacement of ~20% of blood volume with an equal volume of 5% human serum albumin (Alburex® 5%) reduced haemoglobin (13.8±0.8 vs. 11.3±0.6 g/dL; P<0.001) and CaO2 (18.9±1.0 vs 15.0±0.8 mL/dL P<0.001), elevated gCBF (+18±11%; P=0.002), preserved cerebral oxygen delivery (P=0.49), and elevated CO2 washout (+11%; P=0.01). The net cerebral uptake of nitrite (11.6±14.0 nmol/min; P=0.027) at baseline was abolished following haemodilution (-3.6±17.9 nmol/min; P=0.54), perhaps underpinning the conservation of CVR (61.7±19.0 vs. 69.0±19.2 mL/min/mmHg; P=0.23). These findings demonstrate that the cerebrovascular responses to acute anaemia in healthy humans are sufficient to support the maintenance of CVR. ANAEMIA AND CEREBROVASCULAR REACTIVITY 105 5.1 Introduction: Carbon dioxide (CO2) and, to an lesser extent, oxygen (O2) are amongst the most potent regulators of cerebral blood flow (CBF) in humans. (Kety & Schmidt, 1948b; Willie et al., 2014) Increases and decreases in the partial pressure of arterial CO2 (PaCO2) elicit concomitant changes in CBF, (Kety & Schmidt, 1948b; Willie et al., 2012) in order to maintain cerebral tissue “washout” of CO2 and regulate cerebral acid-base balance. (Bradley et al., 1965; Read & Leigh, 1967; Neubauer et al., 1985; Hoiland et al., 2019; Duffin et al., 2021; Carr et al., 2021a) Reductions in the partial pressure of arterial O2 (PaO2) elevate CBF (Kety & Schmidt, 1948b; Willie et al., 2012; Hoiland et al., 2016a) in order to sustain cerebral delivery of O2 (CDO2);(Ainslie et al., 2014) while reductions of arterial O2 content (CaO2: anaemic hypoxia) independent of PaO2, also augment CBF (Paulson et al., 1973b; Hino et al., 1992; Tu & Liu, 1996; Ekelund et al., 2002; Daif et al., 2012) or O2 extraction (Todd et al., 1994; Tomiyama et al., 1999) such that metabolism is maintained. In short, these homeostatic responses to changes in CO2 and O2 act to regulate conditions appropriate for stable cerebral metabolism. Relative to reductions of CaO2 independent of PaO2, anaemia is a hallmark of multiple disease states with well-documented cerebrovascular dysregulation, including sickle cell disease (Nur et al., 2009; Prohovnik et al., 2009) and chronic renal failure. (Kuwabara et al., 2002; Zheng et al., 2016; Sprick et al., 2020) For example, sickle cell disease increases the risk of stroke, which may be related to a reduction in cerebrovascular vasodilatory capacity (Nur et al., 2009; Prohovnik et al., 2009; Kosinski et al., 2017) secondary to elevations in resting CBF. (Nur et al., 2009; Prohovnik et al., 2009; Kosinski et al., 2017) Similarly, CVR is reduced in chronic kidney disease patients with anaemia concomitant to elevations in resting CBF. (Kuwabara et al., 2002) While pre-clinical studies have demonstrated that brain parenchymal O2 tension, and CBF responses to hypercapnia are preserved during subacute anaemia in rats through elevated basal CBF, (Mistry et al., 2018) CDO2 is impaired in human chronic kidney disease patients despite similarly increased CBF. (Zheng et al., 2016) Whether the differences in CBF regulation observed with anaemia are related to anaemia per se or a consequence of the disease state underlying said anaemia remains to be determined. Work in healthy humans assessing the effects of isovolumic haemodilution on functional responses in the peripheral circulation has demonstrated increased endothelium-mediated vasodilation (i.e. brachial artery reactive hyperemia flow-mediated dilation; (Tremblay et al., 2019b; Hoiland et al., 2020c)) and maintained O2 delivery to working skeletal muscles. (Koskolou et al., 1997; Roach et al., 1999) Furthermore, in healthy newborn baboons, CVR varies inversely with hematocrit (Hct) after haemodilution and haemoconcentration. (Raju & Kim, 1991) Whether the slope of the CBF response to changes in CO2 [cerebrovascular reactivity (CVR)] would be steepened during acute anaemia in ANAEMIA AND CEREBROVASCULAR REACTIVITY 106 healthy humans given the absence of pathology associated vascular dysfunction remains unknown. Based on the aforementioned evidence, anaemia - in the absence of pathology related impairments in vasodilatory signalling (Reiter et al., 2002) - may lead to a steepening of CO2 CVR due to a reduction in haemoglobin (Hb)-mediated nitric oxide (NO) scavenging. (Azarov et al., 2005; Tremblay et al., 2019b; Hoiland et al., 2020c) Given the complex roles of NO (Iadecola & Zhang, 1994, 1996; Hoiland et al., 2020a, 2021) and cerebral acid-base balance (Bradley et al., 1965; Read & Leigh, 1967; Neubauer et al., 1985; Hoiland et al., 2019; Duffin et al., 2021; Carr et al., 2021a) in cerebrovascular regulation we quantified blood-borne markers of NO uptake/release, cerebral metabolism, and cerebral CO2 washout to provide insight into the influence of anaemia on CVR. We hypothesized that acute experimental haemodilution in healthy humans would increase CBF and CVR, while CDO2, CO2 washout, and cerebral metabolism would be preserved. 5.2 Methods: Ethical Approval This study was conducted in the Centre for Heart, Lung and Vascular Health at the University of British Columbia Okanagan and adhered to the standards outlined in the Declaration of Helsinki (except registry in a database) and the Canadian Tri-council Policy Statement for Integrity in Research. Institutional approval was received from the University of British Columbia Clinical Research Ethics Board (CREB ID: H16-01028). All participants provided written informed consent prior to participation in this study. Participants Eleven healthy males were recruited to participate in this study (age; 28±7 years: body mass index; 23±2 kg/m2). Participants attended the laboratory at 0600 hr or 1300 hr having abstained from alcohol, caffeine, and exercise for 24 hours, fasted for at least 4 hours, but drank water ad libitum. All participants were free of overt cardiovascular, cerebrovascular, respiratory, neurological, and metabolic disease, and all were non-smokers with no history of smoking, and not taking prescription medicine related to any aforementioned conditions. Experimental Protocol Participants rested in the supine position upon arrival and were instrumented with radial artery, antecubital vein, and internal jugular vein catheters. Using sterile technique, a 20G arterial catheter ANAEMIA AND CEREBROVASCULAR REACTIVITY 107 (Arrow, Markham, ON) was advanced into the left radial artery under local anesthesia (Lidocaine, 1.0%). This technique was assisted via ultrasound guidance. A 13G central venous catheter (Cook Medical, Bloomington, IN) was advanced into the right internal jugular vein under sterile conditions and local anesthesia with ultrasound guidance. The catheter was then advanced up to 15 cm cephalad. (Schell & Cole, 2000) The same technique, performed by our group has been previously demonstrated to lead to catheter tip placement in the jugular bulb, superior to the facial vein. (Ainslie et al., 2014) Further, correct placement was additionally determined by participants noting a sensation in their ear upon full insertion of the catheter. (Schell & Cole, 2000) Fulfilment of these techniques leads to ≤3% contamination by extra-cerebral blood. (Schell & Cole, 2000) Finally, an 18G venous catheter (InsyteTM AutoguardTM, Becton Dickinson, USA) was inserted into the median antecubital vein. Following catheterization, the remainder of the monitoring equipment was set-up while the participant then rested quietly for 20 minutes (see below). Using dynamic end-tidal forcing (DEF), subjects completed a stepwise steady-state hypercapnic test to assess CVR both pre- and post- an isovolumic haemodilution protocol. Following five minutes of steady room-air breathing to assess baseline PETCO2 and PETO2 values, subjects breathed for two minutes with DEF control at baseline end-tidal values. Then PETCO2 was elevated in three sequential step-wise increments to +3, +6, and +9 mmHg PETCO2 above baseline. Each stage of elevated PETCO2 lasted 5-minutes following the attainment of steady end-tidal gases (three breaths within ±1mmHg of target PETCO2) and all cerebrovascular, haemodynamic, cardiorespiratory variables, and arterial blood samples were collected in within the last 1-2 minutes of each stage following attainment of physiologic steady state. (Carr et al., 2021b) Following completion of the test, participants returned to room air breathing. After pre-haemodilution CVR test, to reduce hematocrit, whole blood was transferred into an Anticoagulant Citrate Phosphate Dextrose Solution BLOOD-PACKTM (4R0012MC, Fenwal, USA) through a non-pyrogenic plasma transfer kit (4C2240, Fenwal, USA) to collect up to 450 mL of whole blood per BLOOD-PACK for short term storage at room temperature on an orbital agitator. This blood was replaced with an equal volume of 5% human serum albumin (Alburex® 5%) in two increments that amounted to a total removal and replacement of ~20% of blood volume. The magnitude of haemodilution was verified by measuring the resulting change in hematocrit. Following haemodilution, CVR was re-conducted (see C.1 Supplemental Figure 1 for protocol schematic). ANAEMIA AND CEREBROVASCULAR REACTIVITY 108 Experimental Measures: Cardiorespiratory measures All cardiorespiratory variables were sampled continuously at 1 KHz via an analogue-to-digital converter (Powerlab, 16/30; ADInstruments, Colorado Springs, CO). Heart rate (HR; ADI bioamp ML132), radial artery blood pressures, and jugular venous blood pressures (Truwave Transducer, Edwards Life Sciences) were measured as previously described (Ainslie et al., 2014). The partial pressures of end-tidal CO2 (PETCO2) and O2 (PETO2) were sampled at the mouth and recorded by a calibrated gas analyzer (model ML206, ADInstruments), while respiratory flow was measured by a pneumotachograph (MLT1000L; ADInstruments) paired with a spirometer (FE141; ADInstruments) and connected in series to a bacteriological filter. All data were interfaced with LabChart (Version 7). Cerebral vascular measurements Diameter and blood velocity of the right internal carotid artery (ICA) and left vertebral artery (VA) were measured using a 10 MHz multi-frequency linear array duplex ultrasound (Terason uSmart 3300, Teratech, Burlington, MA). Specifically, B-mode imaging was used to measure arterial diameter, while pulse-wave mode was used to concurrently measure blood velocity. Diameter and velocity of the ICA were measured at least 1.5 cm distal to the common carotid bifurcation to eliminate recordings of turbulent and retrograde flow and non-uniform shear. The VA blood velocity and diameter were measured between C4 and C5 or C5 and C6. Care was taken to ensure that the insonation angle (60°) was unchanged throughout each test. Further, upon acquisition of the first ultrasound image (i.e., resting baseline) there was no alteration of B-mode gain to avoid any artificial changes in arterial wall brightness/thickness. All recordings were made in accordance with published technical recommendations. (Thomas et al., 2015) Volumetric blood flow in the ICA and VA were quantified using the following equation: 𝐼𝐶𝐴 𝑜𝑟 𝑉𝐴 𝑓𝑙𝑜𝑤 = (𝜋 ∙ (𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 (𝑐𝑚)2)2) ∙𝑝𝑒𝑎𝑘 𝑒𝑛𝑣𝑒𝑙𝑜𝑝𝑒 𝑣𝑒𝑙𝑜𝑐𝑖𝑡𝑦 (𝑐𝑚𝑠 )2 ∙60𝑠𝑚𝑖𝑛𝑢𝑡𝑒 Volumetric global cerebral blood flow (gCBF) was subsequently calculated as: 𝑔𝐶𝐵𝐹 = (𝐼𝐶𝐴 𝑓𝑙𝑜𝑤 + 𝑉𝐴 𝑓𝑙𝑜𝑤) ∙ 2 ANAEMIA AND CEREBROVASCULAR REACTIVITY 109 Blood velocity in the right middle cerebral artery (MCAv) and left posterior cerebral artery (PCAv) were measured using a 2 MHz transcranial Doppler ultrasound (TCD; Spencer Technologies, Seattle, WA). The TCD probe was fixed to a headpiece (model M600 bilateral head frame, Spencer Technologies) and secured into place. The MCA and PCA were insonated through the middle trans-temporal window, using previously described location and standardization techniques (Willie et al., 2011). All recordings were screen captured and stored as video files for offline analysis. This analysis involved concurrent determination of arterial diameter and peak blood velocity at 30 Hz, using customized edge detection and wall tracking software (BloodFlow Analysis, Version 5.1) designed to mitigate observer bias. (Woodman et al., 2001) Blood Sampling & Analyses For the measurement of arterial blood gases ~1.0 mL of radial arterial blood was drawn into a pre-heparinized syringe (SafePICO, Radiometer, Copenhagen, Denmark) and analyzed immediately at 37°C using a commercial blood gas analyzer (ABL90 FLEX, Radiometer). This analysis included measurement of the partial pressure of arterial oxygen (PaO2), PaCO2, arterial oxygen saturation (SaO2), arterial oxygen content (CaO2), [Hb] and Hct. Arterial blood was also analyzed for whole blood viscosity. Arterial blood for viscosity measurement was drawn into a Lithium Heparin Vacutainer® (Becton Dickinson, USA). Blood viscosity was measured in duplicate within 15 min of blood sample acquisition at three shear rates (45, 90, and 225 s−1) at 37.0°C with a temperature controlled cone-and-plate viscometer (Model DV2T; Brookfield Amtek, Middleboro, MA, USA). (Baskurt et al., 2009) For NO analyses, arterial and jugular bulb blood samples were drawn into K2EDTA Vacutainers® (Becton Dickinson, USA). Whole blood was then immediately centrifuged at 600 g for 10 minutes at a temperature of 4.0°C. Plasma was aliquoted into cryovials, flash frozen in liquid N2, and stored at -80°C. Ozone-based chemiluminescence (Sievers NOA™ 280i, Analytix Ltd, Durham, UK) was employed to measure plasma NO2− via chemical reagent cleavage. A detailed overview of the methods including principles of detection has previously been published. (Bailey et al., 2017; Hoiland et al., 2020b, 2021) Plasma was mixed with 5% acidified sulphanilamide and left to incubate in the dark at 21º C for 15 min to remove NO2− prior to injection into tri-iodide reagent for direct measurement of S-nitrosothiols (RSNO). A separate sample was also injected into tri-iodide reagent for the combined measurement of NO2− and RSNO with NO2− calculated by subtracting the concentration of RSNO. Signal output (mV) ANAEMIA AND CEREBROVASCULAR REACTIVITY 110 was plotted against time (s) using Origin 8 software (OriginLab Corps, Massachusetts, USA) and smoothed using a 150-point averaging algorithm. The Peak Analysis package was used to calculate the area under the curve (mV/s) and subsequently converted to a concentration, using standard curves of known concentrations of sodium nitrite. The intra- and inter-assay coefficient of variations for all metabolites were <10 %. 5.3 Statistical Analyses: Hematological, blood gas, and cerebral metabolism parameters at rest pre and post haemodilution were compared using two-tailed paired t-tests. Blood gas parameters for the hypercapnia trials pre and post haemodilution were analyzed with linear mixed effects models. Trial (pre/post haemodilution) and stage (baseline, +3, +6, +9 PaCO2) were included as fixed effects while participants were included as a random effect. When significant interaction effects were detected, post-hoc tests were conducted with multiple comparisons adjusted for with the Bonferroni correction. All CBF (VA flow, ICA flow, & gCBF) and shear stress responses to hypercapnia pre- and post-haemodilution were analyzed with linear mixed effects models (Fixed effects: PaCO2 and pre/post haemodilution; Random effect: participants). When significant interaction effects were detected, post-hoc tests were conducted with multiple comparisons adjusted for with the Bonferroni correction. As the haemodilution protocol would be expected to cause a set-point change in absolute CBF due to the well-established relationships between CaO2, [Hb], and CBF (e.g., Brown et al., 1985), we also analyzed CBF responses by assessing the delta changes in CBF from baseline in each trial. This approach was taken to account for the elevation in CBF that occurred secondary to haemodilution, but prior to the hypercapnia. The change in CBF and shear stress from baseline (i.e. delta values) were compared using linear mixed effects model with trial (pre/post haemodilution) and stage (baseline, +3, +6, +9 PaCO2) as fixed effects. Participants were included as a random effect. When significant interaction effects were detected, post-hoc tests were conducted with multiple comparisons adjusted for with the Bonferroni correction. Further, CVR slopes were calculated using linear regression and compared pre- to post-haemodilution with two tailed paired t-tests. To support existing literature on the relationships between CBF and: CaO2, Hb, Hct, PaCO2, pH, and CPP, we conducted repeated measures correlations using the rmcorr package for R (Bakdash & Marusich, 2017) on those variables at three time points (pre-haemodilution baseline, mid-haemodilution check-point, and post haemodilution baseline), to quantify the intra-individual relationships between those variables and CBF. ANAEMIA AND CEREBROVASCULAR REACTIVITY 111 To determine whether significant a-v differences and net exchange (uptake/release) of NO2− was present before or after haemodilution we conducted one-sided two-tailed t-tests (tested against a zero value) within each condition. We also compared the a-v difference and cerebral net exchange of NO2− prior to and following haemodilution using a two-tailed paired t-test. Linear mixed model analyses were performed with the Statistical Package for the Social Sciences (SPSS, Version 24), t-tests were conducted using Excel (Microsoft, Version 16.16.9), and repeated measures correlations were conducted using R. (R Core Team, 2017) All figures were generated using GraphPad (Prism, La Jolla, CA, Version 8.1.1). Shapiro–Wilk normality testing of the fundamental stimuli (PaCO2, PaO2, CaO2) and measured responses upon which conclusions were based (ICA flow, VA flow, gCBF), confirmed that the data were normally distributed. Statistical significance was set a priori at P<0.05 and actual P values are reported herein (unless P<0.001). (Wasserstein & Lazar, 2016; Curran-Everett, 2020) This invasive study resulted in a relatively small sample size, as such there may be an increased risk of type 2 error, and we conduct multiple comparisons which in itself may predispose to an increased risk of type 1 error; so, in addition to significance testing, individual data are displayed for the sake of transparency and improved ease of data interpretation, of particular utility where sample sizes are low and P-values approach the P=0.05 cut-off. Data are presented as means and standard deviations. 5.4 Results: Baseline, pre and post haemodilution: Intervention parameters: Isovolumic haemodilution reduced Hct (P<0.001; Figure 5-1B) and [Hb] by 18±2% (P<0.001; Figure 5-1A). Since PaO2 (P=0.32; Figure 5-1C) and SaO2 (P=0.78; Figure 5-1D) were unchanged, the reduction in [Hb] resulted in an 18±2 % reduction in CaO2 (P<0.001; Figure 5-1E). The PaCO2 was not different between pre- and post-haemodilution baselines (P=0.21; Figure 5-1F), however, haemodilution caused reductions in arterial plasma CO2 content (see C.1 Supplemental materials for calculations) (P<0.001), total arterial content of CO2 (CaCO2) (P<0.001; Figure 5-1G), and arterial [HCO3−] (P<0.001; Figure 5-1H), while increasing arterial [H+] (P=0.001; Figure 5-1I). There was no change in CPP (P=0.84; Figure 5-1K), MAP (P=0.465; Table 5-1), or jugular venous pressure (P=0.810; Table 5-1) with haemodilution. ANAEMIA AND CEREBROVASCULAR REACTIVITY 112 Figure 5-1. Study 3 - Intervention parameters at baseline prior to and following haemodilution. Pre-haemodilution mean and standard deviation data presented in red, post-haemodilution mean and standard deviation data presented in blue, with individual data overlaid for both. A, haemoglobin concentration ([Hb]), B, hematocrit (Hct), C, partial pressure of arterial O2 (PaO2), D, arterial oxygen saturation (SaO2), E, arterial O2 content (CaO2), F, partial pressure of arterial CO2 (PaCO2), G, total arterial content of CO2 (CaCO2), H, arterial bicarbonate ion concentration ([HCO3-]), I, arterial hydrogen ion concentration ([H+]), and K, cerebral perfusion pressure (CPP), depicted at baseline pre- and post- haemodilution. Asterisk (*) symbols indicate a change (P<0.05) from pre to post haemodilution, compared using paired two-tailed t-tests. N=11 Pre Post10121416Hb (g/dL)*Pre Post96.597.097.598.098.5SaO2 (%)Pre Post3035404550Hct (%)*Pre Post363840424446PaCO2 (mm Hg)Pre Post859095100PaO2 (mm Hg)Pre Post14161820CaO2 (mL/dL)*Pre Post54565860626466CaCO2 (mL/dL)*Pre Post232425262728[HCO3-] (mmol)*Pre Post38404244[H+] (nmol)*Pre Post80859095100CPP (mm Hg)A B C D EF G H I KANAEMIA AND CEREBROVASCULAR REACTIVITY 113 Table 5-1. Cerebrovascular parameters during CVR prior to and following haemodilution. Baseline +3mmHg +6mmHg +9mmHg MAP Haemodilution, P=0.465; CO2, P<0.001; Interaction, P=0.860 (mmHg) Pre 97.1±4.8 99.4±4.9 103.1±5.6*† 107.6±8.0*†‡ Post 97.5±5.3 99.8±7.4 104.3±7.9*† 108.6±9.6*†‡ JVP Haemodilution, P=0.810; CO2, P<0.001; Interaction, P=0.185 (mmHg) Pre 6.4±2.0 6.8±1.9 7.2±1.9* 7.8±2.1*†‡ Post 6.5±0.9 6.7±1.0 7.4±1.5* 8.2±1.9*†‡ ICA diameter Haemodilution, P=0.042; CO2, P<0.001; Interaction, P=0.479 (mm) Pre 5.26±0.37 5.35±0.42* 5.47±0.37*† 5.49±0.38*† Post 5.16±0.37 5.26±0.36* 5.34±0.34*† 5.44±0.38*† ICA velocity Haemodilution, P<0.001; CO2, P<0.001; Interaction, P=0.650 (cm/s) Pre 48.1±9.6 55.0±12.7* 64.4±12.8*† 70.7±13.3*†‡ Post 59.2±10.9 68.1±15.0* 78.5±16.0*† 83.6±16.4*†‡ ICA flow Haemodilution, P<0.001; CO2, P<0.001; Interaction, P=0.710 (mL/min) Pre 314.4±65.1 374.2±98.2* 457.8±96.5*† 507.1±108.3*†‡ Post 376.5±89.1 448.0±105.0* 531.6±117.6*† 590.1±144.2*†‡ ICA Shear rate Haemodilution, P<0.001; CO2, P<0.001; Interaction, P=0.602 (s-1) Pre 368±82 414±101* 473±109*† 516±103*†‡ Post 460±84 520±123* 589±128*† 616±124*†‡ ICA Shear stress Haemodilution, P=0.089; CO2, P<0.001; Interaction, P=0.217 (dyne/cm2) Pre 12.9±3.2 14.5±3.8* 16.6±4.2*† 18.1±3.9*†‡ Post 12.4±2.5 14.0±3.4* 15.8±3.6*† 16.5±3.5*†‡ VA diameter Haemodilution, P=0.506; CO2, P<0.001; Interaction, P=0.002 (mm) Pre 4.06±0.31 4.08±0.37 4.14±0.33 4.20±0.34*† Post 3.95±0.23 3.99±0.27 4.13±0.29*† 4.26±0.27*†‡ VA velocity Haemodilution, P <0.001; CO2, P<0.001; Interaction, P=0.640 (cm/s) Pre 23.7±7.6 26.8±8.9 32.3±10.8*† 36.9±12.8*†‡ Post 28.5±7.7 32.0±8.2 38.8±11.8*† 43.7±11.2*†‡ VA flow Haemodilution, P=0.004; CO2, P<0.001; Interaction, P=0.146 (mL/min) Pre 93.4±32.4 105.5±34.2 131.8±46.9*† 157.0±64.2*†‡ Post 105.4±29.9 120.7±34.3 157.9±52.9*† 188.5±54.3*†‡ VA Shear rate Haemodilution, P<0.001; CO2, P<0.001; Interaction, P=0.967 (s-1) Pre 233.7±74.9 264.4±93.9 312.6±106.1*† 349.7±115.6*†‡ Post 289.3±80.9 321.9±83.8 376±114.5*† 411.4±106.6*†‡ ANAEMIA AND CEREBROVASCULAR REACTIVITY 114 Baseline +3mmHg +6mmHg +9mmHg VA Shear stress Haemodilution, P=0.220; CO2, P<0.001; Interaction, P=0.536 (dyne/cm2) Pre 8.1±2.4 9.2±3.1 10.9±3.4*† 12.2±3.9*†‡ Post 7.9±2.5 8.8±2.7 10.3±3.3*† 11.3±3.2*†‡ MCAv Haemodilution, P=0.016; CO2, P<0.001; Interaction, P=1.0 (cm/s) Pre 70.9±12.8 79.4±16.1* 89.2±19.1*† 99.4±17.4*†‡ Post 77.3±14.2 85.8±15.9* 95.8±19.3*† 106.1±21.1*†‡ PCAv Haemodilution, P=0.008; CO2, P<0.001; Interaction, P=0.194 (cm/s) Pre 42.2±7.4 47.0±8.0* 52.1±10.6*† 56.8±12.6*†‡ Post 50.3±14.4 54.6±15.5* 58.2±14.2*† 60.6±13.2*†‡ gCBF Haemodilution, P<0.001; CO2, P<0.001; Interaction, P=0.099 (mL/min) Pre 833.1±147.7 984.9±233.6* 1187.7±252.7*† 1333.2±308.8*†‡ Post 987.1±202.4 1148.5±241.5* 1391.8±268.1*† 1585.8±310.9*†‡ Comparisons conducted using linear mixed-model analyses with Bonferroni adjustments for post-hocs. Asterisk (*) symbols indicate a difference from baseline (P<0.05) in both trials, obelisk (†) symbols indicate a difference from the +3 mm Hg stage (P<0.05) in both trials, and double dagger (‡) symbols indicate a difference from the +6 mm Hg stage (P<0.05) in both trials. Differences between pre and post haemodilution across all stages are identified by bolded type. MAP, mean arterial pressure; JVP, jugular venous pressure; ICA, internal carotid artery; VA, vertebral artery; MCAv, middle cerebral artery blood velocity; PCAv, posterior cerebral artery blood velocity; gCBF, global cerebral blood flow. Viscosity, shear rates and stresses: At all measured shear rates, whole blood viscosity was lower following haemodilution (45s-1; 4.2±0.3 vs. 3.1±0.4 cP, P<0.001: 90s-1; 3.9±0.3 vs. 2.9±0.3 cP, P<0.001: 225s-1; 3.5±0.2 vs. 2.8±0.2 cP, P<0.001). Basal ICA and VA shear rates were both higher post-haemodilution (368.2±82.4 vs. 459.5±84.0 s-1, P<0.001; and 233.7±74.9 vs. 289.3±80.9 s-1, P=0.004, respectively). The decreased viscosity and increased shear rates resulted in ICA and VA shear stress being unchanged from baseline (12.9±3.2 vs. 12.4±2.5 dyne/cm2, P=0.26; and 8.1±2.4 vs. 7.9±2.5 dyne/cm2, P=0.68, respectively). Cerebral blood flow and metabolism parameters: Haemodilution caused an elevation in basal gCBF (833±148 vs. 987±202 ml/min; P=0.002; Figure 5-2A). This increase in gCBF served to maintain CDO2 (P=0.49; Figure 5-2C) in the face of the reduced CaO2 in order to preserve metabolism (cerebral metabolic rate of O2 (CMRO2); P=0.49; Figure 5-2D: cerebral metabolic rate of CO2 (CMRCO2); P=0.57; Figure 5-2G) and oxygen extraction fraction (OEF) (p=0.99; Figure 5-2B). The rise in gCBF and the maintenance of metabolism resulted in elevated CO2 ANAEMIA AND CEREBROVASCULAR REACTIVITY 115 washout (P=0.01; Figure 5-2F), which was also reflected in the decrease of fraction of CO2 inserted into jugular blood (CO2IF; see C.1 Supplemental materials for calculations) (P=0.04; Figure 5-2E). Figure 5-2. Study 3 - Cerebral blood flow and metabolism at baseline prior to and following haemodilution. Pre-haemodilution mean and standard deviation data presented in red, post-haemodilution mean and standard deviation data presented in blue, with individual data overlaid for both. A, global cerebral blood flow (gCBF), B, O2 extraction fraction (OEF), C, cerebral delivery of O2 (CDO2), D, cerebral metabolic rate of O2 consumption (CMRO2), E, CO2 insertion fraction (CO2IF), F, cerebral washout of CO2 content (CO2 washout), G, cerebral metabolic rate of CO2 production (CMRCO2), at baseline pre- and post- haemodilution. Asterisk (*) symbols indicate a change (P<0.05) from pre to post haemodilution, compared using paired two-tailed t-tests. N=9 for A, C, D, F, and G; while n=11 for B and E. NO exchange: There was a positive cerebral a-v difference (25.9±30.1 nM; P=0.023; one-sided t-test) and net uptake (11.6±14.0 nmol/min; P=0.027; one-sided t-test) for plasma 𝑁𝑂2− prior to haemodilution but no a-v difference (-7.2±34.1 nM; P=0.52; one-sided t-test) or net uptake (-3.6±17.9 nmol/min; P=0.54; one-sided t-test) following haemodilution. However, when compared pre- to post-haemodilution there was no statistical difference in the a-v difference (P=0.069; C.1 Supplemental Figure 2A) and net exchange (P=0.098; C.1 Supplemental Figure 2B) of plasma 𝑁𝑂2− (paired t-tests) despite the mean change from Pre Post90120150180210240CDO2 (mL/min) Pre Post5060708090100110CMRCO2 (mL/min)Pre Post400500600700800CO2 washout (mL/min)*Pre Post3040506070CMRO2 (mL/min) Pre Post202530354045OEF (%)Pre Post6810121416CO2IF (%)*Pre Post600800100012001400gCBF (mL/min)*AB C DE F GANAEMIA AND CEREBROVASCULAR REACTIVITY 116 uptake to release for each metric. Therefore, haemodilution appeared to eliminate the net cerebral consumption of plasma 𝑁𝑂2− observed at baseline. Repeated measures correlations: We conducted repeated measures correlations assessing within-participant relationships between gCBF and parameters known to influence CBF, using values taken at baseline, mid-haemodilution checkpoint, and post-haemodilution baseline. There were intra-individual relationships between gCBF and: CaO2 (R2= -0.44, P=0.007), Hb (R2= -0.45, P=0.006), Hct (R2= -0.44, P=0.007), pH (R2= -0.32, P=0.029), and CaCO2 (R2= -0.38, P=0.015). As PaCO2 was clamped between stages and unchanged by haemodilution, there was no relationship with gCBF (P=0.31) or CPP (P=0.43). Cerebrovascular reactivity, pre and post haemodilution: Intervention parameters: By design, PaCO2 was elevated during the CVR test at each stage of hypercapnia (CO2 stage, P<0.001) and each stage was matched between pre- and post-haemodilution (trial, P=0.414; Supplemental Figure 3A). The PaO2 was matched between pre- and post-haemodilution at each of stage of hypercapnia (trial, P=0.342; Supplemental Figure 3B), while CaO2 was lower following haemodilution (trial, P<0.001; Supplemental Figure 3C). During CVR, neither MAP, JVP, nor CPP were different pre- versus post-haemodilution (trial, P=0.465, P=0.810, and P=0.581, respectively), though all were elevated from baseline at the +6 and +9 mmHg PaCO2 stages (CO2 stage, all P<0.001; Table 1). Cerebral blood flow: Hypercapnia led to an increase in gCBF at each PaCO2 stage (CO2 stage, P<0.001; Figure 5-3A). Post-haemodilution, gCBF was elevated at each stage compared to pre-haemodilution (trial, P<0.001; Figure 5-3A and Table 5-1). To account for this is in the subsequent determination of CVR, we also compared gCBF change from baseline, which abolished the gCBF pre- versus post-haemodilution difference (trial, P=0.198, Figure 5-3). Shear stress did not differ between the pre- and post-haemodilution CVR trials (Table 5-1). ANAEMIA AND CEREBROVASCULAR REACTIVITY 117 Figure 5-3. Study 3 - The relationship between global cerebral blood flow and arterial CO2. Mean and standard deviation for pre- (red circles/bar) and post- (blue triangles/bar) haemodilution, with individual data for both. A, global cerebral blood flow (gCBF) at four stages of partial pressure of arterial CO2 (PaCO2), B, gCBF changes from baseline with changes in PaCO2 from baseline, C, cerebrovascular reactivity of gCBF (gCBF CVR) pre and post haemodilution, D, cerebral perfusion pressure (CPP) at four stages of PaCO2, E, cerebral vascular conductance (CVC) at four stages of PaCO2, F, CVC changes from baseline with changes in PaCO2 from baseline. N=9. Cerebrovascular reactivity: While absolute gCBF was elevated throughout post-haemodilution compared to pre-haemodilution, the gCBF CVR response to PaCO2 was not different between trials (P=0.24; Figure 5-3C), nor was gCBF CVR calculated using absolute delta gCBF (P=0.14), or percent delta gCBF (P=0.77). Similarly, CVR did not differ when gCBF was indexed against jugular venous partial pressure of CO2 (PjvCO2; P=0.39), CaCO2 (P=0.10), or jugular venous content of CO2 (CjvCO2) (P=0.87), further demonstrating that CVR was not differentially affected by haemodilution at each stage of PaCO2. Finally, CVR of MCAv and PCAv were also unchanged (P=0.59 and P=0.25, respectively) with haemodilution Table 5-2 presents all CVR slopes indexed against PaCO2. ANAEMIA AND CEREBROVASCULAR REACTIVITY 118 Table 5-2. Cerebrovascular reactivity slopes in response to elevations in PaCO2. Absolute Reactivity ([∆X]/ mm Hg) Absolute delta reactivity (∆[∆X]/ mm Hg) Relative reactivity (%∆[∆X]/ mm Hg) Pre Post P-value Pre Post P-value Pre Post P-value ICA flow (mL/min) 23.9±6.9 24.7±7.9 0.76 23.9±7.7 24.2±10.4 0.92 7.7±2.9 6.4±2.4 0.22 VA flow (mL/min) 7.9±4.2 9.3±4.2 0.26 9.4±7.5 11.6±6.1 0.28 10.4±4.6 11.4±5.4 0.62 gCBF (mL/min) 61.7±19.0 69.0±19.2 0.23 65.7±21.7 74.0±23.6 0.14 7.8±2.2 7.6±2.8 0.77 MCAv (cm/s) 3.6±0.8 3.2±1.7 0.59 3.6±0.6 3.3±1.9 0.76 5.3±0.9 4.8±2.7 0.56 PCAv (cm/s) 1.7±0.8 1.2±0.8 0.25 1.8±1.1 1.1±1.0 0.25 4.1±2.2 2.5±1.7 0.16 gCBF CVC (mL/min/mmHg) 0.51±0.17 0.56±0.17 0.47 0.52±0.21 0.58±0.24 0.15 5.7±2.5 5.5±2.7 0.57 Comparisons conducted using paired two-tailed t-tests. ICA, internal carotid artery; VA, vertebral artery; gCBF, global cerebral blood flow; MCAv, middle cerebral artery blood velocity; PCAv, posterior cerebral artery blood velocity; gCBF CVC, global cerebral blood flow cerebrovascular conductance. ANAEMIA AND CEREBROVASCULAR REACTIVITY 119 Metabolism parameters: The Ca-jvO2 difference was lower post-haemodilution (trial, P<0.001), as a result of the reduction in CaO2, and was narrowed at each step increase in PaCO2 (CO2 stage, P<0.001) as a result of the increase in gCBF at each stage. There was a significant interaction effect (trial*CO2, P=0.012) whereby during both pre- and post-haemodilution Ca-jvO2 was reduced (all P<0.001) at each stage, and post-haemodilution Ca-jvO2 was lower than pre-haemodilution within each CVR stage (all P<0.001; Figure 5-4A). Despite the reduced CaO2, the overall elevation in gCBF post-haemodilution served to maintain CDO2 comparably to pre-haemodilution throughout the CVR test (trial, P=0.260; Figure 5-4B). As such, OEF was also unchanged between trials (trial, P=0.525) and lower at each stage of CVR compared to baseline (CO2 stage, P<0.001; Figure 5-4C). CMRO2 was not different between trials (trial P=0.175) and was lower (CO2 stage, P<0.001; Figure 5-4D) at the +9 mm Hg PaCO2 stage compared to baseline (P=0.002) and +3 mm Hg (P=0.034) stages. When determined as the change from baseline, a difference between trials remained for Ca-jvO2 (trial, P=0.041). During CVR, the Cjv-aCO2 difference was lower post-haemodilution (trial*CO2, P=0.034) at baseline (P=0.004) and +3 mmHg PaCO2 (P<0.001) stages but not +6 or +9 mmHg stages (Figure 5-4E). The overall elevation in gCBF post-haemodilution elevated CO2 washout across all stages (trial, P=0.004), and the expected CVR response elevated gCBF in tandem with the elevated PaCO2 at each stage, thereby increasing absolute CO2 washout at each stage (CO2 stage P<0.001; Figure 5-4F). Correspondingly, CO2IF was lower post-haemodilution (trial, P=0.041) and reduced at each step increase in PaCO2 (CO2 stage, P<0.001; Figure 5-4G). CMRCO2 was maintained between trials (trial P=0.246) and was nominally unchanged with hypercapnia (CO2 stage, P=0.052; Figure 5-4H). When determined as the change from baseline, the interaction effect remained for Cjv-aCO2 (trial*CO2, P=0.026), but the effect of trial was abolished for CO2IF (trial, P=0.385) and CO2 washout (trial, P=0.366). Finally, while PaCO2 and PjvCO2 were both elevated at each stage of CVR (Figure 5-5A), the rise in CaCO2 (Figure 5-5B) was not matched with a corresponding rise in total CO2 content of the cerebral effluent (CjvCO2). Indeed, although CjvCO2 was reduced following haemodilution (trial, P<0.001), it was unchanged across PaCO2 stages in either trial (CO2 stage, P=0.190; Figure 5-5C).ANAEMIA AND CEREBROVASCULAR REACTIVITY 120 Figure 5-4. Study 3 - Cerebral metabolism during hypercapnia prior to and following haemodilution. Pre-haemodilution mean and standard deviation data presented in red, post-haemodilution mean and standard deviation data presented in blue, with individual data overlaid for both. A, cerebral arteriovenous O2 content difference (Ca-jvO2), B, cerebral O2 delivery (CDO2), C, O2 extraction fraction (OEF), D, cerebral metabolic rate of oxygen consumption (CMRO2), E, cerebral veno-arterial CO2 content difference (Cjv-aCO2), F, cerebral washout of CO2 content (CO2 washout), G, CO2 insertion fraction (CO2IF), and H, cerebral metabolic rate of CO2 production (CMRCO2). Comparisons conducted using linear mixed-model analyses with Bonferroni adjustments for post-hocs. Asterisk (*) symbols indicate a difference from baseline (P<0.05) in both trials, obelisk (†) symbols indicate a difference from the +3 mm Hg stage (P<0.05) in both trials, and double dagger (‡) symbols indicate a difference from the +6 mm Hg stage (P<0.05) in both trials. While hash symbols (#) indicate a difference (P<0.05) between pre and post haemodilution in all CVR stages. Where 100200300400CDO2 (mL×min-1) Baseline +3 mm Hg +6 mm Hg +9 mm HgTrial: P=0.260CO2: P<0.001Interaction: P=0.937 † ‡50010001500CO2 washout (mL×min-1)Baseline +3 mm Hg +6 mm Hg +9 mm HgTrial: P=0.004CO2: P<0.001Interaction: P=0.185# † ‡051015CO2IF (%)Baseline +3 mm Hg +6 mm Hg +9 mm HgTrial: P=0.041CO2: P<0.001Interaction: P=0.072# † ‡P=0.051 vs baseline10203040OEF (%)Baseline +3 mm Hg +6 mm Hg +9 mm HgTrial: P=0.525CO2: P<0.001Interaction: P=0.576 † ‡3040506070CMRO2 (mL×min-1) Baseline +3 mm Hg +6 mm Hg +9 mm HgTrial: P=0.175CO2: P=0.003Interaction: P=0.945 †20406080CMRCO2 (mL×min-1)Baseline +3 mm Hg +6 mm Hg +9 mm HgTrial: P=0.246CO2: P=0.052Interaction: P=0.4382468Ca-jvO2 (mL×dL-1)Baseline +3 mm Hg +6 mm Hg +9 mm HgTrial: P<0.001CO2: P<0.001Interaction: P=0.012# # # #† ‡0246810Cjv-aCO2 (mL×dL-1)Baseline +3 mm Hg +6 mm Hg +9 mm HgTrial: P=0.007CO2: P<0.001Interaction: P=0.034# † # ‡a b c de f g hANAEMIA AND CEREBROVASCULAR REACTIVITY 121 an interaction effect is indicated, these symbols refer to changes between stages within trials, and differences between trials within stages. N=11 for A, C, E, and G; while n=9 for B, D, F, and H. Figure 5-5. Study 3 - Cerebral CO2 tensions and contents during hypercapnia prior to and following haemodilution. Solid red circles and blue triangles represent arterial blood pre and post haemodilution, respectively, while empty red circles and blue triangles represent jugular venous blood pre and post haemodilution, respectively. A, global cerebral blood flow (gCBF) against the partial pressures of CO2 in arterial (PaCO2) and jugular venous (PjvCO2) bloods, B, gCBF against the arterial content of CO2 (CaCO2), and C, gCBF against the jugular venous content of CO2 (CjvCO2). All data are mean and standard deviations, with individual data overlaid for B, C, and D. N=9.40 45 50 55500100015002000PCO2 (mmHg)gCBF (mL×min-1)Arterial PostJugular PreJugular PostArterial Pre56 58 60 62 64 66 68 70 72500100015002000CaCO2 (mL×dL-1)gCBF (mL×min-1)56 58 60 62 64 66 68 70 72500100015002000CjvCO2 (mL×dL-1)gCBF (mL×min-1)a b cANAEMIA AND CEREBROVASCULAR REACTIVITY 122 5.5 Discussion: The purpose of this study was to investigate the influence of acute isovolumic haemodilution on CBF and cerebrovascular reactivity to CO2 in healthy humans. Cerebrovascular regulation was preserved following haemodilution, indicated by an unaltered CVR. Furthermore, CBF increased following haemodilution to an extent adequate to preserve CDO2 and thereby maintain the requirements of CMRO2, which also resulted in increased CO2 washout. This preservation of cerebrovascular function coincided with an abolishment of cerebral net uptake of NO2−. Overall, our findings indicate that in the setting of acute experimental anaemia in the absence of pathology, cerebrovascular metabolism and regulation by CO2 are preserved. Along with the principal regulators of CBF [i.e., blood pressure, metabolism, blood gases (Willie et al., 2012, 2014; Iadecola, 2017; Hoiland et al., 2019; Claassen et al., 2021)] a number of other factors have been implicated in CBF control. For example, both animal and human studies have reported effects of changes in blood viscosity, Hct, [Hb], and NO bioavailability on CBF. (Brown & Marshall, 1985; Brown et al., 1985; Tomiyama et al., 2000; Lenz et al., 2001; Rebel et al., 2003; Azarov et al., 2005; Ševerdija et al., 2015; Hoiland et al., 2020c, 2021) Based on these studies, we hypothesized that haemodilution would steepen the CVR response. However, our findings demonstrate CVR is unchanged with haemodilution (Figure 5-3), which is in agreement with previous rodent studies, (Mistry et al., 2018) and with Tu & Liu (Tu & Liu, 1996) who found that following acute isovolumic haemodilution in healthy humans, CBF was elevated while cerebrovascular reactivity to acetazolamide was reduced in the cortex but not in white matter, the putamen, or thalamus (differences in stimulus and mechanisms acknowledged). Below, we place the related findings in the context of the contributory roles of viscosity, Hct, [Hb], and NO and highlight the implications of elevations in CBF on central pH balance. Regulation of CBF in the setting of acute anaemia: Following the removal and replacement of whole blood with albumin (a non-oxygen-carrying protein), Hct was reduced by 20%, which caused an approximate 20% reduction in CaO2 (Figure 5-1). Concomitant with this mild anaemia, gCBF was elevated by approximately 18% (Figure 5-2). While it has been well established that CBF is inversely related to CaO2 (Brown et al., 1985; Hoiland et al., 2016a) indicating the increase in basal CBF following anaemia was likely driven by hypoxic vasodilation, other factors that may concurrently influence CBF cannot be disregarded. Indeed, in addition to the reductions in [Hb] and CaO2 (Figure 5-1A & E, respectively), haemodilution was also associated with an increase in arterial [H+] and a reduction in arterial [HCO3−] (Figure 5-1I & H, respectively). Much work has been presupposed on the notion that arterial [H+] is a predominant driver ANAEMIA AND CEREBROVASCULAR REACTIVITY 123 of CBF, although other work contends that in fact PaCO2 plays the primary role, diffusing across the blood brain barrier to alter perivascular PCO2 and thus [H+]. (Lambertsen et al., 1961b; Kontos et al., 1977b, 1977a; Caldwell et al., 2021b, 2021a) Evidence also suggests that [HCO3−] may independently influence CBF (Boedtkjer et al., 2015; Caldwell et al., 2021b). Our results can neither confirm nor contest this as our study design does not allow for differentiation of the effects of changes in [H+] from the effects of changes in CaO2. However, recent data from our laboratory does indicate that acute changes in arterial [H+] independent of PaCO2 are not associated with concurrent alterations in CVR. (Caldwell et al., 2021b) Nevertheless, while the reduction in CaO2 was likely the primary factor elevating basal CBF following haemodilution, other factors cannot be wholly discounted, as outlined next. As the reduction in CaO2 coincided with reduced Hct, there is further potential for alterations in blood viscosity per se - consequent to the changes in Hct - to alter CBF regulation. In rodents, extreme elevations in whole blood viscosity substantially affect CBF during haemodilution and hypercapnia (Tomiyama et al., 2000) and exhaust available vasodilator reserve, thereby abolishing hypercapnic CVR. (Lenz et al., 2001) Yet, in vivo in humans, viscosity appears to have a lesser role in CBF regulation compared to the effects of changes in CaO2.(Brown & Marshall, 1982; Brown et al., 1985) For example, to isolate the changes in whole blood viscosity from CaO2, Brown and Marshall (Brown & Marshall, 1982, 1985) investigated the influence of a reduction in blood viscosity secondary to plasmapheresis of paraproteinemic patients on CBF. The authors observed no change in CBF following a large reduction in plasma viscosity, and thus whole blood viscosity with which there was no accompanying change in CaO2 ; (Brown & Marshall, 1982, 1985) this is consistent with animal studies showing the greater degree of contribution of CaO2 to changes in CBF during haemodilution.(Tomiyama et al., 1999) Further, haemodilution-induced anaemia has been shown to increase CBF to a much lesser extent than carbon monoxide inhalation, (Paulson et al., 1973a) despite the decreased blood viscosity associated with haemodilution that is absent with carbon monoxide inhalation [acknowledging the potential for the confound of direct vasodilator influences of carbon monoxide (Leffler et al., 2006)]. The majority of studies assessing the effects of hematocrit on CVR have been conducted in animals (Raju & Kim, 1991; Farman et al., 1994; Rebel et al., 2003; Crystal et al., 2014; Mistry et al., 2018) or in humans in the clinical setting of cardiovascular pathology. (Ševerdija et al., 2015) Relative to human investigations, while Ševerdija et al. (Ševerdija et al., 2015) found no difference in hypercapnic CVR between high and low hematocrit groups (≥28 % vs <28 %), varied methods make reconciling the findings of these studies with our own difficult. For instance, Ševerdija et al. (Ševerdija et al., 2015) report CVR as the percent change in cerebral tissue (or rather Hb) oxygen saturation assessed via near-infrared spectroscopy per change in PaCO2 during hypercapnic and hypocapnic steps induced via ANAEMIA AND CEREBROVASCULAR REACTIVITY 124 mechanical ventilation. These CVR tests were conducted during anaesthesia with Propofol and sufentanil, for coronary bypass surgery, in elderly patients. However, it has been demonstrated that there is poor agreement between near-infrared spectroscopy and measures of volumetric CBF during hypercapnia. (Hoiland et al., 2020b) Additionally, the comparisons in that study were between groups, rather than within-individuals, and could be confounded by the between individual variability typical of physiologic measures especially in elderly patients with cardiovascular pathology, hindering the relevance to the current study. Ultimately, however, the findings in the study by Ševerdija et al. (Ševerdija et al., 2015) and our findings are consistent. While analogous considerations apply, previous work in baboons demonstrated that CVR varied inversely with hematocrit ,(Raju & Kim, 1991) supporting our findings that show acute reductions in Hct are associated with elevated gCBF. It is known that Hb is a powerful scavenger of NO, (Jia et al., 1996; Gow & Stamler, 1998; Herold et al., 2001; Azarov et al., 2005) thus reductions in Hb could hypothetically affect CBF through reduced scavenging (Azarov et al., 2005) and improved NO bioavailability. Equivalent findings relative to the peripheral circulation have recently been demonstrated, i.e., NO-dependent shear-mediated dilation of the brachial artery was augmented following a haemodilution induced reduction in Hb. (Tremblay et al., 2019b; Hoiland et al., 2020c) While the notion of a reduction in Hb scavenging of NO was a notable aspect of our hypothesis building, the contribution of NO to CVR remains a contentious topic. Reviews on the summarized evidence of CVR regulatory mechanisms (Tanaka, 1996; Brian, 1998; Toda et al., 2009; Hoiland et al., 2019) and combined recent investigations (White et al., 1998; Ide et al., 2007; Hoiland et al., 2021; O’Gallagher et al., 2021; Carter et al., 2021) suggest that while NO plays a fundamental role in basal CBF regulation, it has a merely permissive rather than obligatory role during hypercapnia in humans (though evidence to the contrary also exists (Schmetterer et al., 1997; Jahshan et al., 2017)). In other words, while hypercapnia stimulates cerebrovascular NO production (Hoiland et al., 2021) and downstream secondary messenger activity, (Irikura et al., 1995) inhibition of this NO production has no influence on the CVR response. (Hoiland et al., 2021) As such, any reductions in Hb NO scavenging may plausibly have had some effect on CBF during baseline, but minimal or no impact on responses during CVR. Indeed, given arterial-to-venous differences of NO2− reflect endothelial NO production and are related to endothelium dependent vasodilation, (Lauer et al., 2001; Hoiland et al., 2021) the abolishment of net cerebral NO2− uptake from pre- to post-haemodilution may represent a reduction in Hb scavenging of NO and mechanism of the increase in CBF post haemodilution. Haemodilution decreased viscosity, and in conjunction with a concomitant increase in shear rate at each stage, shear stress was unaltered pre-to-post-haemodilution at each stage of hypercapnia. While supra-physiologically increased viscosity exhausts available cerebrovascular vasodilator reserve and thereby abolishes CVR in rats, (Lenz et al., 2001) the modest reduction in arterial viscosity per se during haemodilution would be unlikely to steepen CVR responses. Conversely, it may be expected that the ANAEMIA AND CEREBROVASCULAR REACTIVITY 125 anaemia-induced elevation of CBF might blunt CVR through encroaching on cerebrovascular reserve. (Prohovnik et al., 2009) However, CVR was not blunted, and - at least according to our volumetric measures - the haemodilution-induced elevation in gCBF did not exhaust cerebrovascular reserve in the large arteries (e.g. ICA and VA diameters were reduced and unchanged, respectively, following haemodilution; Table 5-1). While anaemia demanded CBF elevation in order to maintain CDO2 (Figure 5-2) presumably via vasodilation at the level of the pial arteries and microcirculation, this was achieved without vasodilation of the large arteries (relative to pre-haemodilution), but rather through faster velocities (Table 5-1) while the decrease in viscosity means that cerebral vascular shear stress was maintained at roughly pre-haemodilution magnitude (Table 5-1). Teleological Perspective The elevation in CBF with haemodilution was associated with an increase in CO2 washout (Figure 5-2), which is relevant to maintaining central pH. (Neubauer et al., 1985; Carr et al., 2021a) From a teleological perspective, in the setting of acute anaemia, elevated basal gCBF suffices to not only maintain CDO2, but also likely maintains central pH. The finding of unchanged CjvCO2 across all stages of CVR during both trials (Figure 5-5) further supports this conception. That total CO2 in the cerebral effluent blood is unchanged with increased PaCO2 is evidence of the efficacy of CVR mechanisms; indeed, it belies the function of CVR to maintain cerebral tissue acid-base balance. Whether or not the impaired CVR observed in anaemic pathologies is related to a disturbance in CO2 washout or central pH remains unknown. In the absence of a concomitant increase in gCBF, the reduction in the CO2 carrying capacity of the blood with isovolumic haemodilution would have resulted in build-up of CO2 causing cerebral tissue acidosis. Further, whether the contemporaneous increase in CO2 washout was merely coincidental or in fact a contributing influence in the increase in basal gCBF is also unknown. 5.6 Experimental considerations: We only studied male participants. Inclusion of both sexes is vital to a full understanding of the roles of NO, Hb, Hct, and viscosity in CBF regulation and for physiological research generally. (Lindsey et al., 2021) Thus the results of the current study cannot be extrapolated to females and future research is needed to address this limitation (Lindsey et al., 2021). Abundant opportunities are available for future research regarding this matter to assess possible sex-related differences and the influence of androgens and estrogens, across the female menstrual cycle, pre versus post menopause, and pre versus post puberty. Furthermore, the sample size was relatively small compared to some of the larger clinical trials assessing the effect of anemia on CBF, and as such may be underpowered for the comparisons of some variables. ANAEMIA AND CEREBROVASCULAR REACTIVITY 126 We acknowledge that as a variable for the assessment of cerebrovascular function, total content of CO2 likely only has significance in settings where some aspect of blood CO2 transport (i.e., Hb, [H+], [HCO3-], etc.) is altered– e.g., anaemia or other pathologies, high altitude, exercise etc. During single CVR tests, calculation of total CO2 content will provide little benefit over PaCO2, however in this study, it provided insight into the underlying factors allowing for the maintenance of CVR. Finally, the degree of anemia achieved in this investigation was relatively mild compared to that found in some severe pathologies, as such our findings may not hold true for more moderate or severe anemias. Nonetheless, mild anemia in clinical populations has been associated with impaired CVR compared to healthy controls, with hemoglobin concentrations comparable to the current study (e.g. 10.3g/dL in chronic renal failure patients; our study, 11.3g/dL post-hemodilution) (Kuwabara et al., 2002). Thus, in those instances of mild anaemia where CO2 reactivity is impaired, our findings suggest it is likely impaired not because of anaemia per se, but other pathologic factors associated with the disease (e.g. perhaps a hemolytic component and cell free Hb are more important in determining impairments in CVR in pathologically chronic anaemia). 5.7 Conclusion: Acute experimental anaemia increased gCBF to maintain CDO2 and preserve CMRO2; this led to – or was in conjunction with – the maintenance of hypercapnic CVR. Therefore, we conclude that in the setting of acute experimental anaemia, absent pathology, cerebral metabolism and the response to reduced CaO2 are robust enough to also allow for normal vascular regulation by CO2. This indicates that reductions of CVR seen in pathology are more likely the result of the direct vascular effects of disease states rather than due to the effects of anaemia per se. .𝛼1 ADRENO-REGULATION OF PERIPHERAL BLOOD FLOW IN HYPERCAPNIA 127 Chapter 6: Study 4A – Brachial artery responses to acute hypercapnia: the roles of shear stress and sympathetic tone Abstract: We aimed to assess the shear stress-dependency of brachial artery (BA) responses to hypercapnia, and the α₁-adrenergic-restraint of these responses. We hypothesized that elevated shear stress during hypercapnia would cause BA vasodilation, but where shear stress was prohibited (via arterial compression) the BA would not vasodilate (study 1); and, in the absence of α₁-adrenergic activity, blood flow, shear stress and BA vasodilation would increase (study 2). In study one, 14 healthy adults (7/7 male/female, 27±4 years) underwent bilateral BA Duplex ultrasound during hypercapnia [partial pressure of end-tidal carbon dioxide, +10.2±0.3 mmHg above baseline, 12 minutes] via dynamic end-tidal forcing, and shear stress was reduced in one BA using manual compression (compression vs. control arm). Neither diameter nor blood flow were different between baseline and the last minute of hypercapnia (P=0.423, P=0.363, respectively) in either arm. The change values from baseline to the last minute, in diameter (%; P=0.201), flow (mL/min; P=0.234), and conductance (mL/min/mmHg; P=0.503) were not different between arms. In study two, 12 healthy adults (9/3 male/female, 26±4 years) underwent the same design with and without 𝛼1 adrenergic receptor blockade (Prazosin; 0.05 mg/kg) in a placebo-controlled, double-blind, and randomized design. BA flow, conductance and shear rate increased during hypercapnia in the Prazosin control arm (interaction, P<0.001), but in neither arm during placebo. Even in the absence of α₁-adrenergic restraint, downstream vasodilation in the microvasculature during hypercapnia is insufficient to cause shear-mediated vasodilation in the brachial artery. 𝛼1 ADRENO-REGULATION OF PERIPHERAL BLOOD FLOW IN HYPERCAPNIA 128 6.1 Introduction: Elevations in the partial pressure of arterial CO2 (PaCO2) elicit vasodilatory effects in the peripheral vasculature (Mcardle et al., 1957; Mcardle & Roddie, 1958; Richardson et al., 1960; Kontos et al., 1967). However, this response is typically masked in normal human physiology by the concomitant sympathetic nervous activity (SNA)-mediated maintenance of vascular tone (Barcroft et al., 1943; Barcroft & Edholm, 1946; Mcardle et al., 1957; Richardson et al., 1960; Kontos et al., 1967) in order to elevate mean arterial pressure (MAP). Despite the increased sympathetic outflow, vasodilation of the brachial artery (BA) (Geijer et al., 2016a, 2016b) and increased BA blood flow (Vantanajal et al., 2007) have been reported during hypercapnia in humans. Vasodilation in the BA during hypercapnia may be shear stress-mediated, occurring due to increased shear rate via increased BA blood flow (Vantanajal et al., 2007) and cardiac output (CO) concomitant with hypercapnia (Davidson et al., 1986; Balanos et al., 2003; Willie et al., 2012) as postulated by Geijer et al., (2016a, 2016b), though this mechanism has not been explored. Hypercapnia also increases sympathetic nervous activity (SNA) (Somers et al., 1989; Shoemaker et al., 2002; Steinback et al., 2009) and circulating catecholamines (Sechzer et al., 1960; Metz, 1969; Davidson et al., 1986; Brofman et al., 1990), which contribute to increased MAP through increases in vascular resistance (Ullian, 1999; Ainslie et al., 2005b). As such, SNA restraint likely influences typical peripheral vascular responses during hypercapnia. The purpose of this investigation, therefore, was to assess the contributions of shear stress and SNA to peripheral vascular control during hypercapnia. We determined BA diameter and flow responses during hypercapnia, while preventing increases in shear stress and blocking α1 adrenergic receptors. Based on the only previous work – to our knowledge – which directly measured human BA diameter during hypercapnia (Geijer et al., 2016a, 2016b), we hypothesized that hypercapnia would cause vasodilation in the control but not the compressed BA. In an effort to explore the sympatho-mediated role in BA blood flow control to hypercapnia, we repeated the experiment in a placebo-controlled, double-blind, and randomized design with 𝛼-1 adrenergic receptor blockade (Prazosin). We hypothesized that Prazosin would prevent any α₁ adrenergic-mediated restraint of vascular tone during hypercapnia (per Richardson et al., 1960; Kontos et al., 1967) and allow for greater presentation of any shear stress- or CO2-mediated vasodilation. We also reasoned that during hypercapnia, the BA in the control arm would dilate to a greater degree than the compression arm. With Prazosin, we hypothesized that BA flow and diameter would be elevated in both arms compared to during placebo, and that during hypercapnia with Prazosin the BA in the control arm would dilate to a greater degree than the compression arm. 𝛼1 ADRENO-REGULATION OF PERIPHERAL BLOOD FLOW IN HYPERCAPNIA 129 6.2 Methods: We conducted two studies on the peripheral vascular responses to hypercapnia using bilateral BA Duplex ultrasound assessment during steady-state iso-oxic hypercapnia using computerized end-tidal forcing. Increases in shear stress in one BA were attenuated via manual arterial compression (Pyke et al., 2008; Tremblay et al., 2019a) while shear stress in the other BA was unhindered, such that each participant was their own control. Study one assessed BA responses bilaterally during hypercapnia with and without control of shear stress. Study two repeated this with and without 𝛼1-adrenergic receptor blockade (Prazosin) in a placebo-controlled, double-blind, and randomized design. The University of British Columbia Clinical Research Ethics Board approved both studies (H20-02716; H19-02355), which conform to the Declaration of Helsinki (except registry in a database). Participants were informed of any potential risks, and written informed consent was given prior to any experimental sessions. Participant characteristics: In study one, 14 healthy adults (7/7 male/female, 27±4 years, 72±13 kg, 173±9 cm) volunteered and participated; while 12 healthy adults (9/3 male/female, 26±4 years, 73±12 kg, 174±8 cm) volunteered for and participated in study two. Only one individual participated in both studies. Exclusion criteria for both studies included the presence or history of cardiovascular, musculoskeletal, or metabolic disease, present smokers or a history of smoking, use of medication (except oral contraceptives), or BMI >30 kg/m2. Phase of menstrual cycle appears to have a small effect on macrovascular endothelium-dependent function, though the full extent of this is unclear (Williams et al., 2020). Therefore, in both studies we attempted to standardize for menstrual phase (Thijssen et al., 2019) by only assessing normally menstruating females during the first seven days of the follicular phase (MacNutt et al., 2012) (n=4), or during the placebo week in those taking oral contraceptives (Minson et al., 2000) (n=4), phases when estradiol is lowest; n=2 females used an intra-uterine device. Experimental protocol: For both studies, participants arrived to the laboratory after a ≥6-hour fast, and having avoided alcohol and vigorous physical activity for 24 hours prior and caffeine for 12 hours prior (Thijssen et al., 2019). Upon arrival, participants body mass was weighed, and heights measured. In study one, participants then rested in a supine position for the following 20 minutes during instrumentation. In study two, following anthropometric measurement, with eyes closed participants ingested 𝛼1 adrenoreceptor blockade (Teva-Prazosin, Teva Canada Limited, Ontario, Canada; ~0.05 mg/kg body mass) in a gelatin capsule or placebo (~0.3 g sugar) in an identical gelatin capsule. Participants rested seated for 45 𝛼1 ADRENO-REGULATION OF PERIPHERAL BLOOD FLOW IN HYPERCAPNIA 130 minutes, then assumed a supine position and instrumentation began, testing then began 90 minutes after ingestion. This dosage of Prazosin has been shown to pharmacokinetically peak in plasma concentration between ~90 and ~180 minutes after ingestion (Hobbs et al., 1978; Bateman et al., 1979). Participant’s laboratory visits were randomized, scheduled 48 hours apart, and began at the same time of day in each participant (either 9 a.m. or 12 p.m.). Five minutes of baseline data was collected to establish baseline PETCO2 and PETO2 values. Participants then begun breathing on the dynamic end-tidal forcing system with PETCO2 and PETO2 maintained at baseline values. Initiation of image recording of both BAs was synchronized, and one minute baseline scans were acquired (baseline). For study one, venous samples were drawn during baseline for viscosity and plasma nitric oxide analyses. Then PETCO2 was elevated to +10 mm Hg above individual baseline and PETO2 was clamped at baseline; these values were maintained for 12 minutes from the onset of hypercapnia. The magnitude and duration of hypercapnia was chosen for direct comparison between these studies and Geijer et al., (2016a, 2016b). In both studies, both BAs were scanned continuously throughout the test from the beginning of baseline to the end of hypercapnia. In the left BA (compressed), in order to maintain shear stress at roughly baseline, the sonographer used manual arterial compression to inhibit any increase in blood velocity (Blair et al., 1959; Pyke et al., 2008; Tremblay et al., 2019a). The right BA (control) was not constrained in any way. For study one, venous blood samples were drawn again during the final two minutes of hypercapnia (last minute). Experimental measures: Cardiorespiratory variables: In both studies, beat-by-beat reconstructed BA blood pressure and computed stroke volume (SV) (ModelFlow: Harms et al., 1999; Leonetti et al., 2004; Bogert & Van Lieshout, 2005) were assessed via non-invasive finger photoplethysmography (Finometer PRO, Finapres Medical Systems, Amsterdam, Netherlands) on the right arm. Absolute correction of Finometer pressure values was based on the average of triplicate BA blood pressure measurements from the right arm using an automated sphygmomanometer (EM-759-E; Omron, Kyoto, Japan). Heart rate (HR) was assessed by lead-II configuration electrocardiogram R-R interval. The fraction of CO2 and O2 were continuously sampled at the mouth by gas analyzer (model ML206, ADInstruments) calibrated prior to each participant. The partial pressures of O2 and CO2 (PETO2 and PETCO2, respectively) were calculated in LabChart using peak detection analysis, with daily correction for barometric pressure. Respiratory flow and inspired and expired volumes were continuously measured via pneumotachometer (model HR 800 L, Hans Rudolph, Shawnee, KS). MAP, HR, SV, minute ventilation (V̇E), PETCO2 and PETO2 were sampled 𝛼1 ADRENO-REGULATION OF PERIPHERAL BLOOD FLOW IN HYPERCAPNIA 131 continuously throughout the protocol, at 1000 Hz via analogue-to-digital converter (PowerLab 16/30, AD Instruments, Colorado Springs, CO) and recorded in LabChart 7 software. Dynamic end-tidal forcing: In both studies, we controlled PETCO2 and PETO2 using a dynamic end-tidal forcing system described previously (Tymko et al., 2015, 2016). This approach allows for computerized breath-by-breath regulation of end-tidal gases, and therefore arterial blood gases, independent of V̇E. Notably, this method of arterial blood gas manipulation provides lesser arterial-to-end-tidal gradients of PETCO2 than other methods of blood gas manipulation (e.g., CO2 inhalation from a reservoir/Douglas bag), furthermore dynamic forcing precludes any influence of V̇E on PETCO2 and PETO2 (Howe et al., 2020), and therefore provides an accurate quantification of PaCO2 stimulus response relationships independent of ventilatory confounds (Fisher, 2016; Fisher et al., 2018). BA ultrasonography: In both studies, bilateral BA blood velocities and vessel diameters were simultaneously measured using two Duplex ultrasound devices (both 10-MHz multi-frequency linear array, Terason uSmart 3300, Teratech, Burlington, MA). Diameter and velocity in each BA were imaged at least 2 cm proximal to the brachial-radial bifurcation to avoid capturing the effects of turbulent flow and nonuniform shear. An insonation angle of 60° was maintained throughout each test. All recordings were made in accordance with published technical recommendations by the same qualified sonographers (Thijssen et al., 2011a). Continuous images were screen captured and stored as video files for offline analysis (see data analysis). Hematological measures: In the first study, venous blood samples were taken from an antecubital vein in the right arm into K2EDTA Vacutainers® (Becton Dickinson, USA), and we conducted analyses of whole blood viscosity and plasma NO biomarkers. Whole blood viscosity was measured within 30 minutes of blood acquisition in duplicate at 37ºC at shear rates of 11.25, 22.5, 45, 90, 225, 337.5 and 450 s-1 using a cone and plate viscometer (DV2T Viscometer, Brookfield Amtek, USA) and circulating heating water bath (TC-150, Brookfield Amtek, USA). The average of the final five seconds of 30 second steps were acquired at each shear rate. When repeated measures exceeded the viscometer’s error for a given shear rate (e.g. >0.1 mPa·s-1 at a shear 𝛼1 ADRENO-REGULATION OF PERIPHERAL BLOOD FLOW IN HYPERCAPNIA 132 rate of 225 s-1), a third viscosity measure was obtained and the average of the two closest measures is reported herein (Tremblay et al., 2020). For NO biomarker analyses in the first study, whole blood in K2EDTA Vacutainers® (Becton Dickinson, USA) was immediately centrifuged at 600 g for 10 minutes at a temperature of 4.0°C. Plasma was aliquoted into cryovials, flash frozen in liquid N2, and stored at -80°C. Samples were later batch analyzed to measure plasma NO2 using tri-iodide reductive ozone based chemiluminescence as previously described by our research group (Bailey et al., 2017; Hoiland et al., 2020d). Samples were rapidly thawed at 37°C for 3-minutes, at which time 450 uL of plasma was mixed with 50 uL of 5% acidified sulphanilamide (9:1 ratio) and left to incubate in the dark at 21°C for 15-minutes to remove NO2 before injection into the tri-iodide reagent for the measurement of s-nitrosothiols (RSNO). During the acidified sulphanilamide/plasma incubation period, two to four 50 uL plasma samples from the original cryovial were injected into the tri-iodide reagent for the combined measurement of NO2- and RSNO. These injections were separated by ≥2min to ensure recordings had reached a new stable baseline between each injection. The average of these two to four injections was calculated. Our coefficient of variation for these measures is <10%. Assessment of millivolt traces and calculation into concentrations (nM) were performed using Origin/Peak Analysis software, and were performed in duplicate by two researchers, our coefficient of variation between observers is <3%. Plasma NO2- was calculated by subtracting the concentration of RSNO from the total NO2-+RSNO value. All chemicals were of the highest purity available from MilliporeSigma. Data analyses: BA diameter, blood velocity, flow, and shear rate (SR) were analyzed with custom wall tracking/edge detection software (BloodFlow Analysis, version 5.1) (Woodman et al., 2001). Designed to mitigate investigator bias (Woodman et al., 2001; Black et al., 2008), the software calculates SR and flow thus: Shear rate (s-1) = 𝑝𝑒𝑎𝑘 𝑒𝑛𝑣𝑒𝑙𝑜𝑝𝑒 𝑏𝑙𝑜𝑜𝑑 𝑣𝑒𝑙𝑜𝑐𝑖𝑡𝑦𝑣𝑒𝑠𝑠𝑒𝑙 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 × 4 Flow (mL/min) = 𝑝𝑒𝑎𝑘 𝑒𝑛𝑣𝑒𝑙𝑜𝑝𝑒 𝑏𝑙𝑜𝑜𝑑 𝑣𝑒𝑙𝑜𝑐𝑖𝑡𝑦2 × [π(0.5 × diameter)2] × 60 Apparent viscosity was used to calculate antegrade, retrograde, and mean shear stresses at baseline and during the last minute of hypercapnia: Shear stress (dyn/cm2) = apparent viscosity (Pa) × SR (s-1) 𝛼1 ADRENO-REGULATION OF PERIPHERAL BLOOD FLOW IN HYPERCAPNIA 133 From ECG-assessed HR and Finometer Modelflow SV, CO was estimated: CO (L/min) = HR (beats/minute) × SV (ml)1000 and TPR was calculated: TPR (mm Hg/L/min) = MAP (mm Hg)CO (L/min) Vascular conductance (VC) in each BA was calculated: VC (mL/min/mm Hg) = BA flow (mL/min)MAP (mm Hg) All BA (i.e., diameter, shear rate, flow, VC) and cardiorespiratory (i.e., MAP, SV, HR, PETCO2, and PETO2) data were time aligned in Microsoft Excel (Microsoft Corporation, Redmond, WA, USA) and averaged in one-minute means for statistical comparisons (minutes baseline, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12). Sample sizes: Study 1: Venous samples were not collected in four participants at their request. In one participant, due to a change in catheter patency, their last minute sample was sufficient only for analysis of viscosity but not NO markers. Finally, in one participant, the baseline sample volume was sufficient only for plasma total NO analysis but not RSNO. Therefore, n=10 for viscosity and shear stress variables; n=9 for total NO; and n=8 for RSNO and NO2- at baseline. Study 2: One participant could not tolerate hypercapnia, their data were only used in baseline comparisons. One participant’s control arm BA image was lost in the final minute of hypercapnia, their data from all other minutes is included in all comparisons while their penultimate minute is taken as the last minute change from baseline comparisons. 6.3 Statistical analyses: Study 1: Linear mixed model analyses were used to compare: 1), all cardiorespiratory variables (i.e., MAP, HR, TPR, SV, CO, PETCO2, PETO2) across all minutes (baseline, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12); 2), all vascular (flow, diameter, VC, and shear rate) variables comparing baseline and minute 12 between 𝛼1 ADRENO-REGULATION OF PERIPHERAL BLOOD FLOW IN HYPERCAPNIA 134 arms; and 3), all vascular variables comparing all minutes between arms. Paired, two-tailed Student’s t-tests were used to compare; 1), all vascular variables comparing the last minute change values between arm; and 2), plasma NO markers (total NO2-, RSNO, NO2-) between pre and post hypercapnia. Study 2: Paired, two-tailed Student’s t-tests were used to compare baseline cardiorespiratory variables (MAP, HR, TPR, SV, CO, PETCO2, PETO2) between conditions (Prazosin vs. placebo). Linear mixed model analyses were used to compare: 1), baseline vascular variables (flow, diameter, VC, and shear rate) between arms and conditions; 2), all cardiorespiratory variables between conditions (Prazosin vs. placebo) and all minutes (baseline, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) ; 3), all vascular variables between arms (control vs. compression) and all minutes, for placebo and separately for Prazosin; and 4), last minute change values for diameter, flow, and VC, between arms and conditions. All linear mixed model analyses were conducted using compound symmetry co-variance and applying Bonferroni post-hoc corrections. For linear mixed model comparisons comparing condition (Prazosin vs. placebo), participants was also included as a random factor. All linear mixed model analyses were performed with the Statistical Package for the Social Sciences (SPSS, V24), all t-tests were conducted with Microsoft Excel (Microsoft Corporation, Redmond, WA, USA), and all figures were created using GraphPad Prism (V8.1.1). P< 0.05 was taken to indicate statistical significance. Sample size: Taking BA diameter changes (%±SD) from a study assessing BA dilation during handgrip exercise (where MAP and HR also increase) with arterial compression after 4.5 minutes (0.80±1.75%) (Pyke et al., 2008), and from a study assessing BA dilation during 12 minutes of hypercapnic stimuli with unimpeded BA shear stress (5.33±3.73%)(Geijer et al., 2016a), we calculated using G*Power that a minimum sample size of n=7 would be sufficient to attain power >0.8 and detect a difference between diameter changes in arms. Additionally, given that previous studies conducted by our group investigating shear-mediated diameter change in the peripheral arteries (Lewis et al., 2014a; Carr et al., 2020) have detected changes with relatively small sample sizes (e.g., n=11 to 19), we determined that a sample size of n=14 should more than suffice to detect changes in BA diameter and differences between arms for study one. 𝛼1 ADRENO-REGULATION OF PERIPHERAL BLOOD FLOW IN HYPERCAPNIA 135 6.4 Results: Study one: Cardiorespiratory variables: PETCO2 was elevated from baseline (minute; P<0.001) by +5.7±32 mm Hg in minute 1, then held steady at +10.2±0.3 mm Hg (mean of minutes 2-12). MAP was elevated (minute; P<0.001) and steady at +10±1 mm Hg (mean of minutes 2-12). HR was raised (minute; P<0.001) by +12±1 bpm (mean of minutes 2-12). SV was unchanged from baseline (minute; P=0.586), while CO was elevated (minute; P=0.001) at minute 2 (P=0.023) and remained so thereafter by +1.2±0.3 L/min (mean of minutes 2-12). There was no change in TPR during hypercapnia (P=0.059; see Table 6-1 for PETCO2, PETO2, MAP, HR, SV, CO, and TPR).𝛼1 ADRENO-REGULATION OF PERIPHERAL BLOOD FLOW IN HYPERCAPNIA 136 Table 6-1. Cardiorespiratory variables during baseline and hypercapnia in study one MINUTE Baseline 1 2 3 4 5 6 7 8 9 10 11 12 PETO2 (mm Hg) 89.9±5.4 91.1±5.7 89±6.3 89.5±5.9 89.9±5.7 89.8±5.8 90.0±5.7 89.8±5.8 89.9±5.6 89.9±5.7 90±5.6 90±5.8 89.7±5.7 PETCO2 (mm Hg) 39.1±2.6 44.9±4.2 50.1±2.7 49.7±2.8 49.4±2.7 49.2±2.7 49.1±2.6 49.2±2.7 49.1±2.6 49.1±2.7 49.1±2.7 49±2.6 49.1±2.7 V̇E (L/min) 13.9±5.2 45.3±13.6 39.1±18.4 45.3±21.8 47.1±25.5 48.2±24.4 49.2±26.5 47.9±26.7 48.6±27.4 49.1±24.5 48.2±24.0 48.1±24.3 48.5±24.1 HR (bpm) 72±14 76±18 81±20 83±20 84±19 85±19 84±18 85±20 85±20 85±18 85±18 85±17 85±16 SV (mL) 108±21 108±24 103±22 106±22 108±21 108±23 109±23 109±23 109±25 110±26 110±27 108±26 108±28 CO (L/min) 7.67±1.43 8.18±1.82 8.12±1.76 8.66±2.01 8.88±1.85 8.93±1.76 8.95±1.83 8.93±1.82 8.95±2.00 9.08±2.05 9.09±2.11 8.98±1.99 8.97±2.13 MAP (mm Hg) 92±8 99±15 103±13 103±13 102±11 101±10 102±10 102±10 101±10 102±10 101±9 101±9 101±10 TPR (mm Hg/L/min) 12±2 13±3 13±3 12±2 12±2 12±2 12±2 12±2 12±3 12±3 12±3 12±3 12±3 HR, heart rate. SV, stroke volume. CO, cardiac output. MAP, mean arterial pressure. TPR, total peripheral resistance. PETO2, partial pressure of end-tidal O2. PETCO2, partial pressure of end-tidal CO2. V̇E, minute ventilation. Bold text means P<0.05 vs baseline. 𝛼1 ADRENO-REGULATION OF PERIPHERAL BLOOD FLOW IN HYPERCAPNIA 137 Brachial artery baseline vs last minute, and across all minutes: Diameter, flow, VC, and mean shear stress were all greater in the control arm than the compression arm at both baseline and during the last minute of hypercapnia (main effect of arm, P<0.001, for all, Figure 6-1A, D, G, and J), while there were no differences between arms in response to hypercapnia for any variable (i.e., no main effects of minute or interaction effects). Likewise, across all minutes of hypercapnia, diameter, flow, VC, and shear rate were greater in control than in compression, but not changed across time (main effect of arm, P<0.001, for all, Figure 6-1B, E, F, and K). Control vs. compression arm – Last minute changes: Control and compression arms were not different in regards to last minute diameter change (%; P=0.201), flow (∆mL/min; P=0.234), VC (∆mL/min/mmHg; P=0.503), or shear stress (∆dyn/cm2; P=0.524; Figure 6-1C, F, I, and L). 𝛼1 ADRENO-REGULATION OF PERIPHERAL BLOOD FLOW IN HYPERCAPNIA 138 Figure 6-1. Study 4A - Brachial artery diameter, flow, conductance, and shear stress/rate. Mean and standard deviation data (black symbols and bars) and individual data (grey symbols) are presented for control (open symbols) and compression arms (closed symbols). Control and compression symbols are offset merely to aid visual differentiation. Baseline 1 2 3 4 5 6 7 8 9 10 11 12020406080MinuteFlow (ml/min)Arm P<0.001Minute P=0.999Interaction P=0.997Baseline 1 2 3 4 5 6 7 8 9 10 11 120.00.20.40.60.8MinuteVC (mL/min/mmHg)Arm P<0.001Minute P=0.981Interaction P=0.998ControlCompress-20020Last minute Δ flow (mL/min) P = 0.234ControlCompress-0.4-0.20.00.2Last minute Δ VC (mL/min/mmHg)P = 0.503Baseline 1 2 3 4 5 6 7 8 9 10 11 120100200300MinuteMean shear rate (1/s)Arm P<0.001Minute P=0.239Interaction P=0.348ControlCompress-4-202Last minute Δ shear stress (dyn/cm2)P = 0.524Baseline Last Minute0255075100Flow (ml/min)Arm P <0.001Minute P = 0.809Interaction P = 0.582Baseline Last Minute0.00.51.0VC (mL/min/mmHg)Arm P <0.001Minute P = 0.363Interaction P = 0.773Baseline Last Minute051015Shear stress (dyn/cm2)Arm P=0.017Minute P = 0.108Interaction P = 0.568Baseline Last Minute2345Diameter (mm)Arm P <0.001Minute P = 0.423Interaction P = 0.815Baseline 1 2 3 4 5 6 7 8 9 10 11 122.53.03.54.04.55.0MinuteDiameter (mm)Arm P<0.001Minute P=0.981Interaction P=1.0ControlCompress-50510Last minute Δ diameter (%) P = 0.201A B CD E FG H IJ K L𝛼1 ADRENO-REGULATION OF PERIPHERAL BLOOD FLOW IN HYPERCAPNIA 139 Viscosity and plasma NO: Viscosity (cP) was elevated from baseline in the last minute (Table 6-2). Antegrade shear stress (dyn/cm2) was lower in the compression arm (arm; P<0.001) and was further reduced in the last minute compared to baseline and control arm (interaction; P=0.009). Retrograde shear stress (dyn/cm2) was lower in compression than control (arm; P=0.026; Table 6-2). Plasma total NO, NO2-, and RSNO (nM) were unchanged from baseline (P=0.66, 0.62, and 0.94, Table 6-2). Table 6-2. Viscosity, shear stress, and plasma NO at baseline and last minute of hypercapnia in study one Where a main effect is reported, bolded terms indicate a main effect or interaction effect. * P<0.05 vs baseline within that arm, and † P< 0.05 Control vs. Compression within that minute Study two: Cardiorespiratory variables: PETCO2 was elevated from baseline (minute; P<0.001; Figure 6-2A) by +9.9±0.4 mm Hg during placebo, and +10.5±0.5 during Prazosin (mean of minutes 2-12). Hypercapnia elevated MAP in both conditions (minute; P<0.001), though to a lesser degree with Prazosin [+7±1 mm Hg during placebo vs Baseline Last Minute Apparent Viscosity (cP) Arm P=0.013, Minute P=0.014, Interaction P=0.997 Control 4.2±0.7 4.4±0.9 Compression 4.5±1.0 4.7±1.1 Antegrade Shear Stress (dyn/cm2) Arm P<0.001, Minute P=0.236, Interaction P=0.009 Control 6.4±1.5† 6.8±2.0† Compression 5.6±2.1 4.5±1.6* Retrograde Shear Stress (dyn/cm2) Arm P=0.026, Minute P=0.063, Interaction P=0.084 Control -1.9±1.1 -2.3±1.3 Compression -1.8±1.2 -1.7±0.9 Baseline Last Minute P values Plasma RSNO (nM) 2.5±1.0 2.5±1.1 0.77 Plasma NO2- (nM) 39.3±24.0 44.1±20.1 0.69 Plasma Total NO (nM) 43.8±24.8 46.8±21.1 0.66 𝛼1 ADRENO-REGULATION OF PERIPHERAL BLOOD FLOW IN HYPERCAPNIA 140 +5±1 mm Hg during Prazosin (mean of minutes 2-12), condition; P=0.004; Figure 6-2C]. HR was elevated from baseline (minute; P<0.001) and higher with Prazosin [+22±5 bpm vs +16±3 bpm (mean of minutes 2-12); condition; P<0.001; Figure 2D]. CO was progressively increased during hypercapnia (minute; P<0.001) and the change was greater with Prazosin (condition; P<0.001). Figure 6-2. Study 4A - Cardiorespiratory variables with Prazosin and placebo Mean and standard deviation data for placebo (black) and Prazosin (red). Asterisks used to indicate post-hoc effects for minute vs baseline only. Baseline 1 2 3 4 5 6 7 8 9 10 11 12354045505560Minute PETCO2 (mm Hg)Condition: P<0.001Minute: P<0.001Interaction: P=0.840*Baseline 1 2 3 4 5 6 7 8 9 10 11 12708090100110120MinuteMAP (mm Hg)Condition: P<0.001Minute: P=0.004Interaction: P=0.993*Baseline 1 2 3 4 5 6 7 8 9 10 11 124681012MinuteCO (L/min)Condition: P<0.001Minute: P<0.001Interaction: P=1.0 *Baseline 1 2 3 4 5 6 7 8 9 10 11 12406080100120MinuteHR (bpm)Condition: P<0.001Minute: P<0.001Interaction: P=0.642 *Baseline 1 2 3 4 5 6 7 8 9 10 11 1280100120140MinuteSV (mL)Condition: P<0.001Minute: P=0.989Interaction: P=0.988A BC DEBaseline 1 2 3 4 5 6 7 8 9 10 11 125101520MinuteTPR (mm Hg/L/min)Condition: P<0.001Minute: P<0.001Interaction: P=1.0*F𝛼1 ADRENO-REGULATION OF PERIPHERAL BLOOD FLOW IN HYPERCAPNIA 141 Brachial artery baseline: At baseline, flow, VC, and shear rate were greater in both arms during Prazosin vs placebo (condition: P<0.001), while baseline diameter was greater in control (right) arm vs compressed (left) arm (arm; P=0.030; Figure 6-3A, D, G, and J). During hypercapnia: During hypercapnia with placebo, there was no differences in diameter between arms (P=0.577) or minutes (P=0.453). While during hypercapnia with Prazosin, control arm diameter was greater than the compressed arm (P<0.001; Figure 6-3B) and there was a main effect of minute (P=0.042), although there was a main effect of minute no single minute was different to any other (all minute vs minute comparisons were P>0.113). During hypercapnia with Prazosin, flow, VC, and shear rate were all increased from baseline in the control arm (interaction effect P<0.001, for all) from minute 4 onwards (post hoc P<0.05 all; Figure 6-3E, H, and K). Last minute changes: Last minute diameter change was not different between control and compression arms (arm; P=0.196) nor between Prazosin and placebo (condition: P=0.543). Last minute flow, VC, and shear rate changes were both greatest in the control arm during Prazosin (interaction; P<0.001 all; Figure 6-3F, I, and L). 𝛼1 ADRENO-REGULATION OF PERIPHERAL BLOOD FLOW IN HYPERCAPNIA 142 Figure 6-3. Study 4A - Brachial artery diameter, flow, conductance, and shear rate with placebo and Prazosin. Mean and standard deviation data (red and blue symbols and bars) and individual data (grey symbols) are presented for control (open) and compression arms (closed), placebo (blue) and Prazosin (red). Symbols are offset merely to aid visual differentiation. Control Compress-8-40481216Last minute Δ diameter (%)Arm: P=0.196Condition: P=0.543Interaction: P=0.651Control Compress050100150Flow (ml/min)Arm: P=0.464Condition: P<0.001Interaction: P=0.649Control Compress2.53.03.54.04.55.0Diameter (mm)Arm: P=0.030Condition: P=0.057Interaction: P=0.164Control Compress0.00.51.01.52.0VC (mL/min/mmHg)Arm: P=0.441Condition: P<0.001Interaction: P=0.583Baseline 1 2 3 4 5 6 7 8 9 10 11 123.03.54.04.55.0MinuteDiameter (mm)PLACEBO:Arm: P=0.577Minute: P=0.453Interaction: P=0.804PRAZOSIN:Arm: P<0.001Minute: P=0.042Interaction: P=1.0Baseline 1 2 3 4 5 6 7 8 9 10 11 12050100150200250MinuteFlow (ml/min)PLACEBO:Arm: P<0.001Minute: P=0.983Interaction: P=0.750PRAZOSIN:Arm: P<0.001Minute: P=0.001Interaction: P<0.001Baseline 1 2 3 4 5 6 7 8 9 10 11 120123MinuteVC (mL/min/mmHg)PLACEBO:Arm: P<0.001Minute: P=0.981Interaction: P=0.858PRAZOSIN:Arm: P<0.001Minute: P=0.007Interaction: P<0.001Baseline Last minuteControl Compress-100-50050100150Last minute Δ flow (mL/min)Arm: P<0.001Condition: P=0.023Interaction: P<0.001Control Compress-1012Last minute Δ VC (mL/min/mmHg)Arm: P<0.001Condition: P=0.219Interaction: P<0.001Prazosin - ControlPlacebo - ControlPlacebo - Compressed Prazosin - CompressedA B CD E FG H IJ LBaseline 1 2 3 4 5 6 7 8 9 10 11 120200400600MinuteMean shear rate (1/s)PLACEBO:Arm: P<0.001Minute: P=0.407Interaction: P=0.820PRAZOSIN:Arm: P<0.001Minute: P=0.012Interaction: P<0.001Control Compress0200400600Shear rate (s-1)Arm: P=0.680Condition: P<0.001Interaction: P=0.689Control Compress-2000200Last minute Δ shear rate (s-1)Arm: P<0.001Condition: P=0.071Interaction: P<0.001K𝛼1 ADRENO-REGULATION OF PERIPHERAL BLOOD FLOW IN HYPERCAPNIA 143 6.5 Discussion: The present studies sought to determine the shear stress and α₁-adrenergic contributions to peripheral conduit artery responses during hypercapnia. The main findings herein are that: 1) hypercapnia does not elicit shear-mediated dilation in the BA; 2) blood flow to the distal tissues during hypercapnia is typically sympathetically-restrained, and 3) even in the absence of this sympatho-mediated tone, BA diameter remains unresponsive to the increased shear rates produced by elevated blood flow. This confirms (Richardson et al., 1960; Kontos et al., 1967) and extends (Geijer et al., 2016a, 2016b) previous findings regarding peripheral vascular responses to hypercapnia, by using direct vascular imaging, within-individuals comparisons, and accounting for shear rate. Altogether, we provide comprehensive assessment of the integration of effects from hypercapnia, shear stress, and sympathetic tone in the BA to show that hypercapnia-induced vasodilation is not shear-mediated. In the following, we outline our findings and discuss putative mechanisms which may contribute to vasomotor control in the peripheral circulation during hypercapnia. In the first study, hypercapnia had no effect on any vascular variable (Figure 6-1). Thus, whatever distal or proximal responses (i.e., flow and diameter, respectively) occurred during hypercapnia, our shear-reduction intervention did not alter the effect in the compressed arm compared to the control arm. Since there was no change in blood flow, VC or diameter in either arm, both constrictor mechanisms (sympathetically-mediated) and dilator mechanisms [non-shear-mediated, other hypercapnia-dependent pathways (e.g., Bergfeld & Forrester, 1992; Ellsworth et al., 1995)] were likely active, resulting in a net status quo. These findings indicate that changes in BA diameter(mild as it is) during hypercapnia are not shear-mediated. Similarly, in the placebo trial of study two, diameter was unaffected by hypercapnia (Figure 6-3B). However, during the Prazosin trial, diameter was mildly greater than baseline by the end of the test (Figure 6-3B), suggesting some sympathetically-mediated restraint of hypercapnic BA vasodilation. Furthermore, despite greater flow, VC, and shear rate in the Prazosin control arm (Figure 6-3F, I, and L) during hypercapnia compared to the Prazosin compressed arm and both placebo arms, the last minute diameter change was not different between either arm or condition (Figure 6-3C). These findings indicate that BA responses during hypercapnia are not shear-mediated, and in fact even when shear rate is vastly increased (through the release of sympathetic restraint) the BA response remains nonetheless unaffected. That the Prazosin control arm did not dilate despite increased shear rate likely indicates that the threshold for shear-mediated dilation was not reached (Wray et al., 2011; Tremblay et al., 2019b). 𝛼1 ADRENO-REGULATION OF PERIPHERAL BLOOD FLOW IN HYPERCAPNIA 144 Hypercapnia increases muscle sympathetic nervous activity (Somers et al., 1989; Shoemaker et al., 2002; Steinback et al., 2009) and circulating catecholamines (Sechzer et al., 1960; Metz, 1969; Davidson et al., 1986; Brofman et al., 1990), which contribute to increased MAP through increases in vascular resistance (Ullian, 1999; Ainslie et al., 2005b) and cardiac output (Davidson et al., 1986; Balanos et al., 2003; Willie et al., 2012). Although a systemic net vasoconstriction could be expected with hypercapnia, the extent of this active constriction varies regionally (Schaefer & Baker III, 1969; Ainslie et al., 2005b; Vantanajal et al., 2007). The clear increase in BA flow during hypercapnia with Prazosin indicates a distal tissue (skeletal muscle and cutaneous) CO2 sensitivity (that is usually restrained by SNA tone), which is supported by animal (Daugherty et al., 1967) and some human (Ito et al., 1989; Sakai et al., 2011; Ogoh et al., 2018) studies. Nevertheless, the current findings confirm the established dominance of sympathetic vascular tone in peripheral arteries (Barcroft et al., 1943) and further suggest that the upper limb peripheral vasculature prioritizes MAP regulation even over distal tissue acid-base balance (given the influence of PCO2 on pH) despite clear CO2 sensitivity. Hypercapnic SNA-mediated increases in vascular resistance in the splanchnic circulation (Gollwitzer-Meier & Bohn, 1930; Mohamed & Bean, 1951; Epstein et al., 1961, 1966; Schaefer & Baker III, 1969; Elam et al., 1981; Davidson et al., 1986; Chapman et al., 2020) likely occurred in the present study and contributed to the increased MAP. So, a degree of hemo-concentration due to hypercapnic SNA-mediated splenic contraction may have contributed to increased blood viscosity (+2.9±2.7 % from baseline at a shear rate of 225 s-1 P=0.008) (Ramlo & Brown Jr, 1959; Lodin-Sundström & Schagatay, 2010; Richardson et al., 2012). The earlier investigations into peripheral CO2-mediated responses (Mcardle et al., 1957; Mcardle & Roddie, 1958; Richardson et al., 1960; Kontos et al., 1967) used venous occlusion plethysmography. This method provides very short duration blood flow measurement and allows for local arterial infusion responses without systemic interference. However, venous occlusion plethysmography lacks fine temporal resolution, while with duplex ultrasound we obtained continuous, beat-by-beat blood velocity and diameter measurements and avoided inducing any metabo-reflex that may have occurred with venous occlusion during hypercapnia. Furthermore, venous occlusion plethysmography precludes discrimination of the contribution of vascular diameter vs blood velocity to increases in flow. These factors confound the combined interpretation with contemporary studies, wherein direct vascular imaging using duplex ultrasound found BA dilation during longer systemic hypercapnic stimuli (e.g., Geijer et al., 2016a, 2016b). Investigating the underlying integration of sympathetic excitation, MAP regulation, and peripheral tissue acid-base balance during hypercapnia is potentially clinically relevant in, for example, patients using 𝛼-blockers for hypertension and with co-morbid pulmonary diseases, or in those with autonomic 𝛼1 ADRENO-REGULATION OF PERIPHERAL BLOOD FLOW IN HYPERCAPNIA 145 dysfunction. While permissive hypercapnia may be a common component of protective lung ventilatory strategies in clinical care (O’Croinin et al., 2005), the use of such ventilator management should be cautioned with added attention to the potential implications for MAP management in those taking 𝛼-blockers, since the inability to vasoconstrict may result in exaggerated peripheral blood flow responses to elevated PaCO2. Furthermore, in those with pulmonary pathology where PaCO2 can be significantly raised, or during sleep (where PaCO2 can often rise by several mm Hg), the use of 𝛼-blockers may likewise allow for uncontrolled blood flow distribution and further MAP challenges. 6.6 Experimental considerations: While we collected venous blood samples in study one, we did not for study two. Measurement of blood viscosity for the calculation of shear stress, along with measurement of hematocrit pre and post hypercapnia, would have allowed for a more complete evaluation of BA hypercapnic responses. However, given that the inclusion of shear stress - as opposed to shear rate – in study one did not change the interpretation, our findings have likely not been altered by the lack of viscosity measurement in study two. There was greater variation of responses within the female cohort than in the male cohort in the first study; e.g., control arm peak diameter change (%) standard deviation: ±4.98 vs ±1.32 in females and males, respectively. The inclusion and comparison of both male and females (as well as differences arising from sexual transition) is integral to comprehensive portrayal of human physiology (Lindsey et al., 2021). As such, we endeavoured to include both males and females in our studies. Despite controlling for stage of menstrual cycle we cannot, however, explain the apparent variation between sexes here since we collected no variables relevant to sex-differences by which we might discern underlying cause, e.g., circulating estrogens, androgens, or core temperature (Krause et al., 2006; Boese et al., 2017; Pabbidi et al., 2018; Williams et al., 2020). The female cohort in the Prazosin study was too few (n=3) to allow for even speculative comparisons between sexes but should be considered in future studies. In study one and the Prazosin arm of study two, BA diameter in the right arm was greater than in the left in all participants; we suggest this may represent the effects of side dominance, however we did not survey participants. Nonetheless, this difference did not impact our or interpretation. There was some between-individual variability in the control and compressed BA responses to CO2, however given that our study utilized a bi-lateral within-individual design, and our statistical model accounted for individuals as a random factor in all linear mixed models, this did not influence our findings. 𝛼1 ADRENO-REGULATION OF PERIPHERAL BLOOD FLOW IN HYPERCAPNIA 146 The use of a single α1-adrenergic blockade without concomitant α2 or β-receptor blockade potentially allowed for upregulation of α2-mediated vasoconstriction (Kiowski et al., 1983) or β-mediated vasodilation (Gomes et al., 1978; Oates & Stoker, 1980). While this may have influenced our findings, the within-subjects design accounts for this. Further, while diameter was greater with Prazosin, we did not see greater vasodilation during hypercapnia, which would have occurred if there was upregulated stimulation of β receptors by hypercapnia-mediated adrenaline output. Finally, although distal to the BA, the radial artery has been shown to be α1 dominated with little β-receptor function (He & Yang, 1998), and this is also likely the case in the BA. 6.7 Conclusion: The current data demonstrate that sustained hypercapnia does not result in vasodilation in the brachial artery, and reducing shear stress does not alter this response. In addition, after administrating an α₁-adrenergic blockade, although hypercapnia resulted in increased BA blood flow, and thus shear stress, this did not translate into changes in arterial diameter. The findings from the presented series of experiments suggest that upper limb peripheral vasculature prioritizes MAP regulation even over distal tissue acid-base balance. 𝛼1 ADRENO-REGULATION OF CEREBRAL BLOOD FLOW IN HYPERCAPNIA 147 Chapter 7: Study 4B – Adrenergic nervous activity in cerebral endothelium-dependent function and reactivity to hypercapnia Key points: • We assessed steady-state hypercapnic cerebrovascular reactivity and cerebral endothelium-dependent function, with and without 𝛼1 adrenergic blockade, in a placebo-controlled, double-blind, and randomized study, in order to assess the contribution of 𝛼1 adrenergic tone to cerebrovascular CO2 regulation. • During Prazosin, cerebrovascular reactivity was not different compared to placebo, while cerebral endothelium-dependent function was reduced due to elevated arterial diameter. • These findings suggest that 𝛼1 adrenoreceptors do not influence the cerebral vascular blood flow response to CO2, but may constrain ICA diameter at rest. Abstract: We assessed hypercapnic cerebrovascular reactivity (CVR) and endothelium-dependent function (cerebral flow-mediated dilation; cFMD) in the internal carotid artery (ICA) with and without systemic 𝛼1 adrenoreceptor blockade (Prazosin). We hypothesized that CVR would be diminished while cFMD would be unchanged during Prazosin compared to placebo. In 15 healthy adults (3 female, 26±4 years) we conducted ICA duplex ultrasound during CVR [target +10mm Hg partial pressure of end-tidal carbon dioxide (PETCO2) above baseline, 5 minutes] and cFMD (+9mm Hg PETCO2 above baseline, 30 seconds) using dynamic end-tidal forcing with and without 𝛼1 adrenergic blockade (Prazosin; 0.05 mg/kg) in a placebo-controlled, double-blind, and randomized design. The CVR in the ICA was not different between placebo and Prazosin (P=0.578). During CVR, the reactivities of mean arterial pressure and cerebrovascular conductance to hypercapnia were also not different between conditions (P=0.921, and P=0.664, respectively). During Prazosin, cFMD was lower (1.1±2.0 % vs 3.8±3.0 %; P=0.032) due to elevated (+0.1±0.2 mm) diameter throughout (condition: P=0.050) and lower (-75±85 s-1) shear rate throughout (condition: P<0.001). We interpret these findings to indicate that the reduction in cFMD is merely the result of greater ICA diameter and not necessarily indicative of any change in endothelium function per se. As such, these findings suggest that 𝛼1 adrenoreceptors do not influence the cerebral vascular blood flow response to CO2, but may constrain ICA diameter at rest. 𝛼1 ADRENO-REGULATION OF CEREBRAL BLOOD FLOW IN HYPERCAPNIA 148 7.1 Introduction: The cerebral vascular tree is richly innervated by both extrinsic and intrinsic nerves (Gezalian et al., 2021; Koep et al., 2022), specifically, large arteries contain a higher 𝛼 receptor density while the α/β ratio tends towards greater β density with more distal vessel branches (Reznikoff et al., 1986). As such, the majority of α1-adrenergic effects on vascular smooth muscle in the cerebral vasculature consist of mild -adrenergic restraint in large extracranial arteries (e.g., Hamner et al., 2010; Fernandes et al., 2016). Following ganglionic blockade or excision in pathology (i.e., complete block of all sympathetic outflow), basal cerebral blood flow (CBF) has been shown to be elevated (Shenkin et al., 1951; Shenkin, 1969; Umeyama et al., 1995; Jeng et al., 1999), though other studies have found no change (Scheinberg, 1950; Ohta et al., 1990). However, the role of α1-adrenergic activity in CBF reactivity to CO2 (CVR) during elevated arterial partial pressure of CO2 (PaCO2) remains controversial (Heistad & Marcus, 1978; Brassard et al., 2017). Hypercapnia causes an increase in CBF through both an increase in MAP and pial vessel myogenic control [reviewed in: (Claassen et al., 2021)]. Inhibition of α-adrenergic signalling (Brassard et al., 2017; Koep et al., 2022) may abolish the CO2-mediated MAP increase (Przybyłowski et al., 2003; Ainslie et al., 2012; Peebles et al., 2012) and/or due to the hypotension-induced alteration of the CBF vs CO2 relationship (Harper & Glass, 1965; Artru & Colley, 1984). Peebles et al. (2012) found that α1-adrenergic blockade with prazosin mildly reduced the middle cerebral artery blood velocity (MCAv) responses to hypercapnia and hypocapnia, though CVR was also further reduced in a time control trial. However, previous investigations in humans addressing CVR with and without α, β, or combined adrenergic blockade have not used measures of volumetric flow with concurrent clamping of the PCO2 stimulus. The former is important due to the evidence that hypercapnia dilates the MCA (Coverdale et al., 2015) and the latter is relevant for optimum control of the CVR stimulus (Duffin, 2011; Fierstra et al., 2013; Fisher, 2016). Additionally, CVR to CO2 is often equated, at least in part, to cerebrovascular endothelial function (Lavallée et al., 2006; Lavi et al., 2006; Ainslie et al., 2007; Iwamoto et al., 2018a, 2020; Suzuki et al., 2020); however, given several confounding factors that hypercapnia induces, more recent evidence is to the contrary (Carr & Ainslie, 2020; Carr et al., 2020; Hoiland et al., 2021). For example, hypercapnia results in transient hypertension (Willie et al., 2012; Regan et al., 2014; Iwamoto et al., 2018a, 2020; Carr et al., 2020), which likely influences vascular tone (via myogenic mechanisms), and therefore, masks the assessment of cerebral endothelial function. As such, the ICA diameter change following short shear stress stimuli may better represent cerebral endothelial function. Our group has developed a technique that may better isolate endothelium-dependent vascular function in the ICA (Hoiland et al., 2017, 2021; Carr et al., 2020), that assesses extracranial cerebral flow-mediated dilation (cFMD). While 𝛼1 ADRENO-REGULATION OF CEREBRAL BLOOD FLOW IN HYPERCAPNIA 149 sympathetic nerve activity (SNA) stimulation using lower body negative pressure attenuates ICA dilation in response to 3-minutes of steady-state hypercapnia (Iwamoto et al., 2018b), and SNA stimulation via cold pressor elicits ICA constriction (Tymko et al., 2017), the effect of a reduction in adrenergic vascular tone on cFMD specifically has not been investigated. The purpose of this investigation therefore, was to determine cFMD and volumetric ICA CVR, while manipulating the influence of 𝛼1-adrenergic innervation. We conducted a placebo-controlled, double-blind, randomized, and counterbalanced study with 𝛼1-adrenergic receptor blockade (Prazosin). Previous work using transcranial Doppler (TCD)-based metrics of CVR in healthy subjects with α −adrenergic (Peebles et al., 2012), combined α+β − adrenergic (Ainslie et al., 2012), and ganglionic blockade (Przybyłowski et al., 2003) has shown that CVR was subsequently reduced secondary due to the hypotension; therefore, we hypothesized that hypercapnic CVR would be reduced in the absence of the typical systemic α-adrenergic SNA-mediated MAP increase. We also hypothesized that cFMD would not be different with and without Prazosin, given that the technique is predicated on eliciting transient increases in cerebral shear stress and blood flow without concurrently effecting MAP through elevations in SNA. 7.2 Methods: We conducted a placebo-controlled, double-blind, and randomized investigation of ICA responses to hypercapnia with and without 𝛼1-adrenergic receptor blockade (Prazosin). The University of British Columbia Clinical Research Ethics Board approved this study (H19-02355), which conformed to the Declaration of Helsinki (apart from database registry). Participants were informed of risks and written informed consent was given prior to any experimental sessions. Participant characteristics: 15 healthy adults (3 female, 26±4 years, 73±11 kg, 176±8 cm) volunteered for and participated in this study. Exclusion criteria included the presence or history of cardiovascular, musculoskeletal, or metabolic disease, present smokers or a history of smoking, use of medication (except oral contraceptives), or BMI >30 kg/m2. We attempted to standardize for menstrual phase by only assessing females during the first seven days of the follicular phase in normally menstruating women (MacNutt et al., 2012) (n=1) or during the placebo week in those taking oral contraceptives (Minson et al., 2000) (n=1) when estradiol is lowest; n=1 participant used an intra-uterine device. 𝛼1 ADRENO-REGULATION OF CEREBRAL BLOOD FLOW IN HYPERCAPNIA 150 Experimental measures: Cardiorespiratory variables: Beat-by-beat reconstructed brachial artery blood pressure was assessed via non-invasive finger photoplethysmography (Finometer PRO, Finapres Medical Systems, Amsterdam, Netherlands). Absolute correction of Finometer pressure values was based on the average of triplicate blood pressure measurements from the same arm using an automated sphygmomanometer (EM-759-E; Omron, Kyoto, Japan). HR was assessed by lead-II configuration electrocardiogram R-R interval. The fraction of inspired and expired CO2 and O2 were continuously sampled at the mouth by gas analyzer (model ML206, ADInstruments). The partial pressures of O2 and CO2 (PETO2 and PETCO2, respectively) were calculated in LabChart using peak and trough detection analysis and atmospheric pressure. Respiratory flow and inspired and expired volumes were continuously measured via pneumotachometer (model HR 800 L, Hans Rudolph, Shawnee, KS). MAP, HR, PETCO2 and PETO2 were sampled continuously throughout at 1000 Hz via analogue-to-digital converter (PowerLab 16/30, AD Instruments, Colorado Springs, CO) and recorded in LabChart 7 software. Dynamic end-tidal forcing: We controlled PETCO2 and PETO2 using a dynamic end-tidal forcing system described previously (Tymko et al., 2015, 2016). This approach allows for computerized breath-by-breath regulation of end-tidal gases, and therefore arterial gases, independent of V̇E. Furthermore, dynamic forcing precludes the influence of V̇E on PETCO2 and PETO2 (Howe et al., 2020) and, therefore, provides an accurate quantification of PCO2 stimulus independent of other confounds (Fisher, 2016; Fisher et al., 2018). Ultrasonography: For both tests, right ICA blood velocity and vessel diameter was measured using Duplex ultrasound (10-MHz multi-frequency linear array, Terason uSmart 3300, Teratech, Burlington, MA). The ICA was imaged at least 2 cm distal to the carotid bifurcation to avoid turbulent flow and nonuniform shear. An insonation angle of 60° was maintained throughout each test and no ultrasound settings were adjusted between tests. All recordings were made in accordance with published technical recommendations (Thomas et al., 2015). Continuous images were screen captured and stored as video files for offline analysis (see data analysis). 𝛼1 ADRENO-REGULATION OF CEREBRAL BLOOD FLOW IN HYPERCAPNIA 151 Transcranial Doppler (TCD): Blood velocities in the right middle cerebral artery (MCAv) and left posterior cerebral artery (PCAv) were measured using transcranial ultrasound (2 MHz, TCD, Spencer Technologies, Seattle, WA). Optimal M-mode imaging was achieved using a headframe (M600, Spencer Technologies) allowing precise alignment of insonation angle and minimal movement artifact. Insonation followed guidelines established previously (Willie et al., 2011). Experimental protocol: Participants arrived to the laboratory after a ≥6-hour fast, and having avoided alcohol and vigorous physical activity for 24 hours prior and caffeine for 12-hours prior (Thijssen et al., 2019). Upon arrival, participant’s body mass was weighed and height measured. Participants then ingested either the 𝛼1 adrenoreceptor blockade (Teva-Prazosin, Teva Canada Limited, Ontario, Canada; ~0.05 mg/kg body mass) in a gelatin capsule or placebo (~0.3 g sugar) in an identical gelatin capsule. This dose of Prazosin has been shown to pharmacokinetically peak in plasma concentration between ~90 and ~180 minutes after ingestion (Hobbs et al., 1978; Bateman et al., 1979). Testing then began 90 minutes after ingestion. Participant’s laboratory visits were scheduled 48 hours apart and began at the same time of day in each participant (either 9 a.m. or 12 p.m.). Eight participants underwent the Prazosin visit first, while seven participants completed the placebo visit first. The order of conditions for each participant was randomized using https://www.randomizer.org/. 90 minutes after drug/placebo administration, five minutes of baseline cardiorespiratory data was collected. Participants then began breathing on the dynamic end-tidal forcing system with PETCO2 and PETO2 maintained at baseline values. All assessments were completed with participants resting in the supine position. Cerebral (ICA) flow-mediated dilation (cFMD): This test has been described in detail elsewhere (Hoiland et al., 2017), briefly, dynamic end-tidal forcing is used to rapidly elevate PETCO2 to 9 mm Hg above participant baseline for 30 seconds. This initiates a transient rise in ICA flow, which is followed over the course of the following ~30-90 seconds by a peak in ICA diameter. This technique has been shown to partially reflect (~37%) endothelium-dependent nitric oxide mediated ICA responses (Hoiland et al., 2021). 𝛼1 ADRENO-REGULATION OF CEREBRAL BLOOD FLOW IN HYPERCAPNIA 152 CVR: Following one minute baseline collection (baseline) PETCO2 was elevated to +10 mm Hg above individual baseline and PETO2 was clamped at baseline; these values were maintained for 5 minutes from the onset of hypercapnia. Data analyses: ICA diameter and blood velocity were analyzed with custom wall tracking/edge detection software (BloodFlow Analysis, version 5.1) (Woodman et al., 2001) designed to mitigate investigator bias (Woodman et al., 2001; Black et al., 2008), the software calculates SR and flow thus: Shear rate (s-1) = 𝑝𝑒𝑎𝑘 𝑒𝑛𝑣𝑒𝑙𝑜𝑝𝑒 𝑏𝑙𝑜𝑜𝑑 𝑣𝑒𝑙𝑜𝑐𝑖𝑡𝑦𝑣𝑒𝑠𝑠𝑒𝑙 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 × 4 Flow (mL/min) = 𝑝𝑒𝑎𝑘 𝑒𝑛𝑣𝑒𝑙𝑜𝑝𝑒 𝑏𝑙𝑜𝑜𝑑 𝑣𝑒𝑙𝑜𝑐𝑖𝑡𝑦2 × [π(0.5 × diameter)2] × 60 cFMD: cFMD is assessed using General Purpose Data Graphing Software (version 1) which smooths the data with a low frequency filter, calculates median baseline diameter and shear rate during the 120 seconds of isocapnic hyperventilation prior to elevation of PETCO2, and subsequently detects thresholds and peaks. This output is then assessed by two independent investigators to ensure that the detected thresholds precede the detected peaks. Of the 30 total cFMD tests during Placebo and Prazosin, 10 files failed the above criteria; specifically, six files were of insufficient image clarity. However, in the remaining four files, despite distinct image clarity, appropriate artery position, and reliable wall detection, peak diameter was nevertheless detected prior to peak shear rate. In these participants, the transient stimulus nonetheless elicited a typical ICA shear rate response comparable with the other participants. This indicates that the change in ICA diameter post-stimulus was no greater than the baseline variation in diameter despite successful elicitation of a transient shear stimulus. Given the satisfactory quality of these scans and transient tests, these four cFMD files are included herein. However, as diameter change in these files could not appropriately be assessed using the method described, all satisfactory quality cFMD files (24 of 30) were instead analyzed thusly: 1. Bloodflow Analysis data was exported at 1Hz and was used to calculate mean baseline diameter, 2. General Purpose Data Graphing Software data was used to attain the time of peak shear rate, 𝛼1 ADRENO-REGULATION OF CEREBRAL BLOOD FLOW IN HYPERCAPNIA 153 3. Diameter during post-hypercapnia was attained as a 60s mean of the second-by-second Bloodflow Analysis data from 30s after each individual’s peak shear rate, as it is within this time that ICA diameter typically peaks, 4. Shear rate area under the curve (SRAUC) was attained from the second-by-second shear rate using the trapezoidal rule, from the onset of CO2 until the beginning of each individual’s diameter average. For comparison with the above manual analysis method, cFMD files that were analyzed using the smoothing analyses were also compared, excluding the 10 files that were of insufficient quality or where smoothing did not detect peak diameters post CO2. Cerebrovascular reactivity to carbon dioxide: All ICA (i.e., diameter, shear rate, flow, CVC) and cardiorespiratory (i.e., MAP, HR, PETCO2, and PETO2) data during CVR were extracted from the blood flow analysis software in one-minute means (baseline and minute four). From the change from baseline to minute four, CVR was calculated thus: Absolute CVR =  ICA flow(mL/min)PETCO2 (mm Hg) Relative CVR= % ICA flow(mL/min)PETCO2 (mm Hg) In order to account for differences in MAP between conditions, we calculated cerebrovascular conductance (CVC), or an index of CVC (CVCi): CVC (mL/min/mm Hg) = 𝑄ICA flow (ml/min)MAP (mm Hg) CVCi (cm/s/mm Hg) = MCAv (cm/s) or PCAv (cm/s)MAP (mm Hg) CVR was also calculated using MCAv, PCAv, ICA CVC, and MAP. Data exclusion: One participant’s ventilatory response to prolonged hypercapnia was such that the CVR test was terminated within ~2 minutes per participant discomfort, and movement artifact confounded ICA analyses in three ICA scans, as such these data are included in baseline comparisons and cFMD (which was tolerated by all participants). Furthermore, logistical issues precluded ICA assessment during CVR in two participants. So, for all ICA variables n=11 at baseline and n=9 in the last minute of CVR. MCAv 𝛼1 ADRENO-REGULATION OF CEREBRAL BLOOD FLOW IN HYPERCAPNIA 154 signal was lost during CVR in three subjects during Prazosin, while PCAv was lost during CVR in one subject during both conditions. As such, during CVR, MCAv n=14 during placebo and 11 during Prazosin; and PCAv CVR n= 13 in both conditions. Six cFMD files were excluded due to either overt angle changes or unclear wall detection. As such, for cFMD analyses n=13 for placebo and n=11 for Prazosin. For paired, two-tailed t tests, only participants where both conditions were available were used for comparisons. 7.3 Statistical analyses: Paired, two-tailed Student’s t-tests were used to compare baseline (5-minute mean) cardiorespiratory variables between conditions (placebo vs. Prazosin). Both hypercapnic tests (cFMD and CVR) have unique baseline periods immediately prior to respective CO2 stimulus, to ensure continuity between baseline and response measures. For cFMD analyses, paired, two-tailed Student’s t-tests were used to compare: SRAUC, MCAv peak responses (%), and ICA diameter responses (%) (participants in whom cFMD data in only one condition was available were excluded from these paired comparisons). Linear mixed model analyses were used to compare shear rate between condition and baseline vs response, and absolute diameter between condition and baseline vs response. We also used linear mixed model analyses to compare the difference between the natural logs of baseline and response diameters with baseline diameter as covariate, and again with baseline diameter and SRAUC as covariates (Atkinson & Batterham, 2013; Atkinson et al., 2013). For CVR analyses, paired, two-tailed Student’s t-tests were used to compare: absolute CVR between conditions (placebo vs. Prazosin), and relative CVR between conditions. Linear mixed model analyses were used to compare: all cardiorespiratory variables between condition and minute (baseline vs minute 4); and all vascular variables (ICA flow, diameter, CVC, and shear rate, and MCAv, MCAv CVC PCAv, PCAv CVC) between condition and minute. Linear mixed model analysis of ICA flow with MAP included as a covariate was also conducted as a second means of assessing the impact of MAP. We tested normality using Shapiro-Wilks Test, baseline PETCO2, MAP, HR, ICA flow, MCAv, PCAv were all normally distributed. All linear mixed model analyses were conducted using compound symmetry co-variance, and applying Bonferroni post-hoc corrections. Participant was included as a random factor. All linear mixed model analyses (and the linear regressions between baseline and peak diameter) were performed with the Statistical Package for the Social Sciences (SPSS, V24), all t tests were conducted with Microsoft Excel (Microsoft Corporation, Redmond, WA, USA), figures were created using GraphPad Prism (V8.1.1). P< 0.05 was taken to indicate statistical significance. 𝛼1 ADRENO-REGULATION OF CEREBRAL BLOOD FLOW IN HYPERCAPNIA 155 7.4 Results: Cardiorespiratory variables at baseline prior to testing: Following supine rest and prior to cFMD and CVR, MAP was lower with Prazosin (P=0.013), while HR and CO were elevated (P=0.002, and P=0.047, respectively), and PETCO2 was -1±1.7 mm Hg lower (P=0.046) following Prazosin (Table 7-1). Table 7-1. Baseline cardiorespiratory variables Placebo Prazosin P-value PETO2 (mmHg) 88.3±4.3 87.8±4.5 0.658 PETCO2 (mmHg) 39.1±1.5 38.1±2.2 0.046 MAP (mmHg) 92±7 82±12 0.013 HR (bpm) 57±9 65±13 0.002 V̇E (l/min) 15.1±2.7 13±4.9 0.181 CO (l/min) 6.2±1.3 6.9±1.7 0.047 MCAv (cm/s) 63.5±11.5 61.2±15.8 0.391 PCAv (cm/s) 41.3±14.0 41.2±10.3 0.594 5-min mean baseline data after 45 minutes of supine rest (90 minutes following administration of placebo or prazosin). Compared with paired, two-tailed Student’s t tests. PETO2, partial pressure of O2; PETCO2, partial pressure of CO2; MAP, mean arterial pressure; HR, heart rate; V̇E, minute ventilation; CO, cardiac output. cFMD: By design, during cFMD PETCO2 was rapidly (~3 breaths) elevated from baseline by +9.6±2.1 mm Hg during placebo, and +8.4±2.3 mm Hg during Prazosin (P=0.16, paired t-test between conditions; Figure 7-1A and B, Table 7-2). While MAP was lower throughout cFMD during Prazosin (condition P<0.001), there was no change in MAP during either condition following transient hypercapnia (minute P=0.134). HR was higher with Prazosin (condition P<0.001), and mildly elevated with hypercapnia in both conditions (minute P=0.046). See Table 7-2 for cardiorespiratory variables during cFMD. 𝛼1 ADRENO-REGULATION OF CEREBRAL BLOOD FLOW IN HYPERCAPNIA 156 Table 7-2. cFMD variables cFMD Placebo Prazosin Baseline During Baseline During Condition P=0.709; Minute P=0.500; Interaction P=0.914 PETO2 (mmHg) 87.2±5.2 88.0±5.6 87.3±5.5 87.9±5.4 Condition P<0.001; Minute P<0.001; Interaction P=0.195 PETCO2 (mmHg) 40.6±1.5 (Peak) 51.4±1.9 39.4±2.2 (Peak) 47.9±2.5 Condition P<0.001; Minute P=0.134; Interaction P=0.725 MAP (mmHg) 96±9 100±13 86±9 89±9 Condition P<0.001; Minute P=0.046; Interaction P=0.799 HR (bpm) 67±13 72±12 73±16 78±17 Cardiorespiratory variables during baseline and. During cFMD, compared using linear mixed model analyses. PETO2, partial pressure of end-tidal O2; PETCO2, partial pressure of end-tidal CO2; MAP, mean arterial pressure; HR, heart rate. The percent change from baseline for diameter and shear rate during cFMD with placebo and Prazosin are depicted in Figure 7-1A and B, respectively. Following the transient CO2 stimulus, response diameters were greater than baseline diameters (minute; P=0.006) and overall, ICA diameter throughout Prazosin cFMD was greater than during placebo (condition; P=0.04986; Figure 7-1C). During cFMD, shear rate increased from 371.7±74.3 at baseline to a peak of 493.4±99.2 1/s during placebo, and from 308.0±93.9 to 370.0±89.7 during Prazosin (minute P<0.001; condition P<0.001, interaction P=0.065; Figure 7-1D). The MCAv peak response (%) was lower during Prazosin (P=0.022; Figure 7-1E). Calculated SRAUC was 31584±9836 1/s during placebo and 28973±12206 1/s during Prazosin (P=0.297). The percent change in ICA diameter during placebo was greater than that during Prazosin (3.8±3.0 % vs 1.1±2.0%, respectively; P=0.032: Figure 7-1F). The cFMD difference between conditions was reduced after accounting for baseline diameter and SRAUC (Figure 7-1G). For comparison with the manual analyses described in the methods section, cFMD analyzed using only the smoothing process (n=6) were compared; Prazosin cFMD (4.1±1.2 %) was not different compared to placebo cFMD (5.5±2.9; P=0.231). 𝛼1 ADRENO-REGULATION OF CEREBRAL BLOOD FLOW IN HYPERCAPNIA 157 Figure 7-1. Study 4B - cFMD A and B, diameter and shear rate changes (%) from baseline (individual raw data averaged in 5 s bins, % change from baseline calculated, mean+SD presented) during placebo (A) and Prazosin (B), respectively, with PETCO2 inlays for both. C, diameter at baseline and 60s average during cFMD response (30s after each individuals’ peak shear rate time). D, shear rate at baseline and at smoothing software detected peak. E, MCAv peak responses (%) at smoothing software detected peak. F, diameter change from baseline (%) using 60s average during cFMD response. G and H, back calculated estimated marginal means (EMM) and standard deviations produced using linear mixed model analyses with covariates for log(baseline) (G), and log(baseline) and SRAUC (H). Data in C, D, E, and F are individual (grey) and means (filled circles and triangles). Data in C and D were compared using linear mixed model analyses. Data in E and F were compared using two-tailed, paired Student’s t-tests. 𝛼1 ADRENO-REGULATION OF CEREBRAL BLOOD FLOW IN HYPERCAPNIA 158 CVR: Cardiorespiratory variables: At baseline and throughout the CVR test, PETCO2 and MAP were lower with Prazosin than with placebo (Table 7-3) and HR was higher with Prazosin (Table 7-3), while PETO2, were not different between conditions (Table 7-3). During the CVR test, PETCO2 was elevated from baseline (minute; P<0.001; Table 7-3) by +10.0±0.7 mm Hg during placebo, and +10.3±1.1 during Prazosin. Hypercapnia elevated MAP in both conditions (minute; P<0.001), by 8±7 mm Hg during Prazosin and 8±5 mm Hg during placebo. Table 7-3. Cardiorespiratory variables during CVR CVR Placebo Prazosin Baseline During Baseline During Condition P=0.431; Minute P=0.568; Interaction P=0.957 PETO2 (mmHg) 86.1±5.0 86.3±4.6 86.6±5.3 87.4±5.7 Condition P<0.001; Minute P<0.001; Interaction P=0.682 PETCO2 (mmHg) 40.6±1.6 50.7±1.5 39.3±2.1 49.7±2.4 Condition P<0.001; Minute P<0.001; Interaction P=0.899 MAP (mmHg) 95±6 103±9 87±10 94±11 Condition P<0.001; Minute P<0.001; Interaction P=0.269 HR (bpm) 61±13 75±14 70±15 86±22 Cardiorespiratory variables during pre-CVR baseline (1 min) and during (minute 4) CVR, compared using linear mixed model analyses. PETO2, partial pressure of end-tidal O2; PETCO2, partial pressure of end-tidal CO2; MAP, mean arterial pressure; HR, heart rate. CVR: Neither absolute nor relative reactivity in the ICA were different between placebo and Prazosin trials (P=0.578, P=0.743, respectively). Reactivity of ICA CVC was also not different (P=0.664). However, relative reactivity of MCAv (P=0.015) and PCAv (P=0.017) was greater with Prazosin than with placebo. MAP reactivity was also unchanged with Prazosin (P=0.921; Figure 7-2 depicts all CVR comparisons). 𝛼1 ADRENO-REGULATION OF CEREBRAL BLOOD FLOW IN HYPERCAPNIA 159 Figure 7-2. Study 4B - Cerebrovascular reactivity to CO2 Individual (grey) and mean (black and red) data from various reactivity during placebo and Prazosin. Comparisons conducted using paired two-tailed t-tests. Cohen’s d effect sizes also presented. ICA, internal carotid artery; MCAv, middle cerebral artery blood velocity; PCAv, posterior cerebral artery blood velocity; MAP, mean arterial pressure. ICA and TCD variables: During Prazosin, ICA flow and shear rate were lower than during Placebo (condition; P=0.012, and P=0.008, respectively) and were increased with hypercapnia in both conditions (minute; P<0.001 both). Diameter and CVC were not different between Prazosin and placebo (condition; P=0.888 and P=0.629, respectively). See Table 7-4 for ICA variables during CVR. The differences in flow were abolished after inclusion of MAP as covariate (minute; P<0.001; condition P=0.527; interaction P=0.894, MAP P=0.004). Neither MCAv, PCAv, MCAv CVCi, nor PCAv CVCi were different between conditions (condition: P=0.530, P=0.160, P=0.300, and P=0.324, respectively; data not shown). PlaceboPrazosin051015ICA flow reactivity ((%mL/min)/mmHg)P=0.743d=0.11PlaceboPrazosin0246MCAv reactivity ((%cm/min)/mmHg)P=0.015d=0.88PlaceboPrazosin02468PCAv reactivity ((%cm/min)/mmHg)P=0.017d=0.81PlaceboPrazosin0510152025ICA flow reactivity (mL/min/mmHg)P=0.578d=0.19PlaceboPrazosin0.00.10.20.3ICA conductance reactivity (mL/min/mmHg)/mmHgP=0.664d=0.15PlaceboPrazosin012345MCAv reactivity ((cm/min)/mmHg)P=0.171d=0.44PlaceboPrazosin0123PCAv reactivity ((%cm/min)/mmHg)P=0.395d=0.26PlaceboPrazosin012MAP (mmHg/mmHg)P=0.921d=0.03A B C DE F G H𝛼1 ADRENO-REGULATION OF CEREBRAL BLOOD FLOW IN HYPERCAPNIA 160 Table 7-4. ICA variables during CVR CVR Placebo Prazosin Baseline During Baseline During Condition P=0.888; Minute P=0.020; Interaction P=0.777 Diameter (mm) 5.05±0.41 5.09±0.32 5.08±0.42 5.22±0.35 Condition P=0.012; Minute P<0.001; Interaction P=0.725 Flow (mL/min) 275.9±64.6 408.7±53.8 240.7±59.2 380.5±76.7 Condition P=0.008; Minute P<0.001; Interaction P=0.607 Shear rate (1/s) 359±55 530±91 313±81 455±101 Condition P=0.629; Minute P<0.001; Interaction P=0.758 CVC (mL/min/mm Hg) 2.9±0.7 3.9±0.6 2.8±0.7 4.0±0.6 ICA variables during pre-CVR baseline (1 min) and during (minute 4) CVR, compared using linear mixed model analyses. CVC, cerebrovascular conductance. 7.5 Discussion: We hypothesized that hypercapnic CVR would be reduced, and that cFMD would be unchanged, in the presence of a systemic α1-receptor blockade. The main findings of this study are that, contrary to our hypotheses, CVR to CO2 was not different between conditions and cFMD was reduced during Prazosin, though this reduction was abolished after accounting for changes in arterial diameter. While the transient CO2 stimulus of the cFMD test was comparable in magnitude between conditions (+9.6±2.1 mm Hg during placebo vs +8.4±2.3 mm Hg during Prazosin; P=0.16), ICA diameter was greater and shear rate was lower during Prazosin at rest and during the test (Figure 7-1C and D). As such, the change in ICA diameter was attenuated by 34% (Cohen’s d = 0.86) in the Prazosin condition compared with placebo. When accounting for baseline diameter using scaling methods suggested by Atkinson & Batterham, (2013) and Atkinson et al., (2013), this difference appears to remain that is, covariation with baseline diameter only ostensibly removes it (estimated marginal means of 3.4±1.8 vs 2.0±1.8; P=0.07). We interpret these findings to indicate that the reduction in cFMD is merely the result of greater ICA diameter and not necessarily indicative of any change in endothelium function per se. Given the greater diameter at rest and therefore lower shear rate, the subsequent increase in shear rate was insufficient to cause further dilation. Previous studies have reported no change in ICA diameter at baseline during Prazosin (Lewis et al., 2014; Fernandes et al., 2016; though Lewis et al. 2014 included background hypoxia). However, 𝛼-receptor density is greatest in the larger cerebral arteries (Forbes & Wolff, 1928; Talbott et al., 1929; Chorobski & Penfield, 1932; Cobb & Finesinger, 1932; Pool et al., 1934; Forbes et al., 1937; James et 𝛼1 ADRENO-REGULATION OF CEREBRAL BLOOD FLOW IN HYPERCAPNIA 161 al., 1969; Reznikoff et al., 1986) with greater extrinsic innervation (i.e., the same as the systemic vasculature rather than from nerves intrinsic to the brain) (Duverger et al., 1987). Additionally, numerous animal studies have reported carotid (common and external) vasoconstriction with 𝛼-adrenergic stimulation and the blockage of these effects with antagonists such as Prazosin (e.g., (Willems et al., 2001, 2003). Therefore, there is support for the contribution of 𝛼-restraint to basal ICA tone. To the best of our knowledge, this study is the first to demonstrate the effect of such systemic removal of ICA 𝛼1-adrenergic restraint in humans. Much work has considered the role of SNA in maintaining tonic vascular tone in the cerebral circulation, though the influences of sympathetic activity per se are challenging to discern in healthy humans [reviewed in: (Ainslie & Brassard, 2014; Brassard et al., 2017)] given the unavoidable integration of CBF with MAP, PaCO2, PaO2, CaO2, and neuronal metabolism. These confounds are notoriously problematic - though not impossible - to circumvent with study design. For example, the present study clamped PETO2 at baseline and eliminated the influence of V̇E on PETCO2 by the use of dynamic end-tidal forcing (Howe et al., 2020). But since 𝛼-adrenergic blockade leads to relative hypotension via removal of normal tonic vascular restraint, the MAP reduction caused by systemic 𝛼1-adrenergic blockade nevertheless remains. As relative hypotension reduces the CBF vs CO2 relationship (Harper & Glass, 1965; Artru & Colley, 1984), we may have expected that CVR would be attenuated in the current study, however, the MAP response to hypercapnia was also unchanged. Given the pivotal role of MAP elevation in CBF responses during CVR (Przybyłowski et al., 2003; Ainslie et al., 2012; Peebles et al., 2012), this comparable MAP increase therefore explains the unchanged CVR. The maintenance of the MAP response may have occurred via upregulation of vascular smooth muscle cell 𝛼2-receptor stimulation during 𝛼1-inhibition (Kiowski et al., 1983; Kanagy, 2005). We attempted to mathematically account for MAP with treatment of CVC, CVCi, and by including MAP as a covariate in the analysis of flow. That CVC, CVCi and MAP-corrected-flow were not different between conditions indicates that flow for a given pressure was unchanged with Prazosin. Other investigations have noted the role of MAP changes in the contribution to CBF responses during hypercapnia (Przybyłowski et al., 2003; Battisti-Charbonney et al., 2011; Willie et al., 2012), particularly during adrenergic (Ainslie et al., 2012) and ganglionic (Przybyłowski et al., 2003) blockade. Since MAP reactivity was not changed herein, any change in CVR would therefore have been representative of an 𝛼-adrenergic role in the typical CBF response to CO2 stimulus. The lack of CVR change therefore indicates that 𝛼1 adrenergic restraint plays little function in the CO2 cerebrovascular response. While cerebral endothelium-dependent function is unchanged during 𝛼1-adrenergic blockade, prolonged reductions in mean shear rate result in impairments in endothelial function in the brachial artery (Tremblay et al., 2019). As such, should similar mechanisms be present in the ICA, the lower shear rate and subsequently reduced requirement for vasodilation may eventually result in 𝛼1 ADRENO-REGULATION OF CEREBRAL BLOOD FLOW IN HYPERCAPNIA 162 downregulation of shear-mediated mechanisms in the ICA in the setting of prolonged 𝛼-adrenergic blocker use. This reduction in the cerebral shear-mediated vasodilator response may have clinical implications for the co-morbid development of, for instance, carotid atheroma in those with hypertension who use MAP reducing medication. 7.6 Experimental considerations: Prazosin and placebo trials were conducted on separate days and without baseline/control comparisons. As such, some degree of the variability in the current findings may arise from between-day differences. An alternative design using pre and post Prazosin testing would have reduced the between day variability, however, previous investigations have noted decrements in CVR following extended periods of rest (Peebles et al., 2012). As such, our protocol benefits from avoiding this issue by only testing participants during both conditions at the same time of day and after the same duration of supine rest (45-minutes). Rather than assessing a transient peak diameter, taking a predetermined time window average of diameter can result in an underestimation of the true FMD (Black et al., 2008) due to averaging reducing the measured diameter. We admit this is a limitation of the data analysis conducted herein compared to true peak detection methods; however, this method was necessitated by the lack of detectable peak diameter in four subjects following cFMD. We aimed to compensate for this by taking the mean diameter window at a time starting 30s after the individual peak shear rate, corresponding to the expected mean time from peak shear to peak diameter (Carr et al., 2020). Taking an average for 30s either side of a peak may have resulted in a reduced cFMD value in some subjects, however, this only means we may have underestimated the true diameter change, and this method allowed for the inclusion of all appropriate quality scans. We also compared cFMD data extracted using only the smoothing process from scans where a peak diameter was detected following the peak shear rate (n=6). Prazosin cFMD (4.1±1.2 %) was not different compared to placebo cFMD (5.5±2.9; P=0.231), which differs from the current main findings, however this comparison is likely far underpowered to detect any changes in cFMD. Due to the combinations of participant intolerance for hypercapnia and insufficient scan quality in some participants, the eventual sample sizes in some comparisons are small, as such some comparisons may be underpowered. Since a posteriori power analyses provide little constructive information in the context of null findings (Levine & Ensom, 2001; Zhang et al., 2019), we calculated Cohen’s d effect sizes which were all small to moderate. The only strong effect sizes were for the relative reactivities of MCAv and PCAv, however, this is merely a mathematical phenomenon due to the absolute change in MCAv and PCAv being similar between conditions while arising from a lower baseline during Prazosin. 𝛼1 ADRENO-REGULATION OF CEREBRAL BLOOD FLOW IN HYPERCAPNIA 163 7.7 Conclusion: The role of 𝛼1-adrenergic receptors in cerebral blood flow control in the extracranial vasculature is minimal, however, removal from 𝛼1 adrenergic restraint resulted in greater internal carotid artery diameter at rest. Given this minor influence, cerebrovascular reactivity to carbon dioxide and cerebral flow-mediated dilation are likely unaffected during 𝛼1-adrenergic receptor inhibition unless mean arterial pressure or internal carotid artery diameter, respectively, are also influenced. DISCUSSION 164 Chapter 8: Discussion The overarching aim of this dissertation was to examine the relationships between endothelium-dependent and -independent vascular function in the cerebral vasculature utilizing hypercapnic challenges 8.1 Main findings: The main findings of the studies above are: Study 1: Internal carotid artery and brachial artery shear-dependent vasodilator function in young healthy humans • Endothelium-dependent responses in ICA are not correlated with endothelium-dependent responses in the BA, nor with CVR. • Assessment of large extracranial cerebral vascular health should be quantified via methods specific to the vessel, not via peripheral endothelial function or CVR. Study 2: Stability of cerebrovascular CO2 reactivity following attainment of physiologic steady-state • In the short-term, once a steady-state PETCO2 is achieved via end-tidal forcing, and provided other relevant variables (e.g., MAP) remain steady, CBF should be stable, and so cerebrovascular CO2 reactivity values extracted from different time points (e.g., 2, 3, 4, or 5-min) should not be different regardless of data extraction length (30s vs 60s). Study 3: O2 and CO2 transport in isovolumic anemia: compensation of cerebral delivery of O2 and maintenance of cerebrovascular reactivity to CO2 • Acute anemia in healthy humans elevates basal CBF which serves to maintain cerebral O2 delivery and elevates cerebral CO2 washout. • Total CO2 content in the cerebral effluent (jugular venous) blood is unaffected by acute hypercapnia, both at rest and during acute anemia. • Findings indicate that acute anemia per se does not impair cerebrovascular function. Study 4A: Brachial artery responses to acute hypercapnia: the roles of shear stress and adrenergic tone • Hypercapnia does not elicit vasodilation in the brachial artery. Even when adrenergic tone is removed, the blood flow response during elevations in CO2 is still insufficient to cause anything more than very mild shear-mediated vasodilation in the brachial artery. DISCUSSION 165 • The upper limb peripheral vasculature prioritizes MAP regulation even over distal tissue acid-base balance. Study 4B: Adrenergic nervous activity in cerebral endothelium-dependent function and reactivity to hypercapnia • Neither cerebrovascular reactivity to CO2 nor cerebral endothelium-dependent function were different in the absence of systemic adrenergic vascular constraint. • Removal from 𝛼-restraint results in greater ICA diameter at rest as it does in arteries elsewhere. 8.2 Contributions to the field: The findings of this dissertation advance the understanding of the integration and regional heterogeneity of vascular endothelium-dependent and -independent responses to arterial CO2 and shear stress in the cerebral and peripheral vasculatures. Specifically, studies have utilized both peripheral FMD and CVR as a means of assessing cerebral endothelium function (Ainslie et al., 2007; Lavi et al., 2006); however, Study 1 demonstrated that peripheral FMD and cerebral endothelium-dependent function (i.e., cFMD) are not correlated; and, likewise, CVR and cFMD are also not correlated. Therefore, while BA FMD and CVR certainly inform both basic science and clinical research, their use is specific to the areas and mechanisms they interrogate, namely peripheral conduit artery shear-mediated vasodilation and cerebral microvascular CO2 sensitivity-mediated vasodilation. As an aside, we acknowledge the associated coronary function indices and cardiovascular health outcomes to which BA FMD is correlated (Anderson et al., 1995; Inaba et al., 2010; Broxterman et al., 2019); and hereby reaffirm that BA FMD should not be used to infer cerebral vascular function (at least in the setting of otherwise healthy volunteers). Study 2 aimed to resolve the stability of the cerebral microvascular CO2 sensitivity responses in light of a publication (Burley et al., 2020) which could have been interpreted as showing physiological instability in CVR, due to not acknowledging the variability inherent in the techniques used (i.e., Douglas bag/use of fixed FiCO2 and repeated testing; Fisher, 2016; Fisher et al., 2018). Study 2 utilized methods that allowed for true stability of the CO2 stimulus, obviating the influence of confounding ventilation. We demonstrated that, indeed, in healthy young humans, CBF is stable if the CO2 stimulus is also stable. Study 3 was the first– to our knowledge – to assess CVR in healthy humans during acute experimental anaemia by haemodilution. Addressing the interaction of both CaO2 and CO2 in CBF regulation, this study added to the extensive literature on healthy CVR by demonstrating that the cerebral vasculature DISCUSSION 166 is able to concurrently compensate for mild reductions (~18 %) in CaO2 and elevations (+9 mm Hg) in CO2. Furthermore, this study utilized the calculation of CO2 content - a previously common (e.g., (van Slyke, 1921; Kety & Schmidt, 1948b; Douglas et al., 1988) but lately either forgotten or overlooked method – to demonstrate the commensurately maintained cerebral CO2 washout. Washout of CO2 from the brain is often cited as the purpose of CVR (Mithoefer & Kazemi, 1963; Severinghaus & Lassen, 1967; Poulin & Robbins, 1998; Peebles et al., 2007, 2012; Duffin, 2007; Ogoh et al., 2008, 2013, 2019; Ainslie & Duffin, 2009; Ogoh & Ainslie, 2009; Fan et al., 2010, 2011; Sato et al., 2012; Skow et al., 2013; Burgess et al., 2014; Hoiland et al., 2018, 2015; Boulet et al., 2016; MacKay et al., 2016; Wilson & Teppema, 2016; Pfoh et al., 2016; Hawkins et al., 2017; Howe et al., 2020; Wenzel et al., 2020; Cleary et al., 2020; Williams et al., 2021; Carr et al., 2021a), and while this can be inferred from jugular venous to arterial PCO2 differences, this study was the first to show that jugular venous CO2 content specifically is maintained even during acute elevations in PaCO2. Thereby, these findings provide direct evidence for the efficacy of CVR in its janitorial role as the agent of CO2 washout. Stduy 4A consisted of two studies originally intended to assess brachial artery responses during systemic hypercapnia, based on publications that showed vasodilation (Geijer et al., 2016a, 2016b) but failed to account for shear stress and, therefore, could not explain the response. The data produced by the first part of Study 4A (bilateral brachial assessment during hypercapnia with shear rate control) showed an absence of significant brachial vasodilation. Given that hypercapnia elevates SNA (Somers et al., 1989; Shoemaker et al., 2002; Steinback et al., 2009), upon completion of the first arm of Study 4A, the clear next step was to repeat the protocol and remove the influence of the adrenergic vascular restrain that likely underpinned the findings. The second part of Study 4A (bilateral brachial assessment during hypercapnia with shear rate control and 𝛼1-adrenergic blockade) demonstrated that indeed it was such 𝛼1-adrenergic restraint that masked peripheral tissue CO2 sensitivity. The second trial of Study 4A provided the opportunity to concomitantly assess ICA flow and diameter with and without 𝛼1-adrenergic blockade (i.e., Study 4B); these data permitted a mechanistic extension of studies 1 and 2, allowing insight into the influence of systemic SNA on cFMD and CVR. Though similar studies have been previously published (Peebles et al., 2012), none have assessed cFMD, and none have utilized volumetric flow measures of CBF nor the use of dynamic end-tidal forcing. Furthermore, the incorporation of cFMD into the study protocol provides the first insight into the unmasking of cerebral endothelium-dependent function from 𝛼1-adrenergic restraint. After administration of Prazosin, cerebrovascular reactivity to CO2 was not different compared to placebo despite lower blood flow, while cerebral endothelium-dependent function was reduced, likely due to elevated baseline internal carotid arterial diameter. These findings suggest that 𝛼1-adrenoreceptor activity does not influence cerebral blood flow regulation to CO2 and cerebral endothelial function. DISCUSSION 167 These novel findings indicate the further capacity of cFMD and CVR to differentiate CBF regulatory mechanisms. 8.3 Integrated summary: Cumulatively, the outlined studies in this dissertation represent an attempt to discriminate vascular responses regulated by local factors (i.e., cFMD in the cerebral, and FMD in the peripheral) and responses regulated by distal or systemic factors (i.e., CVR in the cerebral, and MAP regulation in the peripheral). Combining the interpretations of the above studies shows that in the cerebral vasculature acid-base balance and shear stress are integral to both distal and proximal responses, respectively, while adrenergic activation is implicated only at rest. And conversely, in the peripheral vasculature sympathetic activation dominates while shear stress plays a tonic role and tissue acid-base balance is relegated to the least significance. Specifically, the cerebral vascular distal response to hypercapnia triumphs over adrenergic restraint in the proximal vessels. This observation contrasts the peripheral vasculature, where the distal vascular response to hypercapnia is constrained by adrenergic restraint, normally a tonic presence but also elevated with hypercapnia. While local vascular regulation (i.e. endothelium-dependent function) demonstrates similar patterns of response to shear stress stimuli between the peripheral and cerebral, the magnitudes of these responses differ as the result of differences in systemic acid-base balance, tissue metabolism, and underlying sympathetic nervous activity, amongst others. Fundamentally, end-organ perfusion requirements determine flow - i.e., the brain determines flow responses through the ICA and skeletal and cutaneous tissues determine flow through the BA - while the combination of local and systemic factors govern conduit vessel diameter. 8.4 Strengths of the dissertation: 8.4.1 Cerebral endothelium-dependent function: Measurement of endothelium-dependent function (vascular responses to stimuli that are primarily mediated via a healthily functioning endothelium) in the cerebral vasculature remains somewhat lacking in the field in general - however, using a technique to assess shear-mediated vasodilation in the ICA developed by our group (Hoiland et al., 2017) which appears to be partly eNOS-dependent (Hoiland et al., 2021) – Studies 1 and 4B show that such assessment is possible. This test has implications for possible clinical applications, for example, endothelium-dependent function in the large cerebral arteries may hold predictive power for carotid atherosclerosis or atheroma related ischemic stroke; though much work in healthy humans is required before this becomes a reality (e.g., see Future directions). DISCUSSION 168 8.4.2 Adrenergic restraint of peripheral vascular response to hypercapnia: The series of studies described in Study 4A represent a logical progression of the investigation of the likely most salient factors determining vasodilation in a peripheral conduit artery during hypercapnia. Previous work showing peripheral vascular responses to hypercapnia include seminal works (e.g., Mcardle et al., 1957; Mcardle & Roddie, 1958; Richardson et al., 1960; Kontos et al., 1967) which nonetheless could not discern whether the responses were enacted in the micro or macro-vasculature. Additionally, other studies with seemingly sound study designs (Geijer et al., 2016a, 2016b) were unable to discern the mechanisms responsible. The placebo-controlled and double-blinded study design used for both trials of Study 4A allowed for the comprehensive assessment of brachial hypercapnic responses accounting for shear stress and adrenergic activation. 8.5 Weaknesses of the dissertation/methodological considerations: While dynamic end-tidal forcing allows for rapid and precise control of end-tidal gases with the assumption of minimal end-tidal to arterial gradients, it is known that this technique likely does induce a small end-tidal to arterial CO2 gradient at higher hypercapnia (Tymko et al., 2016). As such, while we use PETCO2 as an index of PaCO2, there is a chance for slight overestimation of the true arterial CO2 stimulus in the hypercapnic challenges. However, even if a ~2 mm Hg end-tidal to arterial CO2 gradient occurred in some participants, the common target of +9-10 mm Hg PETCO2 used in the studies above would nevertheless have produced a ~7-8 mm Hg increase in PaCO2, which is still a robust hypercapnic challenge. Duplex ultrasound provides a means of arterial imaging with excellent temporal resolution and volumetric measures of blood flow, especially pertinent for investigations of the cerebral vasculature. However, this technique is not without pitfalls. It is particularly operator dependent, ideally requiring hundreds of hours of practice and training for technical proficiency. Additionally, the calculation of global CBF from ICA and VA is not only predicated on bi-lateral similarity, but also on the premise that the ICA and VA supply only the cerebral tissues and also that the brain is not supplied by any other vessels. There are in fact several intra-extra cerebral anastomoses (Geibprasert et al., 2009), however, the ICA and VA represent the greatest blood suppliers while the collateral supply is generally negligible in healthy individuals (Zarrinkoob et al., 2015). Finally, Duplex ultrasound is useful for measuring bulk blood flow, however, local changes in flow and tissue metabolism are lost when using this modality, as such other technologies such as magnetic resonance imagining are more appropriate when regional differences are the focus; especially in settings of cerebrovascular pathology where vascular steal and other microvascular nuances are prominent (Duffin et al., 2017; Fisher et al., 2018). DISCUSSION 169 The magnitude of hypercapnia used in Study 3, although greater than any typical PaCO2 elevations within normal circumstances in healthy humans, was perhaps not sufficient to push cerebrovascular reserve to its limits, indicated by the subsequent elevation in CBF at +9 mm Hg PaCO2 during post-hemodilution. Typically, vasodilation in the cerebral vasculature is necessarily limited by cranial volume and intracranial pressure restrictions; as such, had the combination of haemodilution and the CO2 stimulus been sufficiently exhaustive, then perhaps CVR would have been reduced post haemodilution simply due to these limits. Should cerebrovascular reserve have been exhausted, this alone may still not have indicated true CVR mechanism impairment, simply physical limits reached. Since the goal of the study was to assess the mechanisms of CVR during haemodilution, exhausting cerebrovascular reserve may have confounded such interpretation. Furthermore, our volumetric measurements of CBF (Duplex ultrasound) cannot capture intra-regional differences in OEF and CDO2 which may be exacerbated with anemia (Shen et al., 2022), and, as mentioned above, may be better assessed using magnetic resonance imaging modalities. The design of Study 4B (i.e., between day) is the result of the protocol of Study 4A. Between day comparisons for Study 4A were appropriate given the within-individuals bi-lateral design; however, this meant there was no within-day control for the ICA assessments in Study 4A. As such, some of the variation in the responses between conditions (placebo and Prazosin) may have arisen due to natural between-day variability. Alternatively, pre and post Prazosin testing may have reduced the between day variability, however, previous investigations have noted decrements in CVR following extended periods of rest (Peebles et al., 2012), potentially due to some effect of diurnal variation (testing at 9a.m. vs 12p.m.). As such, our protocol benefits from avoiding this issue by testing participants during both conditions at the same time of day and after the same duration of supine rest (45-minutes). 8.6 Future directions: Over the course the studies described above, several other project directions naturally arose, such that each new study design seemed to be the best subsequent option. As such, a number of study designs and protocols were developed and some even given University of British Columbia’s Clinical Research Ethics Board approval, though unfortunately we were unable to conduct them largely due to COVID-19 restrictions. For instance, during data collection for Study 1, an interest in discerning further similarities or differences between the cerebral and peripheral vasculatures lead to speculating that the cerebral vasculature would respond to prolonged shear stress exposure in a similar manner to the peripheral vasculature. From this arose a study design investigating the influence of short term continuous and intermittent hypercapnia on shear-stress-mediated dilation in the cerebral circulation. This study received ethical approval from the University of British Columbia’s Clinical Research Ethics Board (CREB #H19-02595) and initial data collection began in one participant, but collection was DISCUSSION 170 stopped with the onset of COVID lockdown in 2020. See Appendix B.1 for full details. The prospect of studying the cerebral effects of extended exposures to shear stress (using hypercapnia) naturally resulted in further reading and interest in hypercapnia and CO2 per se. After reading a number of inspiring studies from the 20th century on chronic CO2 exposure (e.g., (Consolazio et al., 1947; Schaefer, 1963, 1971, 1979; Gude & Schaefer, 1969), the notion of studying extended hypercapnia while using modern methods spurring the development of an “environmental” focussed study to assess the effects of a range of extended changes in CO2 on cerebral vascular function (see Appendix B.2). Concurrently, though travel was impossible with COVID restrictions, opportunities for future international collaboration stimulated the development of a number of research studies variously addressing cerebral endothelial function in different populations; trained breath-hold divers (see Appendix B.3), in recent stroke patients (see Appendix B.4); and in those with carotid artery atheroma (see appendix B.5) to assess the utility of cFMD to predict stroke risk. Meanwhile, the complex equipment and extensive operator skill needed to conduct cFMD tests precluded the options for location and ease of use of the test; as such, a study design was developed to evaluate other methods of eliciting a cFMD stimulus (see Appendix B.6) This study received ethical approval from the University of British Columbia’s Clinical Research Ethics Board (CREB #H21-01516), though, alas, the study was postponed in favour of conducting Study 4A. Finally, while conducting Study 4A, interest in the relationships between CVR and V̇E-CO2 progressed and a study design to further our understanding of the within-individuals interactions emerged. Specifically, the purpose of this study is to determine the influence of graded step changes in CVR on V̇E-CO2; using pharmacological inhibition of CVR with indomethacin (a cyclooxygenase inhibitor), and (theoretically) augmentation of CVR with sildenafil (a selective phosphodiesterase type 5 inhibitor), we hypothesize that V̇E-CO2 will vary inversely with CVR. While similar studies have been published on the effects of reductions in CVR and seen no relationship with V̇E-CO2 (Fan et al., 2010; Hoiland et al., 2015), these papers have utilized between-individuals correlations on the change values for CVR and V̇E-CO2 when within-individuals repeated measures correlations (Bakdash & Marusich, 2017) are more appropriate, and more importantly, none has assessed CVR augmentation, and so cannot truly speak to within-individual relationships. This study received ethical approval from the University of British Columbia’s Clinical Research Ethics Board (CREB #H21-01516; see Appendix B.7), however, the study was also postponed in favour of Study 4A and also pending amendments to the study design (it was subsequently realised that a Duffin rebreathe test was indispensable yet absent and should be included). It is hoped that these studies will be conducted in the next year or so. DISCUSSION 171 Chapter 9: Conclusion Hypercapnia is a powerful tool for interrogation of some aspects of cerebral vascular function, however it is not particularly useful in the peripheral vasculature where other factors (i.e., shear stress, SNA drive for MAP regulation) prevail. 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APPENDICES - EQUATIONS 229 Appendices Appendix A A.1 Equations: O2 content: CaO2 = 1.36 x [SaO2 / 100] x [Hb] + 0.003 x PaO2 CvO2 = 1.36 x [SvO2 / 100] x [Hb] + 0.003 x PvO2 Where 1.36 is the binding capacity of Hb for O2, arterial O2saturation (%), [Hb] is the concentration of hemoglobin in the blood (g/dL), 0.003 is the solubility of O2 in the blood (ml O2/dl plasma/mm Hg) and PaO2 is the arterial partial pressure of O2 (mm Hg). CO2 content: Plasma content of CO2: PCCO2 = 2.226 ∙ s ∙ PCO2 ∙ (1 + 10pH – pK`) Where s is the solubility coefficient of CO2 and pK` is the apparent pK [both calculated per Kelman (1967), with the assumption that core temperature was 37.5°C and unchanged with hemodilution]. PCCO2 can be calculated for both arterial (PCaCO2) and jugular venous (PCjvCO2) blood using arterial pH, PCO2, and pK` for PCaCO2 and jugular venous pH, PCO2, and pK` for PCjvCO2. Then, CCO2: CCO2 = PCCO2 ∙ (1 – 0.0289 ∙ [Hb]) (3.352 – 0.456 x SO2) ∙ (8.142 – pH) Fick equations: Q = VO2/Cao2-CvO2 VcO2= CvCO2-CaCO2 x Q VO2= CaO2-CvOO2 x Q APPENDICES - EQUATIONS 230 gCBF: Global CBF (gCBF) [acknowledging potential limitations (Friend et al., 2021)]: gCBF (mL/min) = 2 × (ICA flow + VA flow). Cerebral CO2 and O2 kinetics: Though the following equations have been written with cerebral tissues in mind, they are indeed applicable and relevant to any body tissues. Oxygen extraction fraction (OEF) - defined as the fraction of O2 extracted from the arterial blood - is expressed as a percentage, where an increase can reflect either elevated O2 consumption, diminished O2 delivery, or both, and conversely a decrease of OEF can reflect either reduced O2 consumption, increased O2 delivery, or both: OEF (%) = CaO2 (mL/dL) – CvO2 (mL/dL) CaO2 (mL/dL) ∙ 100 Cerebral delivery of oxygen (CDO2): CDO2 (mL/min) = gCBF (mL/min)1000 ∙ [CaO2(mL/dL) ∙ 10] Cerebral metabolic rate of oxygen consumption (CMRO2) was calculated: CMRO2 (mL/min) = gCBF (mL/min) 1000 ∙ {[CaO2 (mL/dL) − CvO2 (mL/dL)] ∙ 10} Since this is a version of the Fick equation (above), it can also be calculated via another rearrangement: VO2 (mL/min) = CDO2 ∙ OEF100 Exchange of the O2 values in the above equations with equivalent CO2 values calculated using content, allows for analogous CO2 related variables. APPENDICES - EQUATIONS 231 CO2 insertion fraction (CO2IF), defined here as the CO2 deposited into the jugular venous blood from the cerebral tissues, expressed as a percentage (analogous and inverse to oxygen extraction fraction). An increase in CO2IF can reflect either elevated CO2 production, diminished CO2 washout, or both; and, conversely, a decrease of CO2IF can reflect either reduced CO2 production, increased CO2 washout, or both: CO2IF (%) = CjvCO2 (mL/dL) – CaCO2 (mL/dL) CjvCO2 (mL/dL) ∙ 100 We also calculated cerebral CO2 washout [in the supine position, jugular venous flow represents ~95% of total cerebral outflow (Manuel Valdueza et al., 2000; Gisolf et al., 2004); as such, we used arterial gCBF (mL/min) in place of global cerebral venous blood flow]: CO2 washout (mL/min) = gCBF (mL/min)1000 ∙ [CjvCO2 (mL/dL) ∙ 10] As well as the cerebral metabolic rate of CO2 production (CMRCO2): CMRCO2 (mL/min) = gCBF (mL/min) 1000∙ {[CjvCO2 (mL/dL) − CaCO2 (mL/dL)] ∙ 10} Finally, these values allow for calculation of respiratory quotients and metabolism indices for the brain using both O2 and CO2 contents: RQ or RER = VCO2 / VO2 Metabolism Ratio = (CjvCO2 – CaCO2) / (CaO2 – CjvO2) APPENDICES – FUTURE DIRECTIONS 232 Appendix B Further details of studies mentioned in 8.6 Future directions section. B.1 Future directions: Influence of short term continuous or intermittent hypercapnia on shear-stress-mediated dilation in the cerebral circulations. PURPOSE • To compare the influence of short term continuous or intermittent hypercapnia on shear-stress-mediated dilation in the extra- and intra-cranial circulation. • HYPOTHESIS • Short term intermittent hypercapnia will elevate shear-stress-mediated dilation in the extra-cranial circulation to a greater extent than continuous hypercapnia. • Continuous hypercapnia will temporarily impair extra- and intra-cranial cerebrovascular reactivity. JUSTIFICATION Cerebrovascular dysfunction is a hallmark of many brain diseases such as stroke (Hu et al., 2017) and dementia (Farkas & Luiten, 2001). For example, in dementia, cerebral blood flow (CBF) is reduced to greater extent than compared to normal healthy aging in various regions of the brain such as the hippocampus, which is notably implicated in memory function (Farkas & Luiten, 2001). Cellular (Iadecola, 2010), animal (Merlini et al., 2016), and recently human (Wolters et al., 2017) data all indicate that chronic hypoperfusion is implicated in the pathogenesis of dementia (Iadecola, 2010); therefore, chronically enhanced cerebral blood flow and increased cerebral tissue perfusion may offset the development of such diseases and may be a direct preventative measure. Favourable blood flow patterns (e.g., antegrade shear stress) evoked via exercise and passive heat stress can acutely (Carter et al., 2013) and chronically (Carter et al., 2014) improve endothelial function (Brunt et al., 2016; Brunt et al., 2018). Moreover, in vitro evidence suggests an increased nitric oxide (NO) bioavailability in cerebral artery cells exposed to shear stress (Mashour & Boock, 1999), which is important to consider as NO related dysfunction precedes cerebral amyloid angiopathy in mouse models of Alzheimer’s disease (Merlini et al., 2016). Additionally, pharmacological blockade of endothelial NO synthase in humans have recently been observed to reduce the CO2-mediated shear stress vasodilation by approx. 50% (Hoiland et al., 2021; see Figure 0-1). Figure 0-1: Appendix B.1 - Internal carotid artery shear-stress mediated dilation during saline infusion and L-NMMA (eNOS blockade) infusion. Demonstrating the importance of eNOS and endothelial NO in vasodilation of the extracranial vasculature. Hypercapnia (increased PaCO2) is a robust challenge to the brain by decreasing tissue pH which affects the NO synthase pathway in vascular smooth muscle cells, promoting vasodilation and giving rise to increases in CBF; this, in turn increases shear-stress on the endothelium thereby causing further NO release and further vasodilation. This increase in CBF aids to mitigate increased PaCO2 and re-balance acid-base homeostasis. The magnitude of the increase in CBF per unit change in PaCO2 (termed cerebrovascular reactivity to carbon dioxide, CO2 CVR) is reduced with aging (Barnes et al., 2012) and in many clinical populations (Brian, 1998; Zimmermann & Haberl, 2003; Peebles et al., 2007; Gupta et al., 2012). To date, our group has contributed to important advances in the field of cerebrovascular response to hypercapnia: we have provided evidence that shear stress regulates cerebral conduit artery vasodilation during hypercapnic challenges even in the absence of sustained increases of PaCO2 (Carter et al., 2016; Hoiland et al., 2017; Hoiland et al., 2019; unpublished), and that the cerebrovasculature is APPENDICES – FUTURE DIRECTIONS 233 more sensitive to changes in PaCO2 and less responsive to sympathetic stimulation, than the peripheral circulation (Ainslie et al., 2005a). Increases in internal carotid artery (ICA) shear stress are strongly correlated to ICA vasodilation (Hoiland et al., 2017); similar shear stress patterns in the extracranial arteries result in similar dilatory responses regardless of the underlying stimulus (Smith et al., 2017); ergo, the haemodynamic forces in the arteries (i.e. shear-stress) that result from physiological challenges are more important for vascular adaptation than the physiological stimulus itself (Hoiland et al., 2017). Moreover, hypercapnia-induced shear-mediated dilation is blunted in the ICA in healthy older adults (Iwamoto et al., 2018a). Since this response is partly mediated via endothelial NO synthase in humans (Hoiland et al., 2019; unpublished), further study is needed to better understand the influences of continuous and intermittent changes in shear patterns on the extra-cranial circulation. Continuous changes in PaCO2 occur during many situations such as changes in posture, sleep and chronic lung disease. Likewise, intermittent changes in PaCO2 occur in both healthy situations (speaking, exercise) as well as pathology (sleep apnea, etc.) Some professions also suffer changes in environmental CO2, aquanauts and astronauts for example (Law et al., 2014). It is known that acute shear stress elicited via hypercapnia causes initial vasodilation of the ICA (Carter, 2016; Hoiland et al., 2017). A prolonged shear stimulus, such as that experienced during moderate-intensity exercise (Smith et al., 2017) may also contribute to regional CBF regulation. It is currently unknown, however, if there is a differential influence of short term continuous or intermittent hypercapnia on cerebrovascular and systemic vascular function. In regards to systemic vascular function, it is known that hypercapnia is a powerful activator of the sympathetic nervous system (Ainslie et al., 2005a). Although it is known that increases in SNA can attenuate shear-stress-mediated dilation in brachial artery, especially in hypoxia (Lewis et al., 2014a, 2017), it is unknown how and if short term changes in PaCO2 can also lead to alterations in endothelial function. The overall goal of the current proposal is to determine the influence of short term continuous or intermittent hypercapnia shear-stress-mediated dilation in the cerebral and systemic circulations. Wolters et al. (2017) found that impaired CO2 CVR is associated with an increased risk of developing dementia in the general population, so investigating how we might either improve CVR or offset the negative consequences of reduced CVR by previously enhanced bulk blood flow is paramount in preventing or inhibiting the onset of disease. In order to determine whether there is scope for potential benefit for those at risk of cerebrovascular degenerative diseases via hypercapnia stimulated vasodilatory adaptation in the extracranial arteries, first, elucidation of whether there is adaptation in healthy cerebral and peripheral arteries is required. Experimental protocols: Control of arterial blood gases: Throughout both trials PETO2 and PETCO2 will be controlled by a portable dynamic end-tidal forcing system (AirForce, GE Foster, Vancouver, BC, Canada)(Tymko et al., 2015, 2016). The end-tidal forcing system uses independent gas solenoid valves for O2, CO2, and N2 for delivery of each gas. The system controls the volume of each gas being delivered to the inspiratory reservoir through a mixing and humidification chamber. PETO2, PETCO2, tidal volume, breathing frequency and minute ventilation are determined for each breath online using specifically designed software (Labview 13.0, National Instruments, Austin, TX, USA). • Transient CO2 Test (cFMD): Following collection of five minutes of resting supine data, participants will breathe simulated room air delivered from the end-tidal forcing system for two minutes. At the two-minute mark, PETCO2 will be abruptly (~5 s) elevated to +9 mmHg above baseline for 30 seconds and then quickly returned to baseline values, which are maintained for three minutes. This procedure has been previously described in detail by our group (Hoiland et al., 2017). Ultrasound recordings are performed throughout the entire test (see below). This test has previously been approved (CREB H16-00342). • Continuous Hypercapnia (CH): Through a mouthpiece connected to a dynamic end-tidal forcing system designed to control end-tidal (and therefore arterial) blood gases on a breath-by-breath basis, after 10 minutes supine rest, participants will breath simulated room air for 5 minutes after which PETO2 will remain unchanged while PETCO2 will be elevated to +9 mmHg PETCO2 above baseline for 60.5 minutes (60.5 minutes was chosen so that elevation of CBF is ended after the same duration during both CH and ITH[next protocol described]). APPENDICES – FUTURE DIRECTIONS 234 • Intermittent Transient Hypercapnia (ITH): After 10 minutes supine rest, participants will breathe on the end-tidal forcing system at simulated room air for 5 minutes. Participants will then pace their breathing at 25 breaths per minute for a 6 minute period. PETCO2 will be quickly (within 2-3 breaths) elevated to +9 mmHg above baseline for 30 seconds, after which time it will be quickly (2-3 breaths) returned to baseline values and maintained for 5.5 minutes. This sequence will be completed 10 times back to back followed by a final 30 seconds of +9 mm Hg PETCO2, the total time of this protocol is 60.5 minutes. • Continuous Shear Match Hypercapnia (CSH): To match the cumulative shear stress caused during the ITH protocol, participants will breathe PETCO2 +9mmHg above baseline continuously for the total time that shear rate was increased above 20% of baseline during the ITH protocol. • Time Control (TC): To account for any normal variation in cerebral vascular function or other factors which may affect vascular function, over the course of ~1 hour supine rest, participants will undergo all testing pre and post 60.5 minutes of poikilo-oxic and poikilocapnic breathing. MEASURES: Respiratory Measures: Ventilatory parameters will be measured continuously by inspiratory and expiratory pneumotachs (Series 3813, Hans Rudolph, Shawnee, KS) on each side of a two-way breathing valve. The flow signal from each pneumotach will be integrated to obtain tidal volume (VT). Breathing frequency will be calculated breath-by breath using an online acquisition system (Labchart 7, AdInstruments, Colorado, US). Volume and frequency loops will be created and presented using a commercially available programing software (Labview 8.0, National Instruments, Texas, USA). Both PETCO2 and PETO2 will be measured using a gas analyzer connected to our data acquisition device (Gas Analyzer, ADInstruments, Colorado, USA). Cardiovascular measures: All cardiovascular variables will be sampled continuously at 1 KHz via an analogue-to-digital converter (Powerlab 16/30, ADInstruments). Heart rate will be measured by 3-lead electrocardiogram (Bioamp ML132, ADInstruments), while beat-to-beat BP will be measured by finger photoplethysmography (Finometer PRO, Finapres Medical Systems). The Finometer reconstructed brachial waveform is used for the calculation of mean arterial pressure after values are back calibrated to the average of three automated brachial BP measurements made over 5 minutes at rest (Tango+, SunTech). Cerebrovascular Measures: • Transcranial Doppler: Blood velocity in the right middle cerebral (MCAv) and left posterior cerebral arteries (PCAv) will be continuously measured by using a 2MHz TCD (Spencer Technologies, Seattle, WA). The TCD probes are fixed to a headpiece (model M600 bilateral head frame, Spencer Technologies) and secured into place. The MCA and PCA are insonated through the middle trans-temporal window by using previously described location and standardization techniques.(Willie et al., 2011) • Extracranial ultrasound: Blood velocity and vessel diameter of the ICA and VA will be measured at discrete times during both protocols by using a 10 MHz multi-frequency linear array duplex ultrasound (Terason 3300 uSmart, Teratech) according to published guidelines.(Thomas et al., 2015) Arterial diameter is measured with B-mode imaging, while pulse-wave mode is used to simultaneously measure blood velocity. The ICA diameter and velocity are measured at least 1.5 cm distal to the common carotid bifurcation to eliminate recordings of turbulent and retrograde flow. These locations are determined on an individual basis in an attempt to select the most reproducible measures, with the same location repeated within participants and between experimental trials. Images are recorded and stored as video files for offline analysis by using automated edge detection software (Woodman et al., 2001) following study completion. A video of no less than one minute is used for the assessment of ICA flow. All recordings will made in accordance with published guidelines (Thomas et al., 2015). Global CBF is estimated as twice the sum of the unilateral ICA flow measurements. APPENDICES – FUTURE DIRECTIONS 235 Blood sampling: Subjects will undergo intravenous catheterization to access blood samples which will be taken at multiple times throughout the protocol. A venous catheter will be placed in the median cubital (median basilic) vein proximal to the elbow crease in order to sample afferent blood. The catheter will be connected to a wasteless sampling system (VAMP, Edwards Life Sciences). Catheterization will be conducted by either Mr. Jay Carr or another qualified member of the research team, who has completed the necessary skills and training to perform these insertions properly. Total blood drawn will not exceed 25mL. Viscosity analysis At each sampling point, 1 mL of venous blood will be collected into SH (sodium heparin) Vacutainers® for blood viscosity analysis, measured by cone and plate viscometer (Brookfield DVST Viscometer) in duplicate at a shear rate of 225 s-1. Biomarker analysis Blood samples will be collected into KEDTA Vacutainers® and SST (serum separation tubes) Vacutainers®. KETDA tubes will be immediately centrifuged for 10-minutes at 4°C and 10,000G. SST tubes will rest for at least 30 minutes before being centrifuged for 10-minutes at 4°C and 10,000G. Plasma, and red blood cell samples will then be aliquoted into 1mL eppendorfs and flash frozen in liquid nitrogen. NO species (nitrite, S-nitrosohemoglobin, S-nitrosothiols) in the plasma and bound to erythrocytes will be measured by using tri-iodide reduction ozone based chemiluminescence, according to published guidelines (Seivers, NOA 280i) (Rogers et al., 2005; MacArthur et al., 2007; Pinder et al., 2009). Experimental design Participants : Twenty healthy young subjects (age: 18-40) will be recruited to participate in this study. Upon receiving written informed consent and completion of the health questionnaire (submitted alongside this CREB application) participants will be familiarized to the measurements and equipment. Non-invasive ultrasound assessment of the individual’s middle cerebral and internal carotid arteries (see below) will be performed to assess adequate measurement clarity. Potential subjects will be excluded from participation if they do not speak English (for safety reasons the participant must be able to communicate with the research team in an instantaneous manner), are taking any medications, are pregnant (self reported – or pregnancy test result), smoked within the previous year, have had, or currently have a history of cardiovascular, metabolic, respiratory or neurological disease, are obese (BMI>30), have had any invasive cerebral surgical procedures, have a cardiac pacemaker, or we are unable to attain reliable ultrasound images of their neck arteries. Based on our previous work in this area, sex differences are unlikely to influence any of the primary outcome variables when the time of testing for females is standardized for the menstrual cycle; therefore, both females and males will be tested. Females will be tested in their self-reported early follicular phase. Contraceptive use will be noted but will not be part of any exclusion criteria. Recruitment: We will recruit through a recruitment poster. Experimental visits: This study will be performed at the Centre for Heart, Lung and Vascular Health at the UBC Okanagan Campus. Following informed consent and familiarization with procedures, participants will visit the laboratory on three occasions with at least 48 hours between visits. Participants will arrive at the laboratory having fasted for ≥4hours and having abstained from alcohol, caffeine, and exercise for ≥24 hours. Upon arrival for the first visit the participant will void their bladder, then lay down for 20-minutes of rest to permit hemodynamic quiescence, during which time experimental set up will take place (see sections: “Cerebrovascular measures”; “Respiratory Measures”; “Cardiovascular measures”; and “Blood sampling”). Resting blood pressure will be measured serially three times. Baseline blood samples will be taken. Following twenty minutes of rest participants will undergo a Transient CO2 Test, after which the participant will undergo one of four separate protocols (see “Continuous Hypercapnia”; “Intermittent Transient Hypercapnia”; “Continuous Shear Match Hypercapnia”; “Time control”) in a quasi-randomized fashion. The order of CH, ITH, and TC visits will be randomized, however CSH will be completed on a visit which by necessity must be subsequent to ITH. Blood sampling and Transient CO2 Test will be completed immediately following each protocol, and again 30 minutes after the end of CO2 exposure (see Figure 0-2). APPENDICES – FUTURE DIRECTIONS 236 For all protocols, cardiovascular, respiratory, and intracranial measures will be recorded continuously from 1 minute prior to the stimulus until 40 minutes after completion. Extracranial ultrasound will be recorded during the Transient CO2 tests; as well as for one minute at minutes 10, 20, 30, 40, 50 and 59.5 during the stimuli. Figure 0-2: Appendix B.1: Timeline of experimental visit Data Analysis Cerebral blood flow: The CBF response to hypercapnia will be scaled to the level of arterial blood gas perturbation to calculate a CVR profile. CVR is the slope response of the CBF increase during an alteration in arterial blood gases, which will be calculated in both absolute (mL · min-1) and relative (%) terms (e.g., 5% ΔCBF · 1 mmHg ΔPaCO2) by using linear regression (Willie et al., 2012). Finally, the ICA diameter change during a transient change in shear stress will be analyzed by using custom in-house software that has been validated and described in detail (Carter et al., 2016; Hoiland et al., 2017). Blood pressure, intracranial cerebral blood velocities (TCD), and the cerebral metabolic rate of oxygen will also be examined. Data from all continuous measures will be extracted from the acquisition system (Labchart 7, AdInstruments, Colorado, US) and input to a Windows Excel spreadsheet for statistical analysis. Blood biomarkers: Changes in systemic circulating biomarkers (e.g., nitrite; Section “Biomarker analysis”) will be calculated. Blood pressure and intracranial cerebral blood velocities (TCD), will also be examined. Statistical Analyses All statistical analysis will be performed with the Statistical Package for the Social Sciences (SPSS, V24). A two factor linear mixed model will be used to analyze ICA Transient CO2 Test dilation (factors: test(CH/ITH/CSH/TH) & time(pre/post)), which will include “subjects” as a random factor to allow for subject drop out. ICA diameter pre, during at 30 minutes, at protocol end, and at 10 minutes and 20 minutes post protocol will be compared with a 3 by 5 factorial repeated measures ANOVA (factors: time & test). Blood concentrations of NO will be compared with 3 by 2 factorial repeated measures ANOVA (factors: time & test). SAMPLE SIZE JUSTIFICATION Based upon previous studies conducted by our laboratory investigating FMD of the peripheral blood vessels (Lewis et al., 2014) and changes in diameter of the internal carotid artery (Lewis et al., 2014; Hoiland et al., 2014; Hoiland, et al., 2016), which used relatively small sample sizes (e.g., n=11), a sample size of 20 should more than suffice to detect changes in internal carotid artery diameter. RISKS Changes in arterial blood gases: There are no major risks associated with the mild changes in PaCO2, however, participants may experience light-headedness or dizziness during increases and decreases in PaCO2, respectively. Upon removal from the end-tidal forcing system, InstrumentationcFMD5 minutes restProtocol: CH, ITH, CSH, or TC ICA diameter minute 10,20,30,40,50,60blood samplingblood sampling5minutes postcFMDcFMDblood sampling30 minutes postAPPENDICES – FUTURE DIRECTIONS 237 these symptoms quickly dissipate. Participants will be closely monitored throughout the protocol, however, and in our experience of conducing >2000 of these tests in our previous CREB approved studies (e.g. # H10-01375; H11-01105; H11-03287; H13-02620; H13-03081; H14-00922; H15-01513) there have been no ill effects reported. Venous catheterization: Venous puncture can cause minimal discomfort and/or bruising. Infection, excess bleeding, clotting, and/or fainting also are possible, although unlikely. Venous catheterization is considered a safe procedure. Venous catheters have previously been safely implemented into our research both at rest and during maximal exercise (refer to H14-00205, H11-03287, and H16-01028). These risks will be minimized by; 1) the use of experienced phlebotomists; 2) lines only being in situ for short periods of time (<3 h); We have extensive experience with these procedures in our laboratory. We have a 100% safe track record of conducting these procedures, including previously approved and completed UBC CREB studies (e.g. CREB ID # H10-01375; H11-01105; H11-03287; H13-02620; H14-00922; H15-01513). Transcranial Doppler and extracranial ultrasound: There are no known health risks associated with transcranial Doppler ultrasound. We have been previously approved to use Transcranial Doppler ultrasound (refer to H16-01028). We have been previously approved to use extracranial ultrasound. IMPLICATONS Assessing the regional vasodilatory capacity for adaptation in “normal or healthy” extra-cranial arteries will aid our understanding of preventative measures for cerebrovascular disease such as Alzheimer’s and dementia, where brain blood flow issues are a known cause and symptom. B.2 Future directions: Effects of extended hypercapnia and hypocapnia on cerebral blood flow and reactivity. PURPOSE To assess the effects of a range of extended (e.g., hours) changes in CO2 on cerebral vascular function. HYPOTHESIS We hypothesize that prolonged (8-hr) hypercapnia will: - Temporarily attenuate endothelium-dependent function in the cerebral conduit vessels, - Temporarily attenuate cerebrovascular reactivity to CO2 - Increase intracranial pressure (as indexed via jugular venous pressure and optical nerve sheath diameter) - Transiently increase, then subsequently decrease circulating biomarkers typically associated with increased vasodilator function (ATP, NO) We hypothesize that prolonged (8-hr) hypocapnia will: - Temporarily attenuate endothelium-dependent function in the cerebral conduit vessels, - Temporarily augment cerebrovascular reactivity to CO2 - Not change circulating biomarkers of nitric oxide - Reduce intracranial pressure (as indexed via jugular venous pressure and optical nerve sheath diameter). JUSTIFICATION Hypercapnia (increased arterial partial pressure of carbon dioxide [PaCO2]) is a robust challenge to vascular control of cerebral blood flow (CBF). In rats, Kondo et al. (1999) found blunted cerebrovascular reactivity to CO2 (CVR) [determined by a reduced effect of CO2 on intra-cranial pressure(ICP)] after chronic hypercapnic (~10% CO2) exposure. However, recently in humans 30 days of exposure to a mildly hypercapnic environment and head-down tilt was shown to have no effect on CVR (Laurie et al., 2020). The latter study was designed by NASA to recreate normal conditions on board the International Space Station (ISS), as such the study utilized a CO2 concentration of merely 0.5%. Extended exposure APPENDICES – FUTURE DIRECTIONS 238 to moderately elevated CO2 is a common occurrence aboard ISS. Indeed, CO2 can reach concentrations three times greater than in the terrestrial atmosphere (Law et al., 2014). There are several systems controlling ISS CO2 regulation (National Aeronautics and Space Administration, 2015); however, in the event that there is an unprecedented rise in CO2 concentration, the physiological state of astronauts during that time would be of critical importance to their survival. Judicious planning and timing during a catastrophic CO2 event should be informed by assessment of cerebrovascular function during heightened CO2 exposure. Even absent such an event, tight-space work or air stagnation can lead to local CO2 accumulation. When the upper hypercapnic bounds of effective CVR are reached and there is maximal vasodilatation, further rises in mean arterial pressure (MAP) begin to elevate cerebral perfusion pressure (CPP) (Czosnyka et al., 1993; Battisti-Charbonney et al., 2011; Willie et al., 2014). Wilkerson et al. (2005) found downregulated cerebral endothelium-dependent function but unchanged CVR after chronically increased intracranial pressure (ICP) in normocapnic rats. The studies of Kondo et al. (1999) and Wilkerson et al. (2005) indicate that chronically elevated hypercapnia and ICP can separately disturb cerebral vascular function, at least in rats, and so pose the question; in humans is CVR or cerebral endothelium-dependent function blunted with hypercapnia and the concomitantly elevated ICP? These studies in rats also include stimuli which are substantially greater in magnitude than those which showed no change to cerebral function in humans, and so may indicate that stimuli of magnitudes mirroring an adverse CO2 event on board ISS may in fact derange ICP and CBF to the detriment of cerebrovascular function, counter to those studies that found no effect. Hypocapnia (reduced PaCO2) is also a profound challenge to regulation of CBF causing significant cerebral vasoconstriction in order to minimize interstitial CO2 “washout” and maintain cerebral tissue pH. In order to assess the effects of reductions in CBF equivalent to that caused by sedentary time or chronic hypotension, we will also assess CVR and endothelium-dependent shear-mediated function before and after prolonged hypocapnia. This study will assess CVR, cerebral endothelium-dependent function, cognition, neuromuscular voluntary activation, and ICP before and after prolonged ICP and CBF challenges, i.e., exposure to a range of PaCO2 above and below the normal terrestrial range. EXPERIMENTAL MEASURES: RESPIRATORY MEASURES: During all measures, ventilatory parameters will be measured continuously by inspiratory and expiratory pneumotachs (Series 3813, Hans Rudolph, Shawnee, KS). The flow signal from each pneumotach will be integrated to obtain tidal volume (VT). Breathing frequency (Fb) will be calculated breath-by breath using an online acquisition system (Labchart 7, AdInstruments, Colorado, US). Minute ventilation VE will be calculated as VT multiplied by Fb. Both PETCO2 and PETO2 will be measured using a gas analyzer connected to our data acquisition device (Gas Analyzer, ADInstruments, Colorado, USA). During hypo and hypercapnia, these measures will be continuously recorded during the first 20 minutes and then will be spot-checked every 30 minutes until completion. CARDIOVASCULAR MEASURES: All cardiovascular variables will be sampled continuously at 1 kHz via an analogue-to-digital converter (Powerlab 16/30, ADInstruments). Electrocardiography will be measured by lead-II electrocardiogram (Bioamp ML132, ADInstruments). Beat-by-beat arterial blood pressure will be monitored via radial artery catheter pressure transducer (see haematological measures: catheterization section below). These measures will be recorded continuously throughout testing and exposure. INTRACRANIAL PRESSURE: Ultrasound measurement of optical nerve sheath diameter (ONSD), and internal jugular venous pressure, will provide discrete indirect and continuous direct measures of ICP, respectively. ONSD has been validated as an effective non-invasive technique in clinical populations and against several other invasive and non-invasive techniques (Hansen & Helmke, 1997; Blaivas et al., 2003; Geeraerts et al., 2008; Kimberly et al., 2008; Horowitz & Bailitz, 2015; Robba et al., 2018; Cardim et al., 2019; Chen et al., 2019). ONSD will be measured at discrete times and according to guidelines adhering to ALARA (as low as reasonably achievable) principles (De La Hoz Polo et al., 2016). ONSD will be measured at least 3 mm proximal to the vitreo-retinal interface. The means of three sagittal plane and three transverse plane ONSD measurements will be taken for both eyes. APPENDICES – FUTURE DIRECTIONS 239 VASCULAR MEASURES: CEREBRAL BLOOD FLOW: Blood velocity in the right middle cerebral artery (MCAv) and left posterior cerebral artery (PCAv) will be measured continuously using a 2MHz transcranial doppler (TCD) (Spencer Technologies, Seattle, WA). TCD probes will be fixed to a headpiece (model M600 bilateral head frame, Spencer Technologies) and secured into place. MCA and PCA will be insonated through the middle trans-temporal window using previously described location and standardization techniques (Willie et al., 2011). Right internal carotid artery (ICA) and left vertebral artery (VA) will be measured at discrete times using a 10 MHz multi-frequency linear array duplex ultrasound (Terason 3300 uSmart, Teratech) according to published guidelines (Thomas et al., 2015). Arterial diameter will be measured with B-mode imaging, while pulse-wave mode will simultaneously measure blood velocity. ICA diameter and velocity will be measured at least 1.5 cm distal to the common carotid bifurcation to eliminate recordings of turbulent and retrograde flow. All images will be recorded and stored as video files for offline analysis by using automated edge detection software (Woodman et al., 2001) following study completion. All recordings will be made in accordance with published guidelines (Thomas et al., 2015). Global CBF will be estimated as twice the sum of ICA + VA flow. HEMATOLOGICAL MEASURES: CATHETERIZATION: Under local anaesthesia (Lidocaine, 1.0%) and careful sterile technique (full surgical scrub, gowns, gloves, clean room), a catheter will be placed in the radial artery of the non-dominant arm and connected to a pressure transducer. The transducer will be levelled with the patient's right atrium, and briefly opened to the atmosphere periodically throughout the recording period to zero at atmospheric reference pressure. Analog output for arterial pressure pressures will transferred to a PC via a commercially available analog to digital signal converter (Biopac). Then, under local anaesthesia (Lidocaine, 1.0%) and careful sterile technique (full surgical scrub, gowns, gloves, clean room), a central venous catheter (Cook Medical, Bloomington, IN, USA) will be placed in the right internal jugular vein and advanced cephalad to the jugular bulb (Schell & Cole, 2000; Ainslie et al., 2014). Our research team was the first to obtain ethical approval for and establish this technique in Canada; we have safely carried out >5 related studies in our laboratory at UBC (Smith et al., 2014; Ainslie et al., 2014; Bain et al., 2015; Willie et al., 2015b; Bain et al., 2016, 2017). The radial and jugular catheters will be connected to a waste-less sampling system (VAMP, Edwards Life Sciences) and connected in series to a pressure monitor (BP amp, ADInstruments). BLOOD GAS ANALYSIS: At each sampling point, 1 mL each of arterial and jugular venous blood will be collected for measurement of blood gases. Blood samples will be immediately analyzed using a commercial blood gas analyzer (ABL90 Flex, Radiometer, Copenhagen, Denmark). Analyzed variables include: PO2, PCO2, pH, [H+], Hct, Hb, [HCO3-], base excess (BE), and oxyhemoglobin saturation (SO2). Body temperature will be assumed to be 37 °C. Total blood drawn will not exceed 30ml. VISCOSITY ANALYSIS: At each sampling time, 1 mLof blood will be collected for viscosity analysis, measured by cone and plate viscometer (Brookfield DVST Viscometer) in duplicate at a shear rate of 225 s-1. BIOMARKER ANALYSIS: Blood samples will be collected pre, during, and post hypercapnia and hypocapnia into KEDTA Vacutainers® and SST (serum separation tubes) Vacutainers®. KETDA tubes will be immediately centrifuged for 10-minutes at 4°C and 10,000G. SST tubes will rest for at least 30 minutes before being centrifuged for 10-minutes at 4°C and 10,000G. Plasma, serum, and red blood cell samples will then be aliquoted into 1mL eppendorfs and flash frozen in liquid nitrogen. Circulating vasodilator biomarkers, adenosine triphophosphate (ATP) and NO species (nitrite, S-nitrosohemoglobin, S-nitrosothiols) in the plasma and bound to erythrocytes, will be measured using tri-iodide reduction ozone based chemiluminescence, according to published guidelines (Rogers et al., 2005; MacArthur et al., 2007; Pinder et al., 2009). Circulating biomarkers of blood brain barrier activity: S100β, ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), and glial fibrillary acidic protein (GFAP), will be measured in the serum. APPENDICES – FUTURE DIRECTIONS 240 CONTROL OF ARTERIAL BLOOD GASES: During the breathing tests described below, PETO2 and PETCO2 will be controlled by dynamic end-tidal forcing system (AirForce, GE Foster, Vancouver, BC, Canada) (Tymko et al., 2015, 2016). The end-tidal forcing system uses independent gas solenoid valves for O2, CO2, and N2 delivery. The system controls the volume of each gas being delivered to the inspiratory reservoir through a mixing and humidification chamber. PETO2, PETCO2, tidal volume, breathing frequency and minute ventilation are determined for each breath online using specifically designed software (Labview 13.0, National Instruments, Austin, TX, USA). CEREBRAL FLOW-MEDIATED DILATION (cFMD): Following collection of five minutes of resting supine data, participants will breathe simulated room air delivered from the end-tidal forcing system for two minutes. At the two-minute mark, PETCO2 will be rapidly (<10 seconds) elevated to 9 mmHg above baseline for 30 seconds then returned to baseline values, which will be maintained for three minutes. ICA ultrasound images will be recorded throughout. This test has previously been approved (CREB H16-00342; H17-01810). CEREBROVASCULAR REACTIVITY TO CARBON DIOXIDE (CVR): Using dynamic end-tidal forcing, baseline PETCO2 and PETO2 will be re-established and maintained for one minute of baseline Duplex imaging, after which PETCO2 will be elevated to 9 mmHg above baseline and maintained for five minutes. ICA ultrasound images will be recorded throughout. EXPOSURE TO ENVIROMENTAL GASES EXTENDED HYPOCAPNIA: Participants will be coached through breathing such that their PETCO2 is reduced by -5 to 10 mm Hg from baseline and then will breath in time with a metronome to maintain that ventilation for a continuous period of eight hours. End-tidal O2 and CO2 concentrations will be continuously analyzed (Gas Analyzer, ADInstruments, Colorado, USA) via sample line and nasal cannula, and displayed on-screen with an acquisition and recording system (Labchart 7, AdInstruments, Colorado, US). EXTENDED HYPERCAPNIA: Under a plastic sheet canopy over the laboratory testing bed, participants will be exposed to a gas mixture of 5% CO2, 20.9% O2, balance N2. The concentration of CO2 and O2 in the local atmosphere will be continuously sampled via sample line to the gas analyzer (Gas Analyzer, ADInstruments, Colorado, USA) via direct sample line, and displayed on-screen with an acquisition and recording system (Labchart 7, AdInstruments, Colorado, US). Participants will be exposed to elevated 5% CO2 (which will elevate PETCO2 roughly +5-10 mmHg above baseline) for a continuous period of eight hours. EXPERIMENTAL DESIGN PARTICIPANTS: 24 healthy participants between 18 and 45 years of age will be recruited. Participants will be excluded from the study if they do not speak English (for safety reasons the participant must be able to communicate with the research team quickly) are taking any medications, are pregnant (self-reported – or pregnancy test result), currently smoke or smoked within the previous year, have had, or currently have a history of cardiovascular, metabolic, respiratory or neurological disease, are obese (BMI>30kg/m2), have had any invasive cerebral surgical procedures, have a cardiac pacemaker, or we are unable to attain reliable ultrasound images of their neck arteries. Based on our previous work in this area, sex differences are unlikely to influence any of the primary outcome variables when the time of testing for females is standardized for the menstrual cycle; therefore, both females and males will be tested. Females will be tested at any stage of their menstrual cycle, phase will be recorded and reported. Contraceptive use will be noted but will not be part of any exclusion criteria. EXPERIMENTAL PROTOCOL: This study will be performed at the Centre for Heart, Lung and Vascular Health at the UBC Okanagan Campus. The study will follow a randomized non-crossover (between participants) design such that 12 participants will undergo hypercapnia and 12 will undergo hypocapnia, in a randomized fashion. Participants will visit the laboratory on two occasions, which should total no more than ~11 hours. The first visit will take roughly 30 minutes. Upon completion of informed written consent and the health questionnaire (submitted alongside this application) participants will APPENDICES – FUTURE DIRECTIONS 241 be familiarized to the measurements and equipment. During this visit, non-invasive ultrasound assessment of the individual’s right ICA and left VA will be performed to assess adequate image clarity. Following the familiarization visit, participants will arrive at the laboratory for the main experimental visit - either hypercapnic or hypocapnic exposure - having fasted for ≥4hours and having abstained from alcohol, caffeine, and exercise for ≥24 hours. Upon arrival the participant will void their bladder, then lay supine during experimental set up, including arterial and jugular catheterization, and for a further 20 minutes of rest to permit hemodynamic quiescence. Baseline normoxic normocapnic blood pressure, ventilation, PETO2, PETCO2, and heart rate will be recorded, and arterial and jugular venous bloods will be sampled for viscosity, blood gases, and biomarkers. Participants will then undergo baseline ONSD, ICA, and VA measurements, followed by a CVR test, 10 minutes of rest, and a cFMD test. The participant will then undergo either hypercapnic or hypocapnic exposure for eight hours. Arterial and jugular bloods will be sampled after five, 30, 60, 120, 240, 360, and 480 minutes. ONSD will be measured at 60, 120, 240, 360, and 480 minutes (see Figure 0-3 for timeline). Figure 0-3: Appendix B.2 - Protocol. Throughout the protocol the participant will be allowed water ad libitum. For comfort during the protocol, after the first 30 minutes of exposure, participants will be allowed to move into a slight semi-supine position, they will be helped to return to a fully supine position at least 30 minutes prior to end of exposure. Following the end of hypercapnia or hypocapnia, subjects will remain supine and at 30 minutes post-exposure blood samples for viscosity, blood gases, and biomarkers will be taken and ONSD, ICA, and VA measurements will be taken, then cFMD and CVR testing. This concludes the experimental protocol; the participant will then be de-instrumented. RISKS: Arterial and venous catheterization: Dr. Mypinder Sekhon will perform this procedure. Jugular vein and radial (or brachial) artery catheterization are considered safe procedures associated with few risks when made by experienced clinicians. The main risks include hematoma formation (6.1%) which is usually minor requiring no specific therapy other than temporary local pressure, infection (0.78%), pneumothorax (0.2%). Another risk with the use of jugular vein catheters is jugular valve incompetence (28%, this is a reduction in how well this valve works). This can potentially lead to back flow of blood, which may be related to short episodes of memory loss and/or headaches. These risks will be further minimized by; 1) the use of ultrasound to image the jugular vein and enable the precise location of the catheter to be quantified; 2) all lines will be inserted under strict aseptic techniques. This includes full surgical scrub, the wearing of a surgical hat, facemask and sterile gowns and the use of chlorohexidine or iodine-based cleaning solutions and sterile towels; 3) risks are also minimized by lines only being in situ for short periods of time (<12 h); 4) as mentioned, oxygen saturation, ECG and blood pressure will be continuously monitored throughout each experimental procedure. Oxygen will be available at all times, as will resuscitation drugs and equipment; and 5) allowing 2-3 hours following completion of the protocol and removal of the catheters to fully assess the participant. We have extensive experience with these procedures in our laboratory. We have a 100% safe track record of conducting these procedures, including previously approved and completed UBC CREB studies (e.g. CREB ID # H10-01375; H11-01105; H11-03287; H13-02620; H14-00922; H15-01513). To further minimize subject risk, patency of the radial and ulnar artery will be confirmed with a negative modified Allen’s test prior to arterial penetration. Transcranial Doppler ultrasound: There are no health risks associated with transcranial Doppler ultrasound. We have been previously approved to use Transcranial Doppler ultrasound (H16-01028). Ocular ultrasonography: 30 60 1205 240 360 480Time (minutes)APPENDICES – FUTURE DIRECTIONS 242 Provided that ALARA principles are followed, that is to say, mechanical and thermal indices of ultrasound power output are attended to and adjusted with consideration to the tissue type under investigation, there are have no been deletriorous effects of ocular ultrasound reported (Hansen & Helmke, 1997; Blaivas et al., 2003; Geeraerts et al., 2008; Kimberly et al., 2008; Horowitz & Bailitz, 2015; Robba et al., 2018; Cardim et al., 2019; Chen et al., 2019). Sterile gel will be used. Changes in arterial blood gases: There are no major risks associated with mild changes in PaCO2, however, participants may experience light-headedness or dizziness during increases and decreases in PaCO2, respectively. Upon removal from the end-tidal forcing system, these symptoms quickly dissipate. Participants will be closely monitored throughout the protocol. In our experience of conducting >2000 of these tests in our previous CREB approved studies (e.g. # H10-01375; H11-01105; H11-03287; H13-02620; H13-03081; H14-00922; H15-01513) there have been no ill effects reported. Data Analysis The CBF response to hypercapnia during CVR tests will be scaled to the level of arterial blood gas perturbation to calculate a CVR profile, i.e. the slope response of the CBF increase during an alteration in arterial blood gases, which will be calculated in both absolute (mL · min-1) and relative (%) terms (e.g., 5% ΔCBF · 1 mmHg ΔPaCO2) using linear regression (Willie et al., 2012). ICA diameter change during cFMD will be analyzed using software that has been validated (Carter et al., 2016; Hoiland et al., 2017). Statistical Analyses Linear mixed model analyses will be used to compare: - ICA and VA diameters and flows - cFMD - CVR - ONSD pre vs post hypercapnia and hypocapnia. Linear mixed model analyses will be used to compare all timepoints for: - ONSD - Arterial blood viscosity - Arterial blood gases - Arterial NO biomarkers - Cerebral arteriovenous differences of all blood biomarkers assessed All statistical analysis will be performed with the Statistical Package for the Social Sciences (SPSS, V24). B.3 Future directions: Effects of breath-hold induced shear stress elevations on the cerebrovascular endothelium in trained divers and untrained controls Rationale: It is well-established that hypoxia causes cerebral vasodilation in order to maintain cerebral oxygen delivery in the face of reduced arterial oxygen content (McDowall, 1966; James et al., 1969; Kogure et al., 1970; Willie et al., 2014, 2015a). In elite divers, during the “struggle phase” of breath-hold dive (BHD) apnea, cerebral blood flow (CBF) also increases as the cerebral vasculature strives to maintain oxygen delivery and carbon dioxide washout in spite of steadily marked hypoxemia, hypercapnia, and transient but profound hypertension (Dujic et al., 2009; Willie et al., 2015a). But, CBF fluctuates near synchronously with cardiac output and blood pressure caused by involuntary breathing movements as the drive to breath develops (Cross et al., 2013). Given such complex and nuanced changes in CBF and oxygen delivery caused by a multitude of systemic influences, the effects that voluntary maximal apneas may have on cerebrovascular endothelial function and shear stress patterns in BHD are uncertain. Transiently increased blood flow can result in temporarily augmented vascular vasodilator function, as is well evidenced in the peripheral arteries with exercise hyperemia and other blood flow increasing stimuli (Tinken et al., 2010; Dawson et al., 2013); as such, intermittent hypoxia-induced increases in blood flow in the coronary and cerebral arteries may also elicit augmented endothelial function in the relevant vessels. The consequences of both arterial desaturation and hypertension are multitude, however, in both animals and humans, recent evidence also indicates that intermittent hypoxia may elicit beneficial adaptations in cardiac health after intermittent ischemic hypoxia of the coronary arteries due to occlusion and reperfusion (Mallet et al., 2018). It is thought that these benefits are related to enhancements in coronary blood flow as there is some evidence of compensatory APPENDICES – FUTURE DIRECTIONS 243 vascular remodeling (Mallet et al., 2018). Whether similar vascular remodelling occurs in BHDs is not well established. Indeed recently, cerebral vasodilator function was shown to be elevated following acute periods (<60 mins) of intermittent hypoxia compared to normoxia (Iwamoto et al., 2020). However, differences between the systemic and hemodynamic characteristics of intermittent hypoxia and voluntary apneas indicate that these findings cannot be applied to the cerebral effects of BHD apneas. For example, throughout intermittent hypoxia in the study by Iwamoto et al. (2020), MAP was unchanged, whereas in a study with a somewhat comparable model using maximum voluntary apnea, MAP remained consistently elevated across all apneas (Baković et al., 2003). During maximal apneas, however, MAP can transiently increase to 150 mmHg without ill-effect (Heusser et al., 2009; Perini et al., 2010). During both intermittent hypoxia and apnea, active splenic contraction ejects red blood cells from the storage pool therein, and in doing so increases arterial oxygen content by elevating hematocrit by 2-10% (Schagatay et al., 2003). However, during apnea, the rise of MAP and CBF are not blunted by ventilatory compensation (such as the case with poikilocapnic hypoxia), translating to an altered hematocrit -driven shear-mediated response. This change in rheological characteristics may lead to improved vascular function in BHDs by altering shear stress engagement of endothelial cells, although previous reports of vascular function in BHD are equivocal (Theunissen et al., 2013; Barak et al., 2020). If chronic vascular remodelling occurs in the cerebrovasculature then it may be that BHDs - in whom cerebrovascular shear stress is frequently elevated repetitively by intermittent hypoxia and hypercapnia during diving and voluntary training apneas - have chronically adapted cerebrovasculature. This may contribute to unimpaired cerebrovascular reactivity to CO2 (CVR) in BHDs (Ivancev et al., 2007) which is nonetheless reduced with prostaglandin inhibition (Ivancev et al., 2009). As such, the degree of endothelium-dependent function would inversely index this adaptation (Tinken et al., 2010) in BHDs (function likely attenuated in the presence of structural adaptation), and CVR would be similar between groups. Purpose: To assess the effects of repeated maximal apneas with and without arterial hypoxemia on cerebrovascular (internal carotid artery “ICA”) vasodilator function and cerebrovascular reactivity to CO2 in BHDs in comparison to a control group of healthy non-divers. Hypothesis: - Endothelium-dependent function in the ICA will be temporarily augmented in a dose dependent manner following repeated shear stress challenges in control volunteers, but in BHD, ICA function will be lower both pre and post apnea protocol. - CVR will be comparable and similar between BHD and control volunteers both before and after apneas. o Collectively, these would indicate maintained cerebrovascular function in spite of reduced endothelium-dependent function, due to substantial contributions of other mechanisms to vascular function, and probable previous vascular structural adaptation in BHDs. Methods: 15 Control and 15 BHD participants will undergo one experimental session, at the beginning of which they will conduct one maximal breath-hold (BH) to establish individual BH time, followed by five trial sequences each with pre and post testing. The first sequence will consist of five voluntary apneas of a duration required to reduce peripheral O2 saturation to 85%, separated by two minutes rest during which participants breath simulated room air (Nx). The order of the following four sequences will be randomized. Two sequences consist of five voluntary apneas each of which will last the same duration as the equivalent apnea during the Nx trial, during the inter-apnea periods participants will either breath hyperoxic gas (Hx) or simulated room air with hyperventilation (Hv) (Bain et al., 2017). Two sequences will simulate the MAP and blood gas changes during the room air apneas, via lower body negative pressure (LBNP) and end-tidal forcing (ETF) respectively (Figure 0-4). In summary, participants will undergo maximal BH, then five Nx apneas, followed by Hx, Hv, LBNP, and ETF trials in random order which will each simulate Nx apneas without the influence of one or more factors contributing to increased CBF (Hx – hypercapnia and MAP increase without hypoxemia; Hv – hypoxemia and MAP increase hypercapnia; LBNP – MAP increase without hypercapnia and hypoxemia; ETF – hypercapnia and hypoxemia without MAP increase). APPENDICES – FUTURE DIRECTIONS 244 Figure 0-4: Appendix B.3 - Protocol schematic CVR and cFMD will be tested pre and post each apnea sequence, at least 20 minutes rest will be allowed between the end post testing and pre testing of the next sequence, to allow for cerebrovascular repose, middle cerebral artery velocity (MCAv) and MAP will be checked for return to baseline values before beginning the next sequence. Inspiratory volumes for each BH will be standardized to 85% of vital capacity, to limit any hyper-inflationary restraint on venous return and cardiac output. Figure 0-5: Appendix B.3 - ICA diameter and shear rate, before, during, and following 30 second bolus of +9 mmHg PETCO2. We will assess endothelium-dependent dilation in the ICA via Duplex ultrasound during transiently (30 seconds) elevated shear stress caused by increased partial pressure of end-tidal carbon dioxide using dynamic end-tidal forcing (+9mmHg; cerebral flow-mediated dilation, cFMD) (Figure 0-5). Cerebrovascular reactivity to carbon dioxide will be assessed during steady-state (five minutes) elevations of carbon dioxide (+9mmHg; CVR). MCAv, posterior cerebral artery velocity, arterial blood pressure, SpO2, end-tidal partial pressures of oxygen and carbon dioxide will be continuously assessed and recorded throughout CVR, cFMD, and the apnea protocol; ICA and vertebral artery (VA) hemodynamics and vessel characteristics will be assessed continuously for 30 seconds leading up to and until the end of each apnea. Arterial blood will be sampled 5 seconds prior to the start of, every 30 seconds during, and at the end of each apnea, and immediately analyzed for blood gases, red blood cell count, hematocrit, and viscosity. Cerebrovascular compliance and resistance will be determined by established methods based on signal processing from the intra-cranial arteries and blood pressure (Chan et al., 2011; Moir et al., 2020) Significance: The implications of this study are two-fold. First, the acute impacts of a single bout of repeated breath-holds on cerebral vascular health and function will be assessed in healthy normal controls and trained breath-holders, providing insight into the potential for cerebrovascular adaptations to repeated shear stress and pressure stimuli. Second, differentiation of CVR and endothelium-dependent function of the large extracranial arteries following breath holding with and without hypoxemia is relevant to the study of diseases such as obstructive sleep apnea and hypertension, wherein cerebral hemodynamics may be variously affected with and without reductions in arterial oxygen. B.4 Future directions: Therapeutic hypercapnia after ischemic stroke Clinical management and treatment following ischemic stroke or other hypoxic ischemic brain injury (e.g., cardiac arrest, traumatic brain injury) requires consideration of the various physiological factors that influence cerebral hemodynamics and safety. Arterial blood pressure, blood gases, cerebral metabolism, and cardiac output all require relative clinical optimization and management, often in conjunction with management of other concurrent injury or disease. Following stroke, hypocapnia (reduced partial pressure of arterial carbon NxHxHvLBNPETFHv – HyperventilationLBNP – Lower body negative pressureETF – End-tidal forcingRandom orderMax BHNx – NormoxiaHx – HyperoxiacFMD + CVRMAPSPO2PaCO2BA ICA BA ICA01020304050Diameter change (%)FMD GTNArtery, P<0.001Test, P<0.001Interaction, P=0.58BA ICA04812Diameter change (%)Scaled for baseline diameter and SRAUCFMD0 200 400 6000102030-5Time (seconds)Diameter change (%)GTN 400ugICABABA ICA01020304050Diameter change (%)Scaled for baseline diameterGTNA B CD E FBA ICA051015Diameter change (%)Scaled for baseline diameterFMD0 60 360 420 480 540-2024604008001200Time (seconds)Diameter change (%)Cuff releaseDiameterShear RateShear rate change (%)0 100 200-202460102030Time (seconds)Diameter change (%)+9mmHgShear rate change (%)DiameterShear RateGBA ICA04812Diameter change (%)Scaled for baseline diameter and DSRAUCFMDH*APPENDICES – FUTURE DIRECTIONS 245 dioxide; PaCO2) has been used since the 1950s to transiently reduce intracranial pressure (ICP) (Lundberg et al., 1959; Neumann et al., 2008). While hypocapnia through mechanical hyperventilation is often applied ‘prophylactically’ in the clinical management of traumatic brain injury and stroke, there are in fact several contra-indications to its use [reviewed in: (Neumann et al., 2008)].7 Conversely, hypercapnia (increased PaCO2) elevates arterial blood pressure and ICP if severe and especially in the event of reduced cerebral compliance (as with cerebral tissue swelling and edema), however hypercapnia also improves cerebral perfusion through increased blood flow. While extreme hypocapnia and hypercapnia may be detrimental - causing ischemia or edema, respectively - maintaining PaCO2 at the upper end within this range above individual baseline normocapnia may be beneficial in maintaining cerebral oxygen delivery and improving oxygen utilization without inducing overt ICP elevations (Sekhon et al., 2017). Mild to moderate hypercapnia following global cerebral ischemia has been shown to be beneficial in animal models (Vannucci et al., 1995, 1997; Kohzuki et al., 2006; Zhou et al., 2010); it is associated with better neurological scores, less histologically indicated brain damage, maintained oxidative glucose metabolism, and reduced cell apoptosis compared to normocapnia and severe hypercapnia (Zhou et al., 2010). Benefits have also been shown in humans; mild hypercapnia during post-resuscitation after cardiac arrest-induced cerebral ischemia provides long term neurological benefits (Schneider et al., 2013; Lee et al., 2014; Vaahersalo et al., 2014). These findings and paradigms have not been replicated in stroke patients, nor have the effects of therapeutic hypercapnia on cerebral endothelial function and cognitive function following ischemic stroke. Purpose: To compare cerebrovascular endothelium-dependent and cognitive function in stroke patients following extended (6 hours) mild hypercapnic exposure compared to normocapnic stroke patients. 7 Hypocapnia can induce; global cerebral under-perfusion and hypoxia (Godoy et al., 2017), blunted dynamic cerebral autoregulation (Nakagawa et al., 2011), worse clinical long-term outcomes (Muizelaar et al., 1991), increased extracellular biomarkers around vulnerable cerebral tissues associated with traumatic ischemia (Marion et al., 2002), higher risk of in-hospital mortality (Hextrum et al., 2020), decreased cerebral oxygen delivery with an increased metabolic rate of oxygen (Coles et al., 2007); eventual loss of effects on ICP due Hypothesis: Degree of endothelial impairment will be associated with degree of cognitive impairment, severity of stroke, and future negative outcome. Stroke patients exposed to 6 hours of mild hypercapnia will demonstrate attenuation of impairment to endothelium-dependent and cognitive function compared to those patients who receive normocapnia. Methods: In 20 patients who recently suffered ischemic stroke or ischemic event of cerebrovascular disease origins, we will non-invasively evaluate large cerebral extra-cranial artery vasoreactivity using three common and complimentary assessments (cerebral flow-mediated dilatation, cFMD; cerebrovascular CO2 reactivity, CVR; and common carotid artery cold pressor reactivity, CAR) and cognitive function (mini-mental state examination, MMSE; critical flicker fusion, CFF) (see experimental methods below). Vasoreactivity and cognitive function will be assessed before and immediately following, as well as 3 and 48 hours following exposure to 6 hours of either normocapnia (35 - 45 mmHg end-tidal pressure of CO2: PETCO2) or hypercapnia (45-50 mm Hg PETCO2). Internal carotid artery cFMD will be assessed via Duplex ultrasound during transiently (30 seconds) elevated shear stress caused by increased inspired carbon dioxide using the Douglas bag method (5% CO2, 21% O2, balance N2), following protocols based on similar previous studies (Carter et al., 2016; Hoiland et al., 2017). Second, CVR (indexed by both middle cerebral artery blood velocity and ICA blood flow) will be assessed during steady-state (5 minutes) elevation of CO2 (5% CO2). Third, CAR will assess CCA reactivity to sympathetic stimulation using cold pressor test (Van Mil et al., 2018). CAR provides a novel strategy to predict cardiovascular events and progression in patients with peripheral artery disease (van Mil et al., 2019). Similar to coronary artery responses to sympathetic stimulation, CAR causes common carotid artery vasoconstriction in the presence to renal pH buffering; rebound hyperemia is also possible after prolonged hyperventilation if PaCO2 rapidly changes back to baseline, causing severe cerebral acidosis, vasodilation, and eventual restoration of elevated ICP as well as worsening of ischemia reperfusion injury (Curley et al., 2010). Neurological and cognitive impairments have also been reported as a result of hypocapnia during operative anesthesia (Wollman & Orkin, 1968; Hovorka, 1982). APPENDICES – FUTURE DIRECTIONS 246 of impaired vascular function indicating increased cardiovascular event risk, compared to those with carotid vasodilation. Middle cerebral artery blood velocity, mean arterial blood pressure, end-tidal partial pressures of oxygen and carbon dioxide will be continuously assessed and recorded throughout CVR, cFMD, and CAR. These measures will be used in conjunction with several other recorded measures, including: CT/MRI and ultrasonographic categorization of carotid stenoses volume, history of cardiovascular risk, and any subsequent adverse events will be recorded. The visual cortex and associated visual processing in the brain is limited by the time required to collect and process, as such, intermittent visual stimuli are perceived as separate events only if observed to occur below a threshold rate, defined as the critical flicker-fusion frequency (CFF). To assess CFF and thereby temporal processing of the visual system in the occipital lobe, we will control the flickering frequency of a LED output in a staircase paradigm, which has been shown to be less time-consuming and more effective than other methods (Eisen-Enosh et al., 2017). Cognitive acuity will be assessed used a mini-mental state examination (Folstein et al., 1975). Significance: Stroke and hypoxic/ischemic brain injury pathophysiology and subsequent management is complex, and considerable risk and damage occurs in the event of secondary insult. The efficacy of interventions aimed at preventing secondary injury in humans have been mostly focused on hyperthermia (Brambrink & Orfanakis, 2010); and while the clinical ramifications of close PaCO2 monitoring are widely understood, post-stroke therapeutic hypercapnia for the optimization of mean arterial pressure, intracranial pressure, and cerebral perfusion has yet to be explored in humans despite showing promise in animals (Vannucci et al., 1995, 1997; Kohzuki et al., 2006; Zhou et al., 2010) and humans following cardiac arrest (Schneider et al., 2013; Lee et al., 2014; Vaahersalo et al., 2014). Differentiation of cerebral endothelial function and cognitive function with therapeutic hypercapnia will aid and inform future decisions of clinical management in the context of ischemic stroke. B.5 Future directions: Assessing endothelial dysfunction in those with carotid atheroma to predict stroke risk PURPOSE To assess large extra-cranial artery endothelium-dependent shear-mediated vasodilation across severities of carotid stenosis, using a comprehensive evaluation of arterial vasomotor function. HYPOTHESIS Large extra-cranial artery endothelium-dependent shear-mediated vasodilation will be associated with volume of carotid stenosis (and/or atherosclerotic burden in those subjects without carotid stenoses but with other risk factors). Shear-mediated vasodilation and sympathetic vasodilation responses will be associated with risk of negative outcome. JUSTIFICATION Cerebrovascular diseases such as ischemic and hemorrhagic stroke, cerebral small vessel disease, carotid artery stenosis, vascular dementia, and Alzheimer’s, are preceded by - and associated with - endothelial dysfunction in the cerebral vasculature (Toda, 2012; Iturria-Medina et al., 2016; Kalaria, 2016; Wolters et al., 2016, 2017). However, clinical assessments of cerebrovascular function currently lack specificity to large extra-cranial artery endothelium-dependent function. In particular, typical measures of cerebrovascular function using MCA velocity (e.g., CO2 reactivity; CVR) assess a number of overlapping and redundant vasodilator pathways (Carr & Ainslie, 2020; Carr et al., 2020), and do not seem to hold strong predictive value (Portegies et al., 2014) in populations other than those with overt steno-occlusive disease (Markus & Cullinane, 2001; Gupta et al., 2012). As such, development of methods that directly assess endothelial dysfunction in the human cerebrovasculature may hold significance in regards to demonstrating in vascular function impairments with disease, and improvements with exercise. Surprisingly, the extent of endothelium-dependent shear mediated vasodilation within patients with various categories of carotid stenoses and atherosclerotic burden are largely unknown. APPENDICES – FUTURE DIRECTIONS 247 Experimental protocols: Control of arterial blood gases: Throughout, PETO2 and PETCO2 will be controlled by either a portable dynamic end-tidal forcing system (AirForce, GE Foster, Vancouver, BC, Canada)(Tymko et al., 2015, 2016) or by Douglas bag, depending on the outcome of another current study (DcFMD). The end-tidal forcing system uses independent gas solenoid valves for O2, CO2, and N2 for delivery of each gas. The system controls the volume of each gas being delivered to the inspiratory reservoir through a mixing and humidification chamber. PETO2, PETCO2, tidal volume, breathing frequency and minute ventilation are determined for each breath online using specifically designed software (Labview 13.0, National Instruments, Austin, TX, USA). Transient CO2 Test (cFMD): Following collection of five minutes of resting supine data, participants will breathe simulated room air delivered from the end-tidal forcing system for two minutes. At the two-minute mark, PETCO2 will be abruptly (~5 s) elevated to +9 mmHg above baseline for 30 seconds and then quickly returned to baseline values, which are maintained for three minutes. This procedure has been previously described in detail by our group (Hoiland et al., 2017). Ultrasound recordings are performed throughout the entire test (see below). This test has previously been approved (CREB H16-00342). MEASURES: Respiratory Measures: Ventilatory parameters will be measured continuously by inspiratory and expiratory pneumotachs (Series 3813, Hans Rudolph, Shawnee, KS) on each side of a two-way breathing valve. The flow signal from each pneumotach will be integrated to obtain tidal volume (VT). Breathing frequency will be calculated breath-by breath using an online acquisition system (Labchart 7, AdInstruments, Colorado, US). Volume and frequency loops will be created and presented using a commercially available programing software (Labview 8.0, National Instruments, Texas, USA). Both PETCO2 and PETO2 will be measured using a gas analyzer connected to our data acquisition device (Gas Analyzer, ADInstruments, Colorado, USA). Cardiovascular measures: All cardiovascular variables will be sampled continuously at 1 KHz via an analogue-to-digital converter (Powerlab 16/30, ADInstruments). Heart rate will be measured by 3-lead electrocardiogram (Bioamp ML132, ADInstruments), while beat-to-beat BP will be measured by finger photoplethysmography (Finometer PRO, Finapres Medical Systems). The Finometer reconstructed brachial waveform is used for the calculation of mean arterial pressure after values are back calibrated to the average of three automated brachial BP measurements made over 5 minutes at rest (Tango+, SunTech). Cerebrovascular Measures: • Transcranial Doppler: Blood velocity in the right middle cerebral (MCAv) and left posterior cerebral arteries (PCAv) will be continuously measured by using a 2MHz TCD (Spencer Technologies, Seattle, WA). The TCD probes are fixed to a headpiece (model M600 bilateral head frame, Spencer Technologies) and secured into place. The MCA and PCA are insonated through the middle trans-temporal window by using previously described location and standardization techniques.(Willie et al., 2011) • Extracranial ultrasound: Blood velocity and vessel diameter of the ICA will be measured at discrete times during both protocols by using a 10 MHz multi-frequency linear array duplex ultrasound (Terason 3300 uSmart, Teratech) according to published guidelines.(Thomas et al., 2015) Arterial diameter is measured with B-mode imaging, while pulse-wave mode is used to simultaneously measure blood velocity. The ICA diameter and velocity are measured at least 1.5 cm distal to the common carotid bifurcation to eliminate recordings of turbulent and retrograde flow. These locations are determined on an individual basis in an attempt to select the most reproducible measures, with the same location repeated within participants and between experimental trials. Images are recorded and stored as video files for offline analysis by using automated edge detection software (Woodman et al., 2001) following study completion. A video of no less than one minute is used for the assessment of ICA flow. All recordings will made in accordance with published guidelines (Thomas et al., 2015). APPENDICES – FUTURE DIRECTIONS 248 EXPERIMENTAL DESIGN Participants: 50 patients with various categories of carotid stenosis (control, 0% ; low, 10-40%; moderate, 50-70%; severe, 70%; NASCET, 1991; Von Reutern et al., 2012) and atherosclerotic burden (various cardiovascular risk factors, obesity, hypertension, etc.) will be recruited to participate in this study. Upon receiving written informed consent participants will be familiarized to the measurements and equipment. Non-invasive ultrasound assessment of the individual’s middle cerebral and internal carotid arteries will be performed to assess adequate measurement clarity. If we are unable to accurately image the participants’ ICA, they will be excluded. Potential subjects will be excluded from participation if they do not speak English (for safety reasons the participant must be able to communicate with the research team in an instantaneous manner), or are pregnant [self reported – or pregnancy test result: improbable given the likely age of the majority of stenoses patients we will have access to (~>50 years)]. We will evaluate large cerebral extra-cranial artery vasoreactivity using three common and integrative assessments (cerebral flow-mediated dilatation, cFMD; CVR; and common carotid artery cold pressor reactivity, CAR). Patients with unstable plaques, stenoses >90%, or with ICA stenoses exceeding ~3cm above the carotid bifurcation will be excluded. First, endothelium-dependent dilation in the ICA (cFMD) will be assessed during transiently (30 seconds) elevated shear stress caused by increased inspired carbon dioxide using the Douglas bag method (5% CO2, 21% O2, balance N2), following protocols based on similar previous studies (Carter et al., 2016; Hoiland et al., 2017; Carr et al., 2020). Second, CVR (indexed by both middle cerebral artery blood velocity and ICA blood flow) will be assessed during steady-state (5 minutes) elevation of CO2 (5% CO2;). CAR provides a novel strategy to predict cardiovascular events and progression in patients with peripheral artery disease (van Mil et al., 2019). Similar to coronary artery responses to sympathetic stimulation, CAR causes common carotid artery vasoconstriction in the presence of impaired vascular function indicating increased cardiovascular event risk, compared to those with carotid vasodilation. All vascular function assessments will be imaged via simultaneous bilateral Duplex ultrasound to assess both left and right ICAs throughout each protocol. In those patients with stenoses but absent stroke, assessment will be conducted on presentation to the TIA clinic, in those with stenoses following stroke, patients will first be cleared for assessment by the PI/MD. Middle cerebral artery blood velocity, mean arterial blood pressure, end-tidal partial pressures of oxygen and carbon dioxide will be continuously assessed and recorded throughout CVR, cFMD, and CAR. These measures will be used in conjunction with several other recorded measures, including: CT/MRI and ultrasonographic categorization of carotid stenoses volume, history of cardiovascular risk, and subsequent adverse events will be recorded. Venous blood gas samples and blood viscosity quantification (at bedside without storage of samples) will be conducted. Twenty healthy young subjects (age: 18-40) will be recruited to participate in this study. Upon receiving written informed consent and completion of the health questionnaire (submitted alongside this CREB application) participants will be familiarized to the measurements and equipment. Non-invasive ultrasound assessment of the individual’s middle cerebral and internal carotid arteries (see below) will be performed to assess adequate measurement clarity. Potential subjects will be excluded from participation if they do not speak English (for safety reasons the participant must be able to communicate with the research team in an instantaneous manner), are taking any medications, are pregnant (self reported – or pregnancy test result), smoked within the previous year, have had, or currently have a history of cardiovascular, metabolic, respiratory or neurological disease, are obese (BMI>30), have had any invasive cerebral surgical procedures, have a cardiac pacemaker, or we are unable to attain reliable ultrasound images of their neck arteries. Based on our previous work in this area, sex differences are unlikely to influence any of the primary outcome variables when the time of testing for females is standardized for the menstrual cycle; therefore, both females and males will be tested. Females will be tested in their self-reported early follicular phase. Contraceptive use will be noted but will not be part of any exclusion criteria. Recruitment: We will recruit through a recruitment poster. Experimental visits: This study will be performed at the Centre for Heart, Lung and Vascular Health at the UBC Okanagan Campus. Following informed consent and familiarization with procedures, participants will visit the laboratory on three occasions with at least 48 hours between visits. Participants will arrive at the laboratory having fasted for ≥4hours and having abstained from alcohol, caffeine, and exercise for ≥24 hours. APPENDICES – FUTURE DIRECTIONS 249 Upon arrival for the first visit the participant will void their bladder, then lay down for 20-minutes of rest to permit hemodynamic quiescence, during which time experimental set up will take place (see sections: “Cerebrovascular measures”; “Respiratory Measures”; “Cardiovascular measures”; and “Blood sampling”). Resting blood pressure will be measured serially three times. Baseline blood samples will be taken. Following twenty minutes of rest participants will undergo a Transient CO2 Test, after which the participant will undergo one of four separate protocols (see “Continuous Hypercapnia”; “Intermittent Transient Hypercapnia”; “Continuous Shear Match Hypercapnia”; “Time control”) in a quasi-randomized fashion. The order of CH, ITH, and TC visits will be randomized, however CSH will be completed on a visit which by necessity must be subsequent to ITH. Blood sampling and Transient CO2 Test will be completed immediately following each protocol, and again 30 minutes after the end of CO2 exposure (see Figure 0-6). For all protocols, cardiovascular, respiratory, and intracranial measures will be recorded continuously from 1 minute prior to the stimulus until 40 minutes after completion. Extracranial ultrasound will be recorded during the Transient CO2 tests; as well as for one minute at minutes 10, 20, 30, 40, 50 and 59.5 during the stimuli. Figure 0-6: Appendix B.5 - Timeline of experimental visit. Data Analysis Cerebral blood flow: The CBF response to hypercapnia will be scaled to the level of arterial blood gas perturbation to calculate a CVR profile. CVR is the slope response of the CBF increase during an alteration in arterial blood gases, which will be calculated in both absolute (mL · min-1) and relative (%) terms (e.g., 5% ΔCBF · 1 mmHg ΔPaCO2) by using linear regression (Willie et al., 2012). Finally, the ICA diameter change during a transient change in shear stress will be analyzed by using custom in-house software that has been validated and described in detail (Carter et al., 2016; Hoiland et al., 2017). Blood pressure, intracranial cerebral blood velocities (TCD), and the cerebral metabolic rate of oxygen will also be examined. Data from all continuous measures will be extracted from the acquisition system (Labchart 7, AdInstruments, Colorado, US) and input to a Windows Excel spreadsheet for statistical analysis. Blood biomarkers: Changes in systemic circulating biomarkers (e.g., nitrite; Section “Biomarker analysis”) will be calculated. Blood pressure and intracranial cerebral blood velocities (TCD), will also be examined. Statistical Analyses All statistical analysis will be performed with the Statistical Package for the Social Sciences (SPSS, V24). A two factor linear mixed model will be used to analyze ICA Transient CO2 Test dilation (factors: test(CH/ITH/CSH/TH) & time(pre/post)), which will include “subjects” as a random factor to allow for subject drop out. ICA diameter pre, during at 30 minutes, at protocol end, and at 10 minutes and 20 minutes post protocol will be compared with a 3 by 5 factorial repeated measures ANOVA (factors: time & test). Blood concentrations of NO will be compared with 3 by 2 factorial repeated measures ANOVA (factors: time & test). InstrumentationcFMD5 minutes restProtocol: CH, ITH, CSH, or TC ICA diameter minute 10,20,30,40,50,60blood samplingblood sampling5minutes postcFMDcFMDblood sampling30 minutes postAPPENDICES – FUTURE DIRECTIONS 250 SAMPLE SIZE JUSTIFICATION Based upon previous studies conducted by our laboratory investigating FMD of the peripheral blood vessels (Lewis et al., 2014) and changes in diameter of the internal carotid artery (Lewis et al., 2014; Hoiland et al., 2014; Hoiland, et al., 2016), which used relatively small sample sizes (e.g., n=11), a sample size of 20 should more than suffice to detect changes in internal carotid artery diameter. RISKS Changes in arterial blood gases: There are no major risks associated with the mild changes in PaCO2, however, participants may experience light-headedness or dizziness during increases and decreases in PaCO2, respectively. Upon removal from the end-tidal forcing system, these symptoms quickly dissipate. Participants will be closely monitored throughout the protocol, however, and in our experience of conducing >2000 of these tests in our previous CREB approved studies (e.g. # H10-01375; H11-01105; H11-03287; H13-02620; H13-03081; H14-00922; H15-01513) there have been no ill effects reported. Venous catheterization: Venous puncture can cause minimal discomfort and/or bruising. Infection, excess bleeding, clotting, and/or fainting also are possible, although unlikely. Venous catheterization is considered a safe procedure. Venous catheters have previously been safely implemented into our research both at rest and during maximal exercise (refer to H14-00205, H11-03287, and H16-01028). These risks will be minimized by; 1) the use of experienced phlebotomists; 2) lines only being in situ for short periods of time (<3 h); We have extensive experience with these procedures in our laboratory. We have a 100% safe track record of conducting these procedures, including previously approved and completed UBC CREB studies (e.g. CREB ID # H10-01375; H11-01105; H11-03287; H13-02620; H14-00922; H15-01513). Transcranial Doppler and extracranial ultrasound: There are no known health risks associated with transcranial Doppler ultrasound. We have been previously approved to use Transcranial Doppler ultrasound (refer to H16-01028). We have been previously approved to use extracranial ultrasound. IMPLICATONS Assessing the regional vasodilatory capacity for adaptation in “normal or healthy” extra-cranial arteries will aid our understanding of preventative measures for cerebrovascular disease such as Alzheimer’s and dementia, where brain blood flow issues are a known cause and symptom. B.6 Future directions: Feasibility and reliability of assessment of cerebral endothelial function using Douglas bag method. PURPOSE To assess the reliability and general feasibility of conducting an established test using the Douglas bag technique rather than dynamic end-tidal forcing. HYPOTHESIS We hypothesize that use of a Douglas bag will provide a sufficient stimulus to assess cerebral endothelial function, and that the Douglas bag method will elicit cerebral endothelial responses comparable in magnitude to end-tidal forcing methods, with both methods being similarly reliable. JUSTIFICATION The partial pressure of arterial CO2 (PaCO2) is the principle factor in the regulation of cerebral blood flow (CBF) and ventilation (V̇E). Vessel diameter changes contributing to CBF responses to changes in PaCO2 occur as far upstream as the large extra-cranial arteries such as the internal carotid arteries (ICA) (Willie et al., 2012; Hoiland et al., 2016b, 2017; Carr et al., 2020) and common carotid arteries (Lavallée et al., 2006; Carter et al., 2016; Iwamoto et al., 2018b, 2018a). Increased blood flow in response to the mechanisms conducted upstream from capillaries described above - upregulates shear stress mediated eNOS activity in larger vessels APPENDICES – FUTURE DIRECTIONS 251 and itself causes dilation independent of hypercapnia per se (Heistad et al., 1978; Lee, 1980; Rubanyi et al., 1990; Fujii et al., 1991, 1992; Mashour & Boock, 1999; Carter et al., 2016; Hoiland et al., 2017; Carr et al., 2020). Thus, measurement of cerebral artery diameter during controlled and transient elevations of cerebral shear stress provides an experimental paradigm for the specific assessment of cerebral endothelial function (Hoiland et al., 2017; Carr et al., 2020). This technique has been demonstrated to produce shear-mediated vasodilation of the internal carotid artery (Figure 0-7A), analogous to traditional flow mediated dilation (FMD) tests of the peripheral large arteries (Figure 0-7B); as such, this technique has been coined the cerebral FMD (cFMD). Shear-mediated vasodilation of arteries constitutes an endothelium-dependent measure of vascular function (Naylor et al., 2011; Green et al., 2017). As such, FMD assessments are widely practiced in peripheral arteries to assess endothelial function (Thijssen et al., 2011a, 2019) yet brachial artery (BA) measures of endothelium-dependent function are not indicative of ICA function (Carr et al., 2020). The ability to produce cFMD responses using cheap and easy-to-operate equipment may enhance the utility of the method, currently the assessment is typically achieved via dynamic end-tidal forcing (DEF). First, quantification of the feasibility and reliability of such a stimulus from other methods is paramount to ensuring that the technique is propagated with the utmost ‘signal’ and minimal ‘noise’. Figure 0-7: Appendix B.6 - Transient increases in shear stress causing internal carotid artery (A) and brachial artery (B) vasodilation. (A) cFMD. Carbon dioxide manipulation via dynamic end-tidal forcing (30 seconds of end-tidal partial pressure of carbon dioxide PETCO2 increased +9 mm Hg above baseline) causes a transient (~30-second) increase in cerebral shear stress which elicits vasodilation of the internal carotid artery. This increase in internal carotid artery diameter occurs following return of carbon dioxide and shear to baseline levels, and follows similar temporal pattern to that of a brachial FMD test (B) FMD. In the brachial artery, release of an arterial occlusion cuff elicits rapid shear rate increase, promptly followed by arterial vasodilatation. While cFMD allows for appropriate assessment and characterization of cerebral larger artery shear-mediated endothelium-dependent function, this test has yet only been characterized in young healthy adults (Hoiland et al., 2017; Carr et al., 2020). Furthermore, the studies that established this technique utilized dynamic end-tidal forcing equipment that is expensive, cumbersome, and requires high levels of operator skill and training. Additionally, mis-identification of ICA vascular responses as representing cerebral endothelium-dependent function is a common issue in the field (Carr & Ainslie, 2020), in part, due to common usage of certain techniques over others. As such, further application of the cFMD technique to clinical and non-specialist arenas requires that the assessment be portable, accessible, and less operator dependent. A potential downside of the technique being applied using Douglas bag methods is that the methods by which PaCO2 is manipulated can contribute considerably to differences in the stimulus efficacy and isolation of responses. In short, hypercapnic PaCO2 challenges can be elicited via breath-holding, fixed increases in the fraction of inspired CO2 (FICO2), rebreathing, modified rebreathing, and computerized end-tidal targeting and forcing (DEF) systems (Robbins et al., 1982; Duffin, 2011; Fierstra et al., 2013). A fundamental consideration is that PaCO2 is determined by both metabolically produced CO2 and alveolar ventilation. Although in healthy individuals at rest metabolically produced CO2 remains relatively constant, tidal volume and alveolar ventilation fluctuate breath-by-breath within individuals and, as such, the pressure of end-tidal CO2 (PETCO2) also changes to a small degree breath-by-breath. This makes the accurate targeting of a specific PaCO2 impossible when using changes in fixed FICO2 (Peebles et al., 2007; Duffin, 2011; Fisher, 2016). We aim to assess Douglas bag cFMD (cFMDD) responses and compare them with dynamical end-tidal forcing cFMD (cFMDDEF). We hypothesize that: 1) hypercapnic exposure via Douglas bag will elicit a transient shear rate increase comparable with that elicited by DEF and, in doing so, will causes the same vasodilation, thereby indicating feasibility of the Douglas bag method for cFMD use; 2), that cFMDD and cFMDDEF will show comparable within-individual variability; and 3), and reliability will also be similar between methods. BA ICA BA ICA01020304050Diameter change (%)FMD GTNArtery, P<0.001Test, P<0.001Interaction, P=0.58BA ICA04812Diameter change (%)Scaled for baseline diameter and SRAUCFMD0 200 400 6000102030-5Time (seconds)Diameter change (%)GTN 400ugICABABA ICA01020304050Diameter change (%)Scaled for baseline diameterGTNA B CD E FBA ICA051015Diameter change (%)Scaled for baseline diameterFMD0 60 360 420 480 540-2024604008001200Time (seconds)Diameter change (%)Cuff releaseDiameterShear RateShear rate change (%)0 100 200-202460102030Time (seconds)Diameter change (%)+9mmHgShear rate change (%)DiameterShear RateGBA ICA04812Diameter change (%)Scaled for baseline diameter and DSRAUCFMDH*APPENDICES – FUTURE DIRECTIONS 252 Experimental measures: Respiratory Measures: Ventilatory parameters will be measured continuously by inspiratory and expiratory pneumotachs (Series 3813, Hans Rudolph, Shawnee, KS). The flow signal from each pneumotach will be integrated to obtain tidal volume (VT). Breathing frequency (Fb) will be calculated breath-by breath using an online acquisition system (Labchart 7, AdInstruments, Colorado, US). Minute ventilation (VE) will be calculated as VT multiplied by Fb. Both PETCO2 and PETO2 will be measured using a gas analyzer connected to our data acquisition device (Gas Analyzer, ADInstruments, Colorado, USA). Cardiovascular measures: All cardiovascular variables will be sampled continuously at 1 kHz via an analogue-to-digital converter (Powerlab 16/30, ADInstruments). Mean arterial pressure (MAP), systolic blood pressure, diastolic blood pressure, will be assessed beat-by-beat via non-invasive finger photoplethysmography (Finometer PRO, Finapres Medical Systems, Amsterdam, Netherlands). Electrocardiography will be measured by lead-II electrocardiogram (Bioamp ML132, ADInstruments). Vascular measures: Cerebral blood flow: Blood velocity in the right middle cerebral artery (MCAv) will be measured a continuously using a 2MHz transcranial doppler (TCD) (Spencer Technologies, Seattle, WA). The TCD probe will be fixed to a headpiece (model M600 bilateral head frame, Spencer Technologies) and secured into place. MCA will be insonated through the middle trans-temporal window using previously described location and standardization techniques (Willie et al., 2011). Right internal carotid artery (ICA) will be assessed throughout using a 10 MHz multi-frequency linear array duplex ultrasound (Terason 3300 uSmart, Teratech) according to published guidelines (Thomas et al., 2015). Arterial diameter will be measured with B-mode imaging, while simultaneously pulse-wave mode will measure blood velocity. ICA diameter and velocity will be measured at least 1.5 cm distal to the common carotid bifurcation to eliminate recordings of turbulent and retrograde flow. All images will be recorded and stored as video files for offline analysis by using automated edge detection software (Woodman et al., 2001) following study completion. All recordings will be made in accordance with published guidelines (Thomas et al., 2015). Control of arterial blood gases: PETO2 and PETCO2 will be controlled by dynamic end-tidal forcing system (AirForce, GE Foster, Vancouver, BC, Canada) (Tymko et al., 2015, 2016) during cFMDDEF, and by Douglas bag during cFMDD. The end-tidal forcing system uses independent gas solenoid valves for O2, CO2, and N2 delivery. The system controls the volume of each gas being delivered to the inspiratory reservoir through a mixing and humidification chamber. PETO2, PETCO2, tidal volume, breathing frequency and minute ventilation are determined for each breath online using specifically designed software (Labview 13.0, National Instruments, Austin, TX, USA). Dynamic end-tidal forcing Cerebral flow-mediated dilation (cFMDDEF): Following collection of five minutes of resting supine data, participants will breathe simulated room air delivered from the end-tidal forcing system for two minutes during which breathing will be coached to ~20 breaths per minute (for the sake of sonographer imaging). At the two-minute mark, PETCO2 will be rapidly (<10 seconds) elevated to 9 mmHg above baseline for 30 seconds then returned to baseline values, which will be maintained for three minutes. ICA ultrasound images will be recorded throughout baseline, CO2 and for 3 minutes following. This test has previously been approved (CREB H16-00342; H17-01810). Douglas bag Cerebral flow-mediated dilation (cFMDD): Participants will breath normal room air through the same valve used for DEF for 1 minute during baseline ICA scan. Beginning 1 minute before initiation of baseline scanning, participants will be coached to breath ~20 breaths per minute (to elicit hypocapnia and mildly reduced CBF). After 1 minute of baseline scan, PETCO2 will be rapidly elevated via inspiration of 5% CO2, 20.9% O2, balance N2 from a Douglas bag reservoir for 30 seconds (~9-10 breaths), participants will then be returned to inspiration of normal room air. ICA ultrasound images will be recorded throughout baseline, CO2 and for 3 minutes following. APPENDICES – FUTURE DIRECTIONS 253 Experimental design Participants: 20 healthy participants - 10 male, 10 female - between 18 and 35 years of age will be recruited for a counterbalanced cross-over design. Participants will be excluded from the study if they: are taking any medications, are pregnant (self-reported – or pregnancy test result), currently smoke or smoked within the previous year, have had, or currently have a history of cardiovascular, metabolic, respiratory or neurological disease, are obese (BMI>30kg/m2), have had any invasive cerebral surgical procedures, have a cardiac pacemaker, we are unable to attain reliable ultrasound images of their neck arteries. Those who do not speak and read English will be excluded as oral communication is essential to participant safety. Based on our previous work in this area, sex differences are unlikely to influence any of the primary outcome variables when the time of testing for females is standardized for the menstrual cycle; therefore, both females and males will be tested. Females will be tested in their self-reported early follicular phase as this is when estradiol concentration is lowest. Contraceptive use will be noted but will not be part of any exclusion criteria. Experimental protocol: This study will be performed at the Centre for Heart, Lung and Vascular Health at the UBC Okanagan Campus. Participants will visit the laboratory on three occasions, which should last no more than ~20 minutes for the first, and ~2 hours each for the second and third visits, all totaling ~4.5 hours altogether. Upon completion of informed written consent and the health questionnaire (submitted alongside this application) participants will be familiarized to the measurements and equipment. Participants will arrive at the laboratory having fasted for ≥6hours and abstained from alcohol, caffeine, and exercise for ≥24 hours. Upon arrival the participant will be weighed and have height measured, then lay supine during experimental set up and for a further 20 minutes of rest to permit hemodynamic quiescence. Baseline normoxic normocapnic blood pressure, ventilation, PETO2, PETCO2, and heart rate will be recorded. Participants will then undergo cFMDD and cFMDDEF tests in randomized counterbalanced order, with at least 30 minutes of rest between each. The participant will then be de-instrumented. This will conclude the first experimental visit. Subsequently, participants will return for a second visit which will follow the same protocol as the first visit. The second visit will be scheduled for 48 hours following the first visit. All pre-testing restrictions will again be observed. Risks: Changes in arterial blood gases: There are no major risks associated with mild changes in PaCO2, however, participants may experience light-headedness or dizziness during increases and decreases in PaCO2, respectively. Upon removal from the end-tidal forcing system, these symptoms quickly dissipate. Participants will be closely monitored throughout the protocol. In our experience of conducting >2000 of these tests in our previous CREB approved studies (e.g. # H10-01375; H11-01105; H11-03287; H13-02620; H13-03081; H14-00922; H15-01513) there have been no ill effects reported. 10% CO2 has been used in multiple animal studies (Wolff & Lennox, 1930; Kliefoth et al., 1979; Adachi et al., 1992), while as high as 8% FICO2 has been well tolerated in human studies (Peebles et al., 2008) and +25 mmHg PETCO2 has been used by our group previously with no ill effects (Willie et al., 2012). Ultrasound: There are no health risks associated with ultrasound. SAMPLE SIZE JUSTIFICATION Based on previous studies conducted by our laboratory investigating cFMD (Lewis et al., 2014; Hoiland et al., 2014; Hoiland, et al., 2016) which used relatively small sample sizes (e.g., n=11), a sample size of 20 should more than suffice to detect the cFMD response, and allow for reliability comparisons between methods. Data Analysis the ICA diameter change during cFMDD and cFMDDEF will be analyzed by using custom in-house software that has been validated and described in detail (Carter et al., 2016; Hoiland et al., 2017; Carr et al., 2020). Data from all continuous measures will be extracted from the acquisition system (Labchart 7, AdInstruments, Colorado, US) and input to a Windows Excel spreadsheet for statistical analysis. APPENDICES – FUTURE DIRECTIONS 254 Statistical Analyses All statistical analysis will be performed with the Statistical Package for the Social Sciences (SPSS, V24) or Prism (Version 7.0, GraphPad Software Inc., La Jolla, CA, USA). Paired, two-tailed t-tests will be used to compare peak change values of PETCO2, MAP, HR, VE, ICA diameter, and ICA shear rate between cFMDD and cFMDD within-day, and between-day for each test. Repeatability of vasodilator characteristics (ICA shear rate, velocity, and diameter) between days within-subjects will be determined using Pearson's (r) and intraclass correlation coefficients (ICC2,1: two-way random effects, absolute agreement, single rater/measurement; (Shrout & Fleiss, 1979; McGraw & Wong, 1996). Within-subjects coefficients of variation will be calculated for between-day assessments of cFMDD and cFMDD (Hopkins, 2000). Finally, Bland-Altman plots will be produced for each variable to assess agreement/systematic bias (Bland & Altman, 1986). Recruitment: We will recruit via advertisements posted around campus, zoom-based classroom scripted advertisement, and emails sent to previous study volunteers who indicated on their consent forms that they were willing to be contacted regarding future studies. B.7 Future directions: Control of ventilatory sensitivity via cerebral blood flow regulation PURPOSE 1. To determine the influence of graded step changes in cerebrovascular reactivity to CO2 (CVR) on ventilatory sensitivity to CO2 (V̇E-CO2), within-individuals. HYPOTHESIS 1. We hypothesize that altered CVR will concomitantly produce inversely altered V̇E-CO2 at each stage within-individuals (i.e., augmented CVR → attenuated V̇E-CO2; and attenuated CVR → augmented V̇E-CO2). Justification: Cerebrovascular reactivity to CO2 (CVR) and ventilatory sensitivity to CO2: The partial pressure of arterial CO2 (PaCO2) is the principle factor in the regulation of both cerebral blood flow (CBF) and ventilation (V̇E). The relationship between acute changes in cerebrovascular reactivity to CO2 (CVR) and ventilatory sensitivity to CO2 (V̇E-CO2) has long been known, demonstrated during transient reductions in CBF in animal models (Hill, 1896; Pike, 1918; Hastings et al., 1921). Here, acute impairments to CVR typically lead to an increased V̇E-CO2. However, a clear relationship between changes in CVR and V̇E-CO2 is lacking, due to the number of mechanisms and redundancies involved in both responses. For example, V̇E-CO2 is indeed altered with acutely decreased CBF and CVR, yet only when CBF is severely reduced, possibly because CBF below some threshold induces failure of the brainstem respiratory control neurons (Chapman et al., 1979). For example, in goats, arterial ligation blunted CVR from 2.45 ml/min/mmHg at normal CBF to 0.78 ml/min/mmHg at 70% of baseline CBF (Chapman et al., 1979), while V̇E-CO2 was unaffected (at least when jugular venous PCO2 (PjvCO2) was taken as the mathematical stimulus); then when CBF was further reduced to 50% of baseline, both CVR and V̇E-CO2 responses were extremely blunted (see Figure 0-8). Dampened CVR responses (at least in the hypercapnic range) during reduced CBF are likely representative of the priority of cerebral perfusion (e.g., for substrate delivery and pressure regulation) over the acute regulation of acid-base balance (Harper & Glass, 1965; Artru & Colley, 1984) as well as physical limitation of cerebrovascular vasodilator reserve (mediated via the myogenic response to reduced CBF). APPENDICES – FUTURE DIRECTIONS 255 Figure 0-8: Appendix B.7 - Cerebrovascular reactivity and ventilatory sensitivity to carbon dioxide with reductions in cerebral blood flow. Cerebral blood flow (CBF) in goats expressed as 100, 70, and 50% of baseline control (A and B, respectively; V̇E-CO2 indexed against PaCO2 and PjvCO2, redrawn and modified from Chapman et al., 1979); and control baseline vs post indomethacin ingestion in humans (C; redrawn and modified from Xie et al., 2006). Note, the data shown in panel C directly supports the present hypothesis, i.e., changes in CVR and changes in V̇E-CO2 are inverse. Within-individual stepped changes with more than a single stage will elucidate this further. In humans, blunted CVR is concomitant with acute CBF reductions, and whatever the mechanism(s) – whether induced pharmacologically or by normal diurnal changes – blunted CVR is often reflected by an increased slope of the V̇E-CO2 response (Xie et al., 2006, 2009; Cummings et al., 2007; Fan et al., 2010, 2011; Hoiland et al., 2015). While CBF certainly contributes to the determination of V̇E-CO2 via modulation of the PbtCO2-PaCO2 gradient at the central chemoreceptors, nonetheless, it seems that minor reductions in basal CBF at rest and without further challenging stimuli result in little to no change in resting V̇E (Fan et al., 2010; Hoiland et al., 2015, 2016b). These studies show that attenuated [H+] washout during reductions in baseline CBF – along with impaired CVR due to encroachment into the vasodilator reserve aimed at re-establishing CBF - causes some degree of agonism of the V̇E-CO2 response. While Hoiland et al (2015) reported a lack of relationship between change in CVR and change in V̇E-CO2, the study utilized between-subjects correlations on single step changes in CVR. Should an investigator be able to modify CVR in a stepwise manner, and then assess the relationship between step-wise changes in CVR and concomitant V̇E-CO2 with methods that allow for CVR/V̇E-CO2 interaction, this should show that within-individuals a change in CVR should result in a concomitant but inverse change in V̇E-CO2. However, given the myriad factors involved in physiologically determining both responses, individuals may have different combinations of CVR and V̇E-CO2 responses (i.e., high CVR + low V̇E-CO2; high CVR + high V̇E-CO2; low CVR + low V̇E-CO2; or low CVR + high V̇E-CO2) and also may have greatly different graduated changes in either response (e.g., large or small effects of pharmacological agents on CVR; large or small effects of changes in CVR on V̇E-CO2), meaning that no two individuals would have the same ∆CVR/∆V̇E-CO2 slope. A useful analogy of the within-individual effect of graduated changes in CVR on V̇E-CO2 may be seen in the menstrual cycle. Here, for instance, female hyperoxic V̇E-CO2 is reduced during the late follicular phase compared to the early follicular and mid-luteal phases (MacNutt et al., 2012), while CVR may be higher during times of elevated estrogen (i.e. late follicular to luteal phase) (Diomedi et al., 2001). Cyclooxygenase: Pharmacologically induced acute reductions of CBF using indomethacin, for example, impairs CVR and enhances V̇E-CO2 without changing resting V̇E; yet, at least in goats acutely reduced CBF through direct ligation appears to blunt both CVR and V̇E-CO2. These two findings indicate that – as discussed above – there may be a crucial threshold below which reduced CBF begins to affect CVR and concomitantly V̇E-CO2, and that pharmacological impairments may blunt CBF responses under challenging stimuli to a degree that crosses that threshold. Additionally, CVR impairment may be dependent on the mechanisms or pathways impaired. For instance, while indomethacin reduces CBF and blunts CVR (Eriksson et al., 1983; Wennmalm et al., 1984; Degiulio et al., 1989; Wang et al., 1993; Markus et al., 1994; St. Lawrence et al., 2002; Shoemaker et al., 2020) via prostaglandin synthesis inhibition, there are marked differences between indomethacin and other cyclooxygenase inhibitors (Markus et al., 1994; Hoiland et al., 2015, 2016b; Hoiland & Ainslie, 2017), indicative of a permissive rather than essential role of cyclooxygenases in CVR. Nevertheless, since indomethacin has little or no effects on cerebral metabolic rate (Kraaier et al., 1992), plasma catecholamines (Wennmalm et al., 1984), arterial blood gases, or the peripheral chemoreceptors and ventilation directly (Xie et al., 2006), it provides an ideal pharmacological tool for interrogation of the CVR versus V̇E-CO2 relationship. 100 70 500123CBF (Δ% of baseline)CVR (Δml/min/ΔmmHg)100 70 500.00.51.01.5CBF (Δ% of baseline)VE-CO2 (ΔL/min/ΔmmHg)PaCO2PJVCO2Control Indomethacin0.00.51.01.52.02.5ResponseCVR (Δcm/s/ΔmmHg)VE-CO2 (ΔL/min/ΔmmHg)A B CAPPENDICES – FUTURE DIRECTIONS 256 Phosphodiesterase type 5: Cyclic guanosine monophosphate (cGMP) is a crucial regulator of vascular smooth muscle cell tone, promoting vasodilation; phosphodiesterases are enzymes that hydrolyze cGMP. As such, inhibition of phosphodiesterases prevents such degradation, allowing for unrestrained cGMP action. Sildenafil (Viagra) is just such a selective phosphodiesterase type 5 (PDE5) inhibitor, the effects resulting in systemically augmented responses to NO-mediated vasodilation. The primary clinical use of Sildenafil is as treatment for erectile dysfunction (Rosen & Kostis, 2003) and pulmonary hypertension (Michelakis et al., 2003). Given that sildenafil acts through potentiation of NO/cGMP-mediated vascular responses – explaining its use in erectile dysfunction and pulmonary hypertension - Sildenafil is thought to augment CVR (Diomedi et al., 2005; Jahshan et al., 2017), though there is conflict in the literature (Kruuse et al., 2009). Kruuse et al., (2009) found no change in blood-oxygen-level-dependent MRI signal during hypercapnia from pre to post sildenafil administration. Sildenafil appears to not cause concomitant effects on potential confounding variables such as baseline CBF, MAP, heart rate (HR), V̇E, or end-tidal PCO2 (PETCO2) or cerebral artery diameter (Kruuse et al., 2002, 2003; Diomedi et al., 2005; Jahshan et al., 2017). Aim: While there is ample evidence (as discussed above) that reduced CVR facilitates a heightened V̇E-CO2 response, there are as yet no studies investigating this in humans using a within-individuals design and progressive step changes in CVR to determine the within-individual CVR - V̇E-CO2 relationship. These two physiological systems of control (CVR and V̇E-CO2) represent crucial aspects of cerebral and systemic homeostasis, and as such, full understanding of the within-individual relationship would progress and contribute to the understanding of numerous situations in which either system is affected, e.g., many pathologies and clinical settings. We aim to assess the direct influence of graded step changes in CVR on V̇E-CO2. We hypothesize that changes in CVR will produce concomitantly and inversely altered V̇E-CO2 at each stage (see Figure 0-9 for hypothetically expected results and Figure 0-10 for visualization of the theorized effects of step changes in CVR). Figure 0-9: Appendix B.7 - Hypothetically expected findings Illustration of hypothetical within-individual CVR vs V̇E-CO2 slopes across altered CVR, and example of repeated measures correlations of the effects of modified CVR on V̇E-CO2, illustration individual data (small colour circles and repeated measures correlations) and overall correlation (black dashed line). CVR VE-CO2CVR VE-CO2APPENDICES – FUTURE DIRECTIONS 257 Figure 0-10: Appendix B.7 - Illustration of the theorized relationship between CVR and ?̇?E-CO2. (A) Theoretical schematic of the relationship between two adjacent fluid compartments separated by a semipermeable membrane (modified from Hoiland et al., 2015). (B) CBF by PaCO2, representative of CVR, with high (red) and low (blue) sensitivity CVR also represented. (C) The implications of both high and low CVR sensitivities on central ventilatory drive. (A) Upon the occurrence of a square-wave change in concentration or pressure of a component substance in one compartment, equilibration up to 95% with the other compartment will occur over three time constants (𝜏). One 𝜏 is the duration required for fluid equal in volume to that of the first compartment to flow through the membrane into the second compartment; as such, if the first compartment is 500ml, then 95% equilibration would require 1.5 l to move across the membrane. Given the compartmentalization of the cerebrovascular circulation and interstitial fluid compartment of the brain, this model can be applied to understand how changes in CBF and/or CVR can determine prevailing central chemoreceptor PCO2 /[H+], and therefore, how CVR can inversely alter V̇E-CO2. Per Eq. 2, PbtCO2 is dictated by PaCO2, medullary CO2 production, and medullary blood flow, and varies proportionally with PaCO2 and inversely with medullary blood flow. However, in order for this theoretic conception to be applied appropriately, several other assumptions need also be taken into consideration, including; 1), tissues are continually producing CO2 via metabolism, 2), blood flow across - and blood volume surrounding - the brainstem increases or decreases with PaCO2, 3), total CSF volume changes reciprocally with CO2 induced cerebral blood volume changes (Grant et al., 1989), 4), central chemoreceptor intracellular PbtCO2 is dependent on CSF PCO2 and as such in reality the system involves two boundaries and three fluid compartments (arterial, CSF, and intracellular), 5), PaCO2 changes only as rapidly as systemic perfusion allows, and 6), cerebral metabolism – and therefore central V̇CO2 – may (Kogure et al., 1975; Kliefoth et al., 1979; Horvath et al., 1994; Yang & Krasney, 1995; Xu et al., 2011) be altered by PaCO2. So, while a rapid change in PbtCO2-PaCO2 gradient can occur (though certainly not a strict square wave change), both the PaCO2-PbtCO2 gradient and the 𝜏 will be in constant flux accordingly due to the aforementioned factors. For ease, one can assume that the true relationship is such that both compartments reside somewhere on the curved dashed line, based on CBF, prevailing PaCO2, and metabolic CO2 production. Higher flow across the compartment membrane would result in a right shift down the curved line and a reduced PaCO2 - PbtCO2 gradient, as would effectively be the case in an individual with highly sensitive CVR (as noted by red line). With a very high CVR, the difference between PaCO2 and PbtCO2 would, in theory, remain unchanged regardless of changes in PaCO2, and as such, no changes in the central chemoreceptor drive to breathe would occur (this conception is purely for the purpose of comprehension of the CVR vs V̇E-CO2 relationship; this degree of CVR is impossible given the limit of vasodilation within the rigid skull). Conversely, with a low or no CVR response (as noted in the blue line), changes in drive to breathe would occur linearly with changes in PaCO2. Experimental measures: Respiratory Measures: Ventilatory parameters will be measured continuously by inspiratory and expiratory pneumotachs (Series 3813, Hans Rudolph, Shawnee, KS). The flow signal from each pneumotach will be integrated to obtain tidal volume (VT). Breathing frequency (Fb) will be calculated breath-by breath using an online acquisition system (Labchart 7, AdInstruments, Colorado, US). Minute ventilation 35 40 45 5050010001500CBF (mL/min)PaCO2 (mm Hg)High CVRLow CVRRegular CVRPbtCO2Low reactivityHigh ReactivityPbtCO2 – PaCO2gradient at constant CBF(no CVR)PaCO2 ! !!Effect of altered CVR on PbtCO2 – PaCO2 gradient35 40 45 500510152025Central chemoreceptor drive to breathe (L/min)PaCO2 (mm Hg)AB CAPPENDICES – FUTURE DIRECTIONS 258 (V̇E) will be calculated as VT multiplied by Fb. Both PETCO2 and PETO2 will be measured using a gas analyzer connected to our data acquisition device (Gas Analyzer, ADInstruments, Colorado, USA). Cardiovascular measures: Mean arterial pressure (MAP), systolic blood pressure, diastolic blood pressure, will be assessed beat-by-beat via non-invasive finger photoplethysmography (Finometer PRO, Finapres Medical Systems, Amsterdam, Netherlands). Heart rate (HR) will be assessed via lead-II electrocardiogram (Bioamp ML132, ADInstruments). All cardiovascular variables will be sampled continuously at 1 kHz via an analogue-to-digital converter (Powerlab 16/30, ADInstruments). Vascular measures: Blood velocity in the right middle cerebral artery (MCAv) and left posterior cerebral artery (PCAv) will be measured a continuously using a 2MHz transcranial doppler (TCD) (Spencer Technologies, Seattle, WA). The TCD probe will be fixed to a headpiece (model M600 bilateral head frame, Spencer Technologies) and secured into place. MCA and PCA will be insonated through the middle trans-temporal windows using previously described location and standardization techniques (Willie et al., 2011). Right internal carotid artery (ICA) and left vertebral artery (VA) will be assessed using a 10 MHz multi-frequency linear array duplex ultrasound (Terason 3300 uSmart, Teratech) according to published guidelines (Thomas et al., 2015). Arterial diameters will be measured with B-mode imaging, while simultaneously pulse-wave mode will measure blood velocities. ICA will be measured at least 1.5 cm distal to the common carotid bifurcation to eliminate recordings of turbulent and retrograde flow. All images will be recorded and stored as video files for offline analysis by using automated edge detection software (Woodman et al., 2001) following study completion. All recordings will be made in accordance with published guidelines (Thomas et al., 2015). Catheterization: In a subset of the sample (n=~6-10), two catheters will be used: 1) under local anaesthesia (Lidocaine, 1.0%) and ultrasound guidance, a 20-gauge arterial catheter (Arrow, Markham, ON, Canada) will be placed in the left radial artery; and 2) under local anaesthesia (Lidocaine, 1.0%) a central venous catheter (Cook Medical, Bloomington, IN, USA) will be placed in the right internal jugular vein and advanced cephalad to the jugular bulb (Schell & Cole, 2000; Ainslie et al., 2014). Our research team was the first to obtain ethical approval for and establish this technique in Canada; we have safely carried out >5 related studies (Smith et al., 2014; Ainslie et al., 2014; Bain et al., 2015, 2016, 2017; Willie et al., 2015b). Arterial and jugular venous samples will be drawn at discrete timepoints during the experimental protocols. Specifically, at baseline and the end of each stage of each test; 11 timepoints in total (22 arterial and jugular venous draws total). Hematological measures: In the catheterized subset, total blood drawn at each sampling timepoint will not exceed ~5mL. At each blood draw ~1.0 mL of arterial blood will be withdrawn into EDTA vacutainers. With this arterial blood sampler, whole blood viscosity will be measured within 30 minutes of acquisition in duplicate at 37ºC at shear rates of 11.25, 22.5, 45, 90, 225, 337.5 and 450 s-1 using a cone and plate viscometer (DV2T Viscometer, Brookfield Amtek, USA) and circulating heating water bath (TC-150, Brookfield Amtek, USA). The average of the final five seconds of 30 second steps were acquired at each shear rate. When repeated measures exceeded the viscometer’s error for a given shear rate (e.g. >0.1 mPa·s at a shear rate of 225 s-1), a third viscosity measure was obtained and the average of the two closest measures reported. Additionally, at each blood draw ~1.0 mL of arterial and venous blood will be drawn into a pre-heparinized syringe (SafePICO, Radiometer, Copenhagen, Denmark) and analyzed immediately at 37°C using a commercial blood gas analyzer (ABL90 FLEX, Radiometer). This analysis includes measurement of the partial pressure of arterial oxygen (PaO2), the partial pressure of arterial carbon dioxide (PaCO2), arterial oxygen saturation (SaO2), arterial oxygen content (CaO2), pH, [Hb], Hct, and calculation of bicarbonate ion concentration [HCO3-]. Control of arterial blood gases: PETO2 and PETCO2 will be controlled by dynamic end-tidal forcing (DEF) system (AirForce, GE Foster, Vancouver, BC, Canada) (Tymko et al., 2015, 2016). The end-tidal forcing system uses independent gas solenoid valves for O2, CO2, and N2 delivery. The system controls the volume of each gas being delivered to the inspiratory reservoir through a mixing and humidification chamber. PETO2, PETCO2, APPENDICES – FUTURE DIRECTIONS 259 tidal volume, breathing frequency and minute ventilation are determined for each breath online using specifically designed software (Labview 13.0, National Instruments, Austin, TX, USA). Additionally, for the single Douglas bag method test, a 200 L Douglas bag will be used. Cerebrovascular reactivity to CO2 (CVR) and ventilatory sensitivity to CO2 (?̇?E - CO2): Three separate tests of CVR will be utilized herein; two which use DEF and one which uses Douglas bag. Participants will breathe simulated room air delivered from the DEF system. Following baseline scan, PETCO2 will be elevated to +3, +6, and +9 mmHg above baseline for 5-minutes steps from attainment of steady-state (determined by PETCO2 within +/-1 mm Hg of target), while PETO2 is elevated to hyperoxic levels (~250 mm Hg) throughout. Following the +9 mmHg stage participants will return to breathing actual room air. ICA and VA ultrasound images will be recorded throughout baseline and during steady state stages of CO2. Throughout, participants will be reminded to breathe at a pace and volume that feels comfortable and necessary, this is so that the V̇E-CO2 response can also be quantified using DEF and during the same stimulus as CVR. DEF separates the influence of V̇E on CVR (Howe et al., 2020), such that changes in V̇E do not influence CVR, this means that the V̇E-CO2 response is unable to correct systemic PaCO2 the way it normally would with environmental stimuli. Should participants voluntarily change their VE during the test, this would not affect the CVR measure, but would change the calculated V̇E-CO2. The reflexive unaltered V̇E response to hypercapnia however, still represents V̇E-CO2, as such, participants will be reminded to breathe ad libitum throughout. Figure 0-11: Appendix B.7 - CVR and ?̇?E-CO2 using DEF Note that blood sampling will only occur in the catheterization subset. DEF for a single 5 minute step of baseline to +9 mm Hg. Figure 0-12: Appendix B.7 - Single stage CVR and ?̇?E-CO2 using DEF Note that blood sampling will only occur in the catheterization subset. CVR using a Douglas Bag with 6% fraction of inspired CO2 (FICO2) will also be conducted to assess CVR and V̇E-CO2 using methods which: 1), allow V̇E to reflexively influence PaCO2 (to abolish the limitation that DEF precludes V̇E influence on PaCO2), and 2), are commonly utilized worldwide for the sake of accessibility and applicability to other studies. For CVR/V̇E-CO2 assessment via Douglas bag technique, a bag will be prepared prior to use, with 6% CO2, balance CVR2 min baseline5 minutes +9 mmHg5 minutes +6 mmHg5 minutes +3 mmHg1 min scan 1 min scan 1 min scanA and V blood drawsCVR2 min baseline5 minutes +9 mmHg1 min scanA and V blood drawsAPPENDICES – FUTURE DIRECTIONS 260 O2, to elicit hyperoxia, thus meaning that the V̇E response represents almost entirely the response of the central chemoreceptors. Figure 0-13: Appendix B.7 - Single stage CVR and ?̇?E-CO2 using Douglas bag Note that blood sampling will only occur in the catheterization subset. Cerebral flow-mediated dilation (cFMD): Participants will breathe simulated room air delivered from the end-tidal forcing system for two minutes during which breathing will be coached to ~20 breaths per minute (for the sake of sonographer imaging). At the two-minute mark, PETCO2 will be rapidly (<10 seconds) elevated to + 9 mmHg above baseline for 30 seconds then returned to baseline values, which will be maintained for three minutes. ICA ultrasound images will be recorded throughout baseline, CO2 and for three minutes following. This test has previously been approved (CREB H16-00342; H17-01810). Figure 0-14: Appendix B.7 - Cerebral flow-mediated dilation (cFMD) Note that blood sampling will only occur in the catheterization subset. Experimental design Participants: 26 healthy participants - 13 male, 13 female - between 18 and 35 years of age will be recruited. Participants will be excluded from the study if they are taking any medications, are pregnant (self-reported – or pregnancy test result), currently smoke or smoked within the previous year, have had, or currently have a history of cardiovascular, metabolic, respiratory, or neurological disease, are obese (BMI>30kg/m2), have had any invasive cerebral surgical procedures, have a cardiac pacemaker, or we are unable to attain reliable ultrasound images of their neck arteries. Based on our previous work in this area, sex differences are unlikely to influence any of the primary outcome variables provided that the time of testing for females is standardized for the menstrual cycle; therefore, both females and males will be tested. Females will be tested in their self-reported early follicular phase as this is when estradiol concentration is lowest and hormonal effects on CVR will be least. Contraceptive use will be noted but will not be part of any exclusion criteria. Pharmacology: Indomethacin: We will give a single oral dose administration of indomethacin (1.2 mg kg-1), which requires ~90 minutes to elicit peak cerebrovascular effects, as suggested by other human studies (Xie et al., 2006, 2009; Hoiland et al., 2015). Sildenafil: We will give a single oral dose of Sildenafil (100 mg), which requires ~60 minutes to achieve maximum plasma concentration (Jackson et al., 1999), used previously by our group (Stembridge et al., 2019) and others (Diomedi et al., 2005). Experimental protocol: This study will be performed at the Centre for Heart, Lung and Vascular Health at the UBC Okanagan Campus. Participants will visit the laboratory on three occasions, the first of which should last ~30 minutes and the second two of which should last no more than ~4 hours each, altogether totalling ~8.5 hours. The first visit will consist of a familiarization visit wherein participants will be given all information regarding the study, and should they give informed consent, will then CVR2 min baseline5 minutes 6% FiCO21 min scanA and V blood drawsA and V blood drawscFMD30 seconds +9 mmHg2 minbaseline 3 min post-bolus scanAPPENDICES – FUTURE DIRECTIONS 261 have both ICAs and both VAs scanned using duplex ultrasound to assess patency of reliable images. All participants will undergo both experimental sessions, in randomized, counterbalanced order. Upon completion of informed written consent and the health questionnaire (submitted alongside this application) participants will be familiarized to the measurements and equipment. The two visits will be separated by ≥48 hours and ≤1 week (except in the catheterization subset, whose visits will need to be separated by ≥4 weeks for participant comfort). Figure 0-15: Appendix B.7 - Experimental protocol Indomethacin visit: Participants will arrive at the laboratory having fasted for ≥6hours, abstained from caffeine for ≥12 hours, and abstained from alcohol, and exercise for ≥24 hours. Upon arrival the participant will be weighed and have height measured, then lay supine during experimental set up for a minimum of 20 minutes to permit hemodynamic quiescence before baseline normoxic normocapnic MAP, V̇E, PETO2, PETCO2, and HR are recorded and baseline blood samples drawn. Participants will then undergo baseline testing, with at least 20 minutes of rest between each. Once these tests are complete, the participant will ingest indomethacin (1.2 mg kg-1), then 90 minutes later tests will be conducted again. This will conclude this experimental visit. Sildenafil visit: Participants will arrive at the laboratory having fasted for ≥6hours, abstained from caffeine for ≥12 hours, and abstained from alcohol, and exercise for ≥24 hours. Upon arrival the participant will be weighed and have height measured, then lay supine during experimental set up for a minimum of 20 minutes to permit hemodynamic quiescence before baseline normoxic normocapnic MAP, V̇E, PETO2, PETCO2, and HR are recorded and baseline blood samples drawn. Participants will then undergo testing in randomized order, with at least 20 minutes of rest between each. Once these tests are complete, the participant will ingest sildenafil (100 mg). 60 minutes after sildenafil ingestion, tests will be conducted again. This will conclude this experimental visit. The second visit will be scheduled ≥48 hours and ≤1 week (except in the catheterization subset, whose visits will need to be separated by ≥4 weeks for participant comfort). In females, testing will be conducted within the same phase of menstrual cycle [or on the same day of the next menstrual cycle in those with arterial and jugular venous catheters (as assessed by self-report)]. All pre-testing restrictions will again be observed. Participants will not be informed of which drug was received during which session until after the completion of the entire experimental study. 26 young healthy participants (13M/13F)Instrumentation:Transcranial Doppler UltrasoundFinometer PhotoplethysmographerHans Rudolph PneumotachographGas analyzerElectrocardiographCatheterization subset: Radial artery + jugular venousInstrumentationIndomethacin90 min wash-in1.2 mg kg−1Baseline testing90 min wash-inInstrumentationSildenafil60 min wash-in100 mg60 min wash-inINDOSildenafilVisits randomized and counterbalanced orderTestingTestingBaseline testingAPPENDICES – FUTURE DIRECTIONS 262 Risks: Changes in arterial blood gases: There are no major risks associated with mild changes in PaCO2, however, participants may experience light-headedness or dizziness during increases and decreases in PaCO2, respectively. Upon removal from the end-tidal forcing system, these symptoms quickly dissipate. Participants will be closely monitored throughout the protocol. In our experience of conducting >2000 of these tests in our previous CREB approved studies (e.g. CREB ID # H10-01375; H11-01105; H11-03287; H13-02620; H13-03081; H14-00922; H15-01513; H19-02595; H20-02716; H20-03708; H21-01516) there have been no ill effects reported. Upto 10% FICO2 has been used in multiple animal studies (Wolff & Lennox, 1930; Kliefoth et al., 1979; Adachi et al., 1992), while as high as 8% FICO2 has been well tolerated in reported human studies (Peebles et al., 2008) and +25 mm Hg PETCO2 has been used by our group previously with no ill effects (Willie et al., 2012). Additionally, currently pilot work from our lab indicates that PETCO2 upto +30 mm Hg can also be tolerated. Pharmacological agent: Indomethacin: We will administer a single oral dose of indomethacin (1.2 mg kg-1). Indomethacin is a non-steroidal anti-inflammatory drug, which has been extensively used and characterized previously in both healthy humans and patients with cardiovascular disease. Indomethacin has been used extensively in CVR and V̇E-CO2 related studies for >35 years in humans (Eriksson et al., 1983; Wennmalm et al., 1984; Wagerle & Degiulio, 1994; St. Lawrence et al., 2002; St Lawrence et al., 2003; Rasmussen et al., 2004; Xie et al., 2006, 2009; Fan et al., 2010; Hoiland et al., 2015, 2016c; Peltonen et al., 2015; Mikhail Kellawan et al., 2020). The acute oral dose of indomethacin to be used in this study poses minimal risk to young healthy individuals. The primary associated risks are: • 3-9% incidence: Nausea, heart burn, dizziness • 1% incidence: diarrhea, abdominal pain, constipation, headache, vertigo, drowsiness, depression, fatigue Further, it is important to be aware that Indomethacin does pose several other serious side effects that occur with less than 1% incidence. These include, but are not limited to, gastro-intestinal bleeding, ulcertive collitis, anxiety, confusion, Stevens-Johnsons syndrome, and chest pain. Additional factors that will be avoided in our rigorous screening include: usage of antidepressants, beta-adrenergic blockers, heart failure disease, hypertension and related allergies to this drug (i.e., sulfate allergies). Our group has successful utilized indomethacin previously (e.g. CREB ID # H11-03287; H11-02657). Pharmacological agent: Sildenafil: We will administer single oral dose of sildenafil (100 mg). Side effects of sildenafil are generally transient and mild to moderate in nature. In clinical flexible-dosed, placebo-controlled, and randomized trials, side-effects included headache, facial flushing, dyspepsia, nasal congestion, and respiratory tract infection (percentage of patients reporting events 4.2 -15.8 %, n=734). Less common adverse reactions include abnormal electrocardiogram, angina pectoris, and arrhythmia, among others, though this was in <2% of patients in phase II/III controlled clinical trials - where a causal relationship is uncertain (see attached product monograph detailing all reported side-effects and adverse reactions). We have safely used sildenafil in previous CREB approved research (e.g. H11-03287). Arterial and jugular venous catheterization: Dr. Mypinder Sekhon will perform these procedures. Jugular vein and radial artery catheterization are considered safe procedures associated with few risks when made by experienced clinicians. The main risks include hematoma formation (6.1%) which is usually minor requiring no specific therapy other than temporary local pressure, infection (0.78%), pneumothorax (0.2%). Another risk with the use of jugular vein catheters is jugular valve incompetence (28%, this is a reduction in how well this valve works). This can potentially lead to back flow of blood, which may be related to short episodes of memory loss and/or headaches. These risks will be further minimized by; 1) the use of ultrasound to image the jugular vein and enable the precise location of the catheter to be quantified; 2) all lines will be inserted under strict aseptic techniques. This includes full surgical scrub, the wearing of a surgical hat, facemask and sterile gowns and the use of chlorohexidine or iodine-based cleaning solutions and sterile towels; 3) risks are also minimized by lines only being in situ for short periods of time (<6 h); 4) as mentioned, oxygen saturation, ECG and blood pressure will be continuously monitored throughout each experimental procedure. Oxygen will be available at all times, as will resuscitation drugs and equipment. We have extensive experience with these APPENDICES – FUTURE DIRECTIONS 263 procedures in our laboratory. We have a 100% safe track record of conducting these procedures, including previously approved and completed UBC CREB studies (e.g. CREB ID # H10-01375; H11-01105; H11-03287; H13-02620; H14-00922; H15-01513). To further minimize subject risk, patency of the radial and ulnar artery will be confirmed with a negative modified Allen’s test prior to arterial penetration. Transcranial Doppler ultrasound: There are no health risks associated with transcranial Doppler ultrasound. We have been previously approved to use transcranial Doppler ultrasound (e.g. CREB ID # H16-01028). Data Analysis For each CVR test for each individual, ICA and VA flow responses, and MCA and PCA velocity responses to hypercapnia will be scaled to the level of arterial blood gas perturbation to calculate a CVR profile. CVR is the slope response of the CBF increase during an alteration in arterial blood gases, which will be calculated in both absolute (mL · min-1) and relative (%) terms (e.g., 5% ΔCBF · 1 mmHg ΔPETCO2) using linear regression (Willie et al., 2012). Similarly, for each test, V̇E responses to hypercapnia will be scaled to the level of arterial blood gas perturbation to calculate a V̇E-CO2 profile. Pharmacologically induced changes in CVR and V̇E-CO2 from the baseline responses will be calculated (sildenafil ΔCVR and ΔV̇E-CO2, and indomethacin ΔCVR and ΔV̇E-CO2) Finally, the ICA diameter change during a transient change in shear stress (cFMD) will be analyzed using custom in-house software that has been validated. Trans-cerebral gradients for PaCO2 and pH will be calculated. The relationship between trans-cerebral exchange of these markers and the concurrent changes in CBF and V̇E will be examined. Blood pressure and the cerebral metabolic rate of oxygen will also be examined. Statistical Analyses Differences in both CVR and V̇E-CO2 between visits will be examined using linear mixed modal analysis [factor: trial (baseline-1, baseline-2, sildenafil, indomethacin)]. Between-day variability will be examined using paired two-tailed T-tests (baseline-1 vs baseline-2). Repeatability between days within-subjects will be determined using Pearson's (r) and intraclass correlation coefficients (ICC2,1: two-way random effects, absolute agreement, single rater/measurement; (Shrout & Fleiss, 1979; McGraw & Wong, 1996). Within-subjects coefficients of variation will be calculated for between-day assessments (Hopkins, 2000). For the main hypothesis of the study the slope of the ΔCVR/ΔV̇E-CO2 relationship will be examined within-individuals with repeated measures correlations using the rmcorr package for R to determine the influence of intraindividual changes in CVR on V̇E-CO2 (Bakdash & Marusich, 2017). These tests will be repeated separately for each CVR test. Each individual’s CVR, V̇E-CO2, and ΔCVR/ΔV̇E-CO2 slope will be compared between the three CVR tests using linear mixed modal analyses [factor: test (DEF-3-stage, DEF-1-stage, Douglas Bag)]. Finally, cFMD responses will be examined using linear mixed modal analysis [factor: trial (baseline-1, baseline-2, sildenafil, indomethacin)]. Sample Size Justification Based on our previous study using indomethacin (Hoiland et al., 2016b), we estimate that we will be adequately powered to see reductions in CVR from baseline with a sample size of n=~15. Based on other groups’ previous studies using sildenafil (Diomedi et al., 2005; Jahshan et al., 2017), we estimate that we will be adequately powered to see augmentations in CVR from baseline with a sample size of n=~15-20. Therefore, we estimate that a target sample size of n=26 should sufficiently allow for some (n=~3-5) participant drop-out while still allowing us to demonstrate the required changes in CVR. Appendix C Supplementary material to study chapters C.1 Study 3 – Supplementary materials Calculations Volumetric cerebral blood flow was calculated as follows: APPENDICES – FUTURE DIRECTIONS 264 Equation 1: ICA or VA flow (mL/min) = (12 ∙Peak Envelope Velocity) × (π (12 ∙diameter)2) x 60 Global CBF (gCBF) was calculated as twice the sum of our unilateral ICA and VA measurements, acknowledging potential limitations (Friend et al., 2021): Equation 2: gCBF (mL/min) = 2 × (ICA flow + VA flow). CVR was determined by the slope of the relationships between gCBF and PaCO2 (repeated with MCAv and PCAv). Shear stress was calculated as the product of shear rate and whole blood viscosity measured at 225 s-1: Equation 3: shear stress (dyne/cm2) = shear rate (s-1) ∙ (whole blood viscosity (cP) / 100). To account for cerebral perfusion pressure (CPP = MAP – jugular venous pressure) in our analyses of the CBF responses, cerebrovascular conductance (CVC) was subsequently calculated: Equation 4: CVC (mL/min ∙ mm Hg-1) = gCBF (mL/min)CPP (mm Hg) CVR of CVC was determined by the slope of the relationship between CVC and PaCO2. Oxygen extraction fraction (OEF) - defined as the fraction of O2 extracted from the arterial blood - is expressed as a percentage, where an increase can reflect either elevated O2 consumption, diminished O2 delivery, or both, and conversely a decrease of OEF can reflect either reduced O2 consumption, increased O2 delivery, or both: Equation 5: OEF (%) = CaO2 (mL/dL) – CvO2 (mL/dL) CaO2 (mL/dL) ∙ 100 Cerebral delivery of oxygen (CDO2): Equation 6: CDO2 (mL/min) = gCBF (mL/min)1000 ∙ [CaO2(mL/dL) ∙ 10] Cerebral metabolic rate of oxygen consumption (CMRO2) was calculated: Equation 7: CMRO2 (mL/min) = gCBF (mL/min) 1000 ∙ {[CaO2 (mL/dL) − CvO2 (mL/dL)] ∙ 10} CO2 in blood is transported in three main modes; freely dissolved (i.e. that which is measured as PCO2), and bound as both bicarbonate (HCO3−), and as carbamate to Hb (HbCO2), and it is the latter two by which the majority of CO2 is carried (van Slyke, 1921; Margaria, 1931, 1957; Roughton, 1935; Geers & Gros, 2000). As such, isovolumic haemodilution would certainly cause not only reduced CaO2, but also the reduction of total CO2 carrying capacity due to reduced [HCO3−] and both erythrocyte-bound and cell-free Hb. Furthermore, at sites such as the capillaries where CO2 diffuses freely across the vascular wall the appropriate measure is surely as a partial pressure, however, since dehydration and rehydration of CO2 is catalytically accelerated by carbonic anhydrase to ~2 milliseconds (Geers & Gros, 2000), at sites such as large vessels where blood is conveyed away from tissues and diffusional areas, the partial pressure of CO2 represents only a fraction of the total CO2 being transported. As such, we assessed cerebral CO2 parameters by calculating total arterial and cerebral venous CO2 content (CCO2) per Douglas et al. (1988) for comprehensive blood gas interpretation of the changes incurred by haemodilution. First, we calculated the plasma content of CO2 (PCCO2): Equation 8: PaCCO2 or PvCCO2 = 2.226 ∙ s ∙ PCO2 ∙ (1 + 10pH – pK`) Where s is the solubility coefficient of CO2 and pK` is the apparent pK [both calculated per Kelman (1967), with the assumption that core temperature was 37.5°C and unchanged with haemodilution]. PCCO2 was calculated for both arterial (PCaCO2) and jugular venous (PCjvCO2) blood using arterial pH, PCO2, and pK` for PCaCO2 and jugular venous pH, PCO2, and pK` for PCjvCO2. Then, CCO2: Equation 9: CaCO2 or CvCO2 = PCCO2 ∙ (1 – 0.0289 ∙ [Hb]) (3.352 – 0.456 x SO2) ∙ (8.142 – pH) Where SO2 is O2 saturation. CCO2 was calculated for both arterial (CaCO2) and jugular venous (CjvCO2) blood, using arterial Hb, SO2, and pH for CaCO2 and jugular venous Hb, SO2, and pH for CjvCO2. Then, manipulation of the Fick equation with substitution of CaO2 and CjvO2 with CjvCO2 and CaCO2, respectively, allowed for the calculation of the CO2 insertion fraction (CO2IF), defined here as the CO2 deposited into the jugular venous blood from the cerebral APPENDICES – FUTURE DIRECTIONS 265 tissues, expressed as a percentage (analogous and inverse to oxygen extraction fraction). An increase in CO2IF can reflect either elevated CO2 production, diminished CO2 washout, or both; and, conversely, a decrease of CO2IF can reflect either reduced CO2 production, increased CO2 washout, or both: Equation 10: CO2IF (%) = CjvCO2 (mL/dL) – CaCO2 (mL/dL) CjvCO2 (mL/dL) ∙ 100 We also calculated cerebral CO2 washout [in the supine position, jugular venous flow represents ~95% of total cerebral outflow (Manuel Valdueza et al., 2000; Gisolf et al., 2004); as such, we used arterial gCBF (mL/min) in place of global cerebral venous blood flow]: Equation 11: CO2 washout (mL/min) = gCBF (mL/min)1000 ∙ [CjvCO2 (mL/dL) ∙ 10] As well as the cerebral metabolic rate of CO2 production (CMRCO2): Equation 12: CMRCO2 (mL/min) = gCBF (mL/min) 1000∙ {[CjvCO2 (mL/dL) − CaCO2 (mL/dL)] ∙ 10} As another indicator of cerebral CO2 status, we took CjvCO2 to be an index of cerebral tissue CO2. Although the majority of work published to date has utilized PCO2 values [whether jugular venous, the cerebral a-v difference, or the arithmetic mean+1 (Fencl, 1986)] as indicators of cerebral tissue or cerebrospinal fluid CO2, given that cerebral tissue CO2 status will change not only with PaCO2, but also with CBF, intracellular [H+], [HCO3-], and tissue metabolism, and also for the reasons given above for calculation of CO2 content, we believe that CO2 content in the cerebral effluent is a suitable marker of cerebral tissue CO2 in this particular setting. Given the previous work demonstrating changes in [Hb] may influence NO bioavailability / signal transduction (Azarov et al., 2005; Tremblay et al., 2019b; Hoiland et al., 2020c), we also calculated trans-cerebral exchange of plasma NO2−: Equation 13: Plasma NO2− exchange (nmol/min) = gCBF (mL/min)1000∙ (1-Hct100) ∙ [arterial NO2− (nM) – jugular venous NO2 (nM)] Where positive values indicate cerebral net uptake, and negative values indicate net release. Supplemental figures: Figure 0-16. Supplemental Figure 1. Protocol schematic A, B, C, and D represent aspects of instrumentation and measurement (A, placement of radial arterial catheter, B placement of internal jugular venous bulb catheter, C and D are screen captures of internal carotid artery and vertebral artery respectively). E, F, and G represent the protocol; i.e., cerebrovascular reactivity to CO2 (3 stages of elevated PaCO2) followed by haemodilution via blood removal and replacement with Albumin, targeting a ~20% reduction of blood volume, followed by post-haemodilution CVR. Aspects of the diagram have been reproduced with permission from (Hoiland et al., 2021). Figure 0-17. Supplemental Figure 2. Trans-cerebral plasma nitrite exchange prior to and following haemodilution. HaemodilutionCBF+3 mmHg PaCO2+6+9CBFInternal Carotid ArteryVertebral Artery+3 mmHg PaCO2+6+9-18.1±2.2 % HctA B CDE F GUltrasound and a-v samplePre post-40-2002040Net NO2- exchange (nmol/min)pre post-80-4004080a-v NO2- (nmol)P=0.069 P=0.098† †a bUptakeReleaseUptakeReleaseAPPENDICES – FUTURE DIRECTIONS 266 Panel A depicts the arterial-to-jugular venous (a-v) difference for plasma nitrite (𝑁𝑂2−), while Panel B depicts the cerebral net exchange for plasma 𝑁𝑂2−. An obelisk (†) symbol reflects a significant a-v difference or net exchange of plasma 𝑁𝑂2− (one-sided t-test). Pre- to post-haemodilution comparisons were conducted with paired t-tests. N=10. Figure 0-18. Supplemental Figure 3. Blood gases during hypercapnia prior to and following haemodilution. Pre-haemodilution mean and standard deviation data presented in red, post-haemodilution mean and standard deviation data presented in blue, with individual data overlaid for both. A, partial pressure of arterial CO2 (PaCO2), B, partial pressure of arterial O2 (PaO2), C, arterial O2 content (CaO2). Comparisons conducted using linear mixed-model analyses with Bonferroni adjustments for post-hocs. Asterisk (*) symbols indicate a difference from baseline (P<0.05) in both trials, obelisk (†) symbols indicate a difference from the +3 mm Hg stage (P<0.05) in both trials, and double dagger (‡) symbols indicate a difference from the +6 mm Hg stage (P<0.05) in both trials. While hash symbols (#) indicate a difference (P<0.05) between pre and post haemodilution in all CVR stages. N=11 8090100110PaO2 (mm Hg)Baseline +3 mm Hg +6 mm Hg +9 mm HgTrial: P=0.342CO2: P<0.001Interaction: P=0.612 †3540455055PaCO2 (mm Hg)Baseline +3 mm Hg +6 mm Hg +9 mm HgTrial: P=0.414CO2: P<0.001Interaction: P=0.807 † ‡A B1416182022CaO2 (mL/dL)Baseline +3 mm Hg +6 mm Hg +9 mm HgTrial: P<0.001CO2: P=0.001Interaction: P=0.840# †C
Thesis/Dissertation
eng
Kinesiology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Ainslie, Philip N., 1974-
Cerebral and peripheral vascular responses to hypercapnia
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