Health and Social Development, Faculty of (Okanagan)
Health and Exercise Sciences, School of (Okanagan)
DSpace
UBCO
Howe, Connor A.
Doctor of Philosophy - PhD
University of British Columbia
Hypoxic environments elicit multiple physiological responses in order to maintain sufficient oxygen delivery, and ultimately, maintain function. The brain is particularly susceptible to hypoxic environments as it has essentially no metabolic storage capacity; therefore, it relies on a constant, well titrated supply of oxygen to match metabolic demand. The aim of this thesis was to investigate cerebral blood flow (CBF) regulation under acute and chronic severe environmental hypoxic conditions. Study 1 investigated regional and global patterns of CBF and oxygen delivery (CDO₂) in Andean and Sherpa high-altitude populations and in lowlanders, all residing at an altitude of ~5000m. The findings showed that Sherpa possess a unique CBF phenotype whereby CDO₂ is markedly lower compared to Andeans and lowlanders. Andeans, however, presented with a CBF pattern more similar to lowlanders, but with larger extracranial vessels. In Study 2, it was explored how children and adolescents residing in the highest city in the world (La Rinconada, Peru; 5100m) regulate CBF and whether it differed when compared to Sherpa living at 3800m, and lowlanders living in Wales, UK. The findings revealed that despite Andean children being more hypoxemic, their global CBF was not different compared to the other populations; however, their posterior CBF was ~24% lower. Finally, Study 3 investigated the effect of mild-hypothermia on CBF, CDO₂, cerebral metabolic rate of oxygen (CMRO₂), and blood brain biomarkers in healthy humans during progressive maximal hypercapnic hypoxia, simulating avalanche burial. The main findings were that 1) mild hypothermia reduced CBF during room-air breathing, primarily due to reductions in the partial pressure of arterial CO₂; and 2) CMRO₂ was not different with hypothermia under room-air conditions and during progressive hypercapnic-hypoxia; however, blood brain biomarkers of neuronal and axonal damage, and blood brain barrier permeability were reduced, indicating some selected neuroprotection. This dissertation furthers our understanding of how populations indigenous to high-altitude have divergent adaptations within the cerebrovasculature despite living under a similar environmental stressor. Additionally, it was shown for the first time that mild hypothermia does not alter cerebral metabolism during room-air breathing or during progressive hypercapnic-hypoxia, but may potentialy contribute to some unique aspects of neuroprotection.
Cerebrovascular function under extreme environmental hypoxia by Connor A. Howe B.Sc., University of British Columbia Okanagan, 2018 M.Sc., University of British Columbia Okanagan, 2020 A DISSERTATION 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) September 2024 © Connor Howe, 2024 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, the dissertation entitled: Cerebrovascular function under extreme environmental hypoxia submitted by Connor A. Howe in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Kinesiology Examining Committee: Dr. Phil Ainslie, School of Health and Exercise Sciences Supervisor Dr. Jonathan Little, School of Health and Exercise Sciences Supervisory Committee Member Dr. Justin Lawley, Institute of Sports Science Supervisory Committee Member Dr. Chris West, Faculty of Medicine University Examiner Dr. Kate Thomas, Department of Surgical Sciences External Examiner iii Abstract Hypoxic environments elicit multiple physiological responses in order to maintain sufficient oxygen delivery, and ultimately, maintain function. The brain is particularly susceptible to hypoxic environments as it has essentially no metabolic storage capacity; therefore, it relies on a constant, well titrated supply of oxygen to match metabolic demand. The aim of this thesis was to investigate cerebral blood flow (CBF) regulation under acute and chronic severe environmental hypoxic conditions. Study 1 investigated regional and global patterns of CBF and oxygen delivery (CDO2) in Andean and Sherpa high-altitude populations and in lowlanders, all residing at an altitude of ~5000m. The findings showed that Sherpa possess a unique CBF phenotype whereby CDO2 is markedly lower compared to Andeans and lowlanders. Andeans, however, presented with a CBF pattern more similar to lowlanders, but with larger extracranial vessels. In Study 2, it was explored how children and adolescents residing in the highest city in the world (La Rinconada, Peru; 5100m) regulate CBF and whether it differed when compared to Sherpa living at 3800m, and lowlanders living in Wales, UK. The findings revealed that despite Andean children being more hypoxemic, their global CBF was not different compared to the other populations; however, their posterior CBF was ~24% lower. Finally, Study 3 investigated the effect of mild-hypothermia on CBF, CDO2, cerebral metabolic rate of oxygen (CMRO2), and blood brain biomarkers in healthy humans during progressive maximal hypercapnic hypoxia, simulating avalanche burial. The main findings were that 1) mild hypothermia reduced CBF during room-air breathing, primarily due to reductions in the partial pressure of arterial CO2; and 2) CMRO2 was not different with hypothermia under room-air conditions and during progressive hypercapnic-hypoxia; however, blood brain biomarkers of neuronal and axonal damage, and blood brain barrier permeability were reduced, indicating some selected neuroprotection. This dissertation furthers our understanding of how populations indigenous to high-altitude have divergent adaptations within the cerebrovasculature despite living under a similar environmental stressor. Additionally, it was shown for the first time that mild hypothermia does not alter cerebral metabolism during room-air breathing or during progressive hypercapnic-hypoxia, but may potentialy contribute to some unique aspects of neuroprotection. iv Lay Summary In situations where the available oxygen is low, such as at high-altitude or when an individual is buried in snow, the body must rapidly adapt and compensate at multiple system levels to maintain energy and oxygen delivery. How brain blood flow is altered under some of the most profound low-oxygen environments that humans may experience was examined in the following studies: 1) how populations that live at high-altitude have different patterns of brain blood flow and oxygen delivery; 2) how children that live in the highest city in the world have different patterns of brain blood flow compared to other population living at lower altitudes; and 3) whether hypothermia may be protective of the brain under conditions of avalanche burial when humans are exposed to a combination of environmental stressors. v Preface The studies included in this thesis were approved by the University of British Columbia Clinical Research Ethics Board (CREB ID#s: H16-01028 and H21-00773) Chapter 1. Written by Connor Howe and edited by Prof. Phil Ainslie Chapter 2. Written by Connor Howe and edited by Prof. Phil Ainslie Chapter 3. In review at the Journal of Applied Physiology C.A.H., C.K.W., S.V. and P.N.A. conceived the study design. C.A.H., A.P., C.K.W., R.L.H., B.C., E.S., S.D., A.P., J.B., B.D., I.H., S.V., and P.N.A. were involved in data collection. C.A.H., A.P., and P.N.A. were involved in data analyses and interpretation. C.A.H., and P.N.A. drafted the manuscript. All authors critically reviewed and approved the final version of this manuscript. Chapter 4. In review at the Journal of Cerebral Blood Flow and Metabolism C.A.H., S.V., A.M. M.S. and P.N.A. conceived the study design. C.A.H., S.V., D.F., J.S.T., B.C., E.S., S.D., I.H., S.H., A.M., M.S., and P.N.A. were involved in data collection. C.A.H., D.F., J.S.T A.M., M.S., and P.N.A. were involved in data analyses and interpretation. C.A.H. and P.N.A. drafted the manuscript. All authors critically reviewed and approved the final version of this manuscript. Chapter 5. In preparation for submission to the Journal of Physiology vi C.A.H. and P.N.A. conceived the study design. C.A.H., R.L.H., T.D.G., A.R.S., J.M.J.R.C., G.A.V., M.M.T., T.S., V.C.C., A.L.M., T.A.D., M.S.S., and P.N.A. were involved in data collection. C.A.H., R.L.H., and P.N.A. were involved in data analyses and interpretation. C.A.H. and P.N.A. drafted the manuscript. All authors critically reviewed and approved the final version of this manuscript. Chapter 6. Written by Connor Howe and edited by Prof. Phil Ainslie Statement on generative artificial intelligence No generative artificial intelligence was used in any aspect of this dissertation. vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ........................................................................................................................ vii List of Tables ................................................................................................................................ xi List of Figures .............................................................................................................................. xii Acknowledgements .................................................................................................................... xiv Dedication ................................................................................................................................... xvi Chapter 1: Introduction ................................................................................................................1 1.1 Thesis objectives ............................................................................................................. 6 1.2 Individual study aims and hypotheses ............................................................................ 6 1.2.1 Study 1: Cerebral blood flow and oxygen delivery in Sherpa, Andeans, and lowlanders at 5000m. .............................................................................................................. 6 1.2.2 Study 2: Cerebral blood flow in Andean children and adolescents living above 5000m. .................................................................................................................................... 7 1.2.3 Study 3: Cerebral blood flow and metabolism under conditions of simulated avalanche burial in humans: the triple H syndrome................................................................ 7 Chapter 2: Literature Review .......................................................................................................8 2.1 Mechanisms of cerebral blood flow regulation .............................................................. 8 2.1.1 Oxygen ........................................................................................................................ 8 2.1.1.1 Adenosine triphosphate ..................................................................................... 11 viii 2.1.1.2 Adenosine ......................................................................................................... 11 2.1.1.3 Nitric oxide ....................................................................................................... 12 2.1.1.4 Prostaglandins ................................................................................................... 12 2.1.1.5 Potassium channels ........................................................................................... 13 2.1.2 Carbon dioxide .......................................................................................................... 13 2.1.3 Blood pressure and autoregulation............................................................................ 14 2.1.4 Metabolism ............................................................................................................... 16 2.1.5 Autonomic activity.................................................................................................... 17 2.2 Cerebral blood flow regulation in children and adolescents ......................................... 18 2.3 High-altitude ................................................................................................................. 19 2.3.1 Acclimatization to high-altitude ............................................................................... 22 2.3.2 Cerebral blood flow regulation at high-altitude ........................................................ 25 2.3.3 Indigenous high-altitude populations ........................................................................ 28 2.3.4 Cerebral blood flow regulation in indigenous high-altitude populations ................. 30 2.3.4.1 Cerebral blood flow regulation in children and adolescents at high-altitude ... 32 2.4 Hypothermia ................................................................................................................. 33 2.4.1 Systemic physiological responses to cold stress and hypothermia ........................... 35 2.4.1.1 Peripheral vasoconstriction ............................................................................... 35 2.4.1.2 Shivering and non-shivering thermoregulation ................................................ 38 2.4.1.3 Metabolism ....................................................................................................... 40 2.4.1.4 Hemoconcentration ........................................................................................... 40 2.4.1.5 Ventilation......................................................................................................... 42 2.4.2 Cerebrovascular responses to cold stress and hypothermia ...................................... 42 ix 2.4.3 Avalanche burial ....................................................................................................... 45 2.4.3.1 The physiology of avalanche burial .................................................................. 46 Chapter 3: Study 1 – Cerebral blood flow and oxygen delivery in Sherpa, Andeans, and lowlanders at 5000m ....................................................................................................................52 3.1 Introduction ................................................................................................................... 52 3.2 Methods......................................................................................................................... 54 3.3 Results ........................................................................................................................... 58 3.4 Discussion ..................................................................................................................... 65 3.5 Conclusion .................................................................................................................... 68 Chapter 4: Study 2 – Cerebral blood flow in Andean children and adolescents living above 5000m ............................................................................................................................................69 4.1 Introduction ................................................................................................................... 69 4.2 Methods......................................................................................................................... 71 4.3 Results ........................................................................................................................... 74 4.4 Discussion ..................................................................................................................... 80 4.5 Conclusion .................................................................................................................... 83 Chapter 5: Study 3 – Cerebral blood flow and metabolism under conditions of simulated avalanche burial in humans: the triple H syndrome ................................................................84 5.1 Introduction ................................................................................................................... 84 5.2 Methods......................................................................................................................... 87 5.3 Results ........................................................................................................................... 93 5.4 Discussion ................................................................................................................... 104 5.5 Conclusion .................................................................................................................. 110 x Chapter 6: Conclusion ...............................................................................................................112 6.1 Main findings .............................................................................................................. 112 6.2 Contributions to the field ............................................................................................ 112 6.3 Weaknesses of the dissertation/methodological considerations ................................. 114 6.4 Future directions and studies ...................................................................................... 116 6.4.1 Long term high-altitude adaptation in lowlanders .................................................. 116 6.4.2 Cerebrovascular phenotype of Ethiopian highlanders ............................................ 117 6.4.3 Longitudinal cerebrovascular measures in Andean children and adolescents ........ 118 6.4.4 Jugular bulb temperature measures in healthy humans .......................................... 118 Bibliography ...............................................................................................................................119 xi List of Tables Table 1-1. Morricocha study of hypoxic/anoxic exposure in Andeans .......................................... 3 Table 2-1. Categorization of hypothermia .................................................................................... 34 Table 3-1. Participant characteristics ............................................................................................ 56 Table 3-2. Arterial blood gas and blood pressure ......................................................................... 59 Table 3-3. Regional and global cerebral blood flow ..................................................................... 60 Table 3-4. Regional and global cerebral blood flow with covariate analysis ............................... 63 Table 4-1. Participant demographics and cardiorespiratory variables .......................................... 75 Table 4-2. Cerebral blood flow measures in Andean, Sherpa, and Lowlander Children and Adolescents ................................................................................................................................... 76 Table 4-3. Pregnancy and birth metrics for Andean participants ................................................. 79 Table 5-1. Cardiorespiratory variables during room air breathing and maximal hypercapnic-hypoxia under normothermic and hypothermic conditions .......................................................... 95 Table 5-2 Hematological and blood gas variables during room air breathing and maximal hypercapnic-hypoxia under normothermic and hypothermic conditions ..................................... 96 Table 5-3. Cerebrovascular variables during room air breathing and maximal hypercapnic-hypoxia under normothermic and hypothermic conditions .......................................................... 98 Table 5-4. Blood-brain biomarker and cytokine variables during room air breathing and maximal hypercapnic-hypoxia under normothermic and hypothermic conditions ................................... 101 xii List of Figures Figure 1-1. The Red Wing Study. ................................................................................................... 2 Figure 1-2. Time of consciousness in lowlanders and highlanders during acute hypoxia/anoxia .. 4 Figure 2-1. CBF and CDO2 reactivity to reductions in CaO2 ......................................................... 9 Figure 2-2. Erythrocyte mediated pathways of hypoxic cerebral vasodilation ............................ 10 Figure 2-3. The contemporary autoregulatory plateau ................................................................. 15 Figure 2-4. Cerebral glucose transporters ..................................................................................... 17 Figure 2-5. Changes in CBF across the lifespan ........................................................................... 19 Figure 2-6. Population estimates from high-altitude regions........................................................ 21 Figure 2-7. Phases of respiratory, and hematological acclimatization at high-altitude ................ 22 Figure 2-8. Changes in blood volume at high-altitude ................................................................. 25 Figure 2-9. Cerebral blood flow, oxygen content and oxygen delivery during ascent and acclimatization at high-altitude in lowlanders .............................................................................. 27 Figure 2-10. Estimations of paths and dates of human migration to high-altitude ....................... 29 Figure 2-11. Cerebral blood flow, oxygen content and oxygen delivery in Andeans and Sherpa at high-altitude .................................................................................................................................. 32 Figure 2-12. Mechanisms of reflex and local cold induced vasoconstriction ............................... 37 Figure 2-13. Reductions in CBF reported in a classic animal model, a clinical patient population, and in healthy humans. ................................................................................................................. 44 Figure 2-14. Avalanche burial survival curves ............................................................................. 46 Figure 2-15. Causes of death as they relate to duration of burial ................................................. 48 Figure 2-16. Relationship between PaCO2 and CMRO2 .............................................................. 50 xiii Figure 3-1. Regional cerebral blood flow ..................................................................................... 61 Figure 3-2. Oxygen content, global cerebral blood flow, and oxygen delivery ........................... 62 Figure 3-3. Cerebral blood flow with and without covariate analysis .......................................... 64 Figure 4-1. Regional and global cerebral blood flow ................................................................... 77 Figure 5-1. Hypercapnic-hypoxic breathing protocol. .................................................................. 89 Figure 5-2. Core body temperature ............................................................................................... 94 Figure 5-3. Regional and global cerebral blood flow ................................................................. 99 Figure 5-4. CDO2 and CMRO2 ................................................................................................... 100 Figure 5-5. Neurovascular unit brain biomarkers ....................................................................... 102 xiv Acknowledgements I’d like to start off by thanking my supervisor Professor Phil Ainslie. It’s been over 10 years since I first stumbled into your lab and I am forever grateful that you let me stick around. Thank you for being patient and understanding when I fail to respond to emails or take an unbearable amount of time to finish a manuscript. I am extremely appreciative of the countless opportunities and guidance you have provided me over these years. Beers on me. Thank you to my committee members Drs. Justin Lawley and Jonathan Little for providing your time and knowledge through my comprehensive exams and thesis defense. Thank you to my external examiner Dr Kate Thomas and University examiner Dr. Chris West for providing your time and insight. Beers on Phil. Ryan - Thank you for recruiting me as a participant all those years ago. I’d never have imagined the road that it would take me down, or more importantly, the people I would meet. Thank you for always being a role model in academia and life. Boom. Still got it. Michael - Boss, thank you for bringing me along with you on your business adventures and providing an outlet for me to learn, build, and procrastinate on writing. Soon the world will be covered in LBNP boxes. Travis - Thank you for always managing to get me excited about research and setting the example for running cool, hardcore studies. And more importantly, for teaching me how to brew beer. Jay - It’s wild thinking back to when you first came here in 2018. We didn’t really know what the deal was or who you were, but to see the researcher you became has been truly inspirational and I’m extremely grateful to call you my friend. Your random thought provoking theories always make those long testing days so enjoyable and I am looking forward to experiencing the next chapter of this journey in academia with you and Nique. xv Joshie - Thank you for being my friend. Your straight forwardness in science and life is always appreciated. Cody (and Watson) – You have been such an important friend to me and Hannah over these years. From climbing and outdoor adventures to adventures in life and academia in Copenhagen. Thank you for always being there for both of us. Especially Watson. Andrew… Rest of the Ainslie lab, past and present - Kurt, Tony, Chris, Daniella, Nia, Kevo, Matt, Hansen, Geoff, Alex, Courtney, Gus, Madden. There are far too many people to be able to thank you all properly. Everyone that has come through the lab are the reason I am here today. You have all been the greatest colleagues and friends that I could have asked for. Thank you to the members of the 2019 and 2020 Expedition 5300 team, Sam and co. A large portion of the work I am able to present within this thesis is thanks to you all accepting me into the research team, despite my extremely limited French vocabulary. Thank you to my family for your continued love and support through this wild ride. You never stopped supporting me through the twists and turns this life in academia has taken me and Hannah, and for that I am truly grateful. Han - thank you for your endless love and support, being an absolute academic superstar that I continue to look up to and admire, and for being my soup snake. xvi Dedication To excellence1 Chapter 1: Introduction Although a cliché statement for those in the field of cerebrovascular physiology, it is impossible to discuss regulation of blood flow to the brain without highlighting that despite only accounting for a rather miniscule proportion of total body mass (~2%), the brain receives ~15-20% of cardiac output (Williams & Leggett, 1989; Xing et al., 2017) and accounts for 20-30% of total oxygen consumed (Cheinberg & Stead, 1949; Reinmuth et al., 1965) under resting conditions. In addition to its high flow to volume ratio at rest, uninterrupted blood flow is a necessity due to the brain’s lacking of energetic storage capacity (Gjedde, 2002) whereby cessation of flow, even briefly, results in immediate loss of function. This loss of function was demonstrated in the ethically questionable Red Wing Study Investigations. Here, methodology was developed whereby a cuff was placed around the neck of schizophrenia patients, prison inmates, and young offenders, and inflated to 600mmHg to completely and rapidly occlude cerebral blood flow (CBF) (Rossen et al., 1943). This resulted in loss of consciousness in subjects within 4-10 seconds, highlighting how important coupling of oxygen and substrate delivery is to cerebral function, and by consequence, whole body function. 2 Figure 1-1. The Red Wing Study. Panel A: the custom made occlusion cuff which could either be uni-laterally or bi-laterally inflated to ~600mmHg to rapidly occlude cerebral blood flow and cerebral venous drainage. Panel B: John P Anderson, inventor of the cuff, 1942. Images from (Smith et al., 2011) The study of acute severe hypoxic and anoxic exposure would continue as technological advancements within the military sector during and following WW2 meant pilots were faced with an increased likelihood of experiencing profound levels of hypoxia previously only encountered by mountaineers or balloon aviators (LUFT et al., 1949; Velasquez, 1959; Ernsting & McHardy, 1960, 1963; Ernsting et al., 1960; Ernsting, 1963). These studies helped establish the limits in which the brain could maintain function under rapid hypoxic exposures and provided some of the first evidence that populations indigenous to high-altitudes may present with adaptations that are protective of the cerebrovasculature against hypoxic challenges. In 1959, Velasquez published work conducted in Peruvian Andean residents of Morricocha Peru (located at 4,540m) in which participants were rapidly exposed to multiple simulated altitudes between 30,000ft to 40,000ft using a hypobaric chamber (Velasquez, 1959) (Table 1-1). 3 Table 1-1. Morricocha study of hypoxic/anoxic exposure in Andeans Altitude Inspired PO2 Time of consciousness Feet Meters mmHg seconds 32,000 9,754 33.2 354 34,000 10,363 29.4 218 36,000 10,973 25.8 136 38,000 11,582 22.6 96 40,000 12,192 19.6 88 Andean residents of Morricocha Peru were exposed to simulated altitudes of 32,000ft to 40,000ft with duration of consciousness at each altitude recorded in seconds. Participants breathed through an oxygen mask as the chamber depressurized. Once the chamber reached the desired altitude the mask was removed and recording started. Data in these high-altitude residents was then compared to measures obtained in lowlander pilots under similar near anoxic conditions (Figure 1-2). The findings showed that Andeans living at 4,540m in Morricocha had a longer time of consciousness at each altitude and were able to tolerate even higher simulated altitudes compared to lowlanders. 4 Figure 1-2. Time of consciousness in lowlanders and highlanders during acute hypoxia/anoxia Mean duration of consciousness (seconds) in high-altitude Andean residents of Morricocha Peru (4540m) and in two sets of lowlander subjects (lines 1 and 2) between simulated altitudes of 30,000-40,000ft (9144-12,192m). Modified from (Velasquez, 1959). These findings highlighted that Andeans possessed some form of adaptation, resultant from either their time spent at altitude or related to their ancestry that provided them with a greater tolerance to hypoxia (Velasquez, 1959). Since these early studies, numerous investigators have studied populations that are indigenous to high-altitude including the Tibetan (Sherpa), Andean, and Ethiopian high-altitude populations. These studies have aimed to understand the different pathways by which humans adapt to maintain and preserve oxygen delivery under chronic hypoxic exposure. Much of this work has been conducted by - and highlighted in - the publications of Dr. Cynthia Beal (Beall et al., 1997, 2001, 2002; Beall, 2000, 2006, 2007; Erzurum et al., 2007; Hoit et al., 2011; Alkorta-Aranburu et al., 2012; Cheong et al., 2017) who described Andeans and Sherpa as possessing differing adaptations across a wide range of physiological systems (Beall, 2000, 2007). This work has been reviewed elsewhere (Luks et al., 2021). 5 While we continue to progressively tease apart these different pathways of adaptation in high-altitude populations, a number of important questions and areas of research remain to be answered, particularly related to the brain. As technology has developed and methods of assessing CBF have progressively become more portable and robust, quantification of the profile of cerebrovascular function in Andeans and Sherpa residing at high-altitude has been reported in various studies (Jansen et al., 1999, 2000, 2014; Appenzeller et al., 2004, 2006; Claydon et al., 2008; Jansen & Basnyat, 2011; Hoiland et al., 2019b; Stacey et al., 2023). Unfortunately, this is not the case regarding children and adolescents living at high-altitude. Most high-altitude research in Andean and Sherpa populations are limited to a snapshot of adaptation in young to middle aged adults with little understanding regarding CBF control during the early stages of life. Whether the adaptations that have been previously reported in Andean and Sherpa populations develop before, during, or after childhood and adolescents is largely unknown. Studying these cerebrovascular responses at high-altitude provides an opportunity to examine the pathways and mechanisms by which cerebral oxygen delivery is regulated and maintained under clinically significant levels of hypoxia, without the associated comorbidities which commonly occur with hypoxia resigning from or along aside illness and disease. Further, populations indigenous to high-altitude such as Sherpa and Tibetans may have developed differing adaptations to alter CDO2 which could serve as potential pathways to target pharmaceutically in various patient groups to modulate CDO2. Over the past century, the field of hypoxia research has grown both within the clinical and molecular scope, as well as in the integrative physiology field (Prabhakar, 2020). However, the effect of combined physiological stimuli and cross adaptation are much less understood (Lee et al., 2019). The term cross adaptation is often used to describe physiological effects resultant from one stimuli or stressor that elicit an adaptation that is beneficial under a separate stimuli or stressor. The lack of research investigating how combined and realistic environmental stressors affect physiological processes is somewhat ironic when justification for a majority of the work within the field of high-altitude physiology is based on the large number of individuals either living in these regions (Tremblay & Ainslie, 2021), or traveling for military service (Muza, 2007), religious pilgrimage (Basnyat, 2014), mountaineering, and trekking activities. Under these conditions, hypoxia is hardly ever acting as an isolated stimulus and understanding the combined impact of 6 environmental stressors such as hypoxia, hypothermia, hyperthermia, air pollution and UV exposure are highly relevant. The conditions in which individuals are exposed to during avalanche burial are perhaps some of the most extreme combination of environmental stressors (Falk et al., 1994a). This is especially the case for the cerebrovasculature which, in addition to being highly susceptible to anoxic damage, is highly sensitive to changes in CO2 (Kety & Schmidt, 1948; Hoiland et al., 2019a). The addition of hypothermia with profound hypoxia and hypercapnia during burial is particularly unique. Hypothermia represents a stimulus that is sometimes both used as a clinical tool to protect the cerebrovasculature (Bernard et al., 2002) and as an environmental stressor that can cause cardiovascular instability resulting in death (Paal et al., 2016). 1.1 Thesis objectives The aim of this dissertation was to evaluate how CBF is regulated under profound environmental stressors such as at high-altitude and during simulated avalanche burial. The two main objectives included herein aimed to address the following: 1. To understand whether populations indigenous to high-altitude have/or do not have differential regulation of CBF under hypoxic environments. 2. Determine how hypothermia may cause differential regulation of CBF and metabolism under conditions of simulated avalanche burial. 1.2 Individual study aims and hypotheses 1.2.1 Study 1: Cerebral blood flow and oxygen delivery in Sherpa, Andeans, and lowlanders at 5000m. In this study, regional and global CBF and cerebral oxygen delivery (CDO2) were compared between Andeans, Sherpa, and Lowlanders at a closely matched altitude of ~5000m. It was hypothesized that: 1) Sherpa would present with lower CBF and CDO2 compared to lowlanders at matched altitudes, suggestive of a unique protective physiological adaptation, and; 2) Andeans and lowlanders will present with similar CBF and CDO2. 7 1.2.2 Study 2: Cerebral blood flow in Andean children and adolescents living above 5000m. To expand our understanding of CBF across the lifespan in high-altitude populations, CBF was quantified in children and adolescents of Andean descent living at 5100m and compared to age, sex, maturity and body mass-matched Sherpa children living at high-altitude (3800m) and lowlander children living at sea-level. It was hypothesized that despite the higher altitude of residence, Andean children living at 5100m would present with similar CBF compared to Sherpa and lowlander children. 1.2.3 Study 3: Cerebral blood flow and metabolism under conditions of simulated avalanche burial in humans: the triple H syndrome. The purpose of this study was to determine the effects hypothermia on CBF, CDO2, cerebral metabolism, and neurovascular unit brain biomarkers during room air breathing and during progressive combined hypercapnic-hypoxia simulating conditions of avalanche burial. It was hypothesized that 1) during room-air breathing, hypothermia would elicit decreases in both CBF and CMRO2; and 2) during hypothermic hypercapnic-hypoxia, CBF, cerebral metabolism and circulating blood-brain biomarkers would be lower compared to normothermic hypercapnic-hypoxia. 8 Chapter 2: Literature Review 2.1 Mechanisms of cerebral blood flow regulation As outlined in Chapter 1, CBF is modulated in order to sustain sufficient O2 and substrate delivery to support neuronal function. This delivery occurs through multiple regulator pathways that respond to changes in the partial pressures of oxygen (PaO2) and carbon dioxide (PaCO2) within local tissue and vascular beds; alterations in neuronal activity and metabolic demand; perfusion pressure; and, sympathetic and parasympathetic activity. Within these regulatory pathways there appears to be multiple, often redundant mechanisms for modulating CBF. These primary mechanisms of CBF regulation will be discussed herein. 2.1.1 Oxygen Under most conditions, the cerebral vasculature is only somewhat sensitive to changes in PaO2 or, more precisely, arterial oxygen content (CaO2). This sensitivity, however, occurs at and below the steeper section of the oxygen–hemoglobin dissociation curve in which PaO2 is ~55 mmHg and where further reductions in PaO2 lead to an accelerated reduction in SaO2 [reviewed in:(Willie et al., 2014b; Hoiland et al., 2016; Carr et al., 2023; Mascarenhas et al., 2024)]. This response characteristic is due to the shape of the oxyhemoglobin dissociation curve and its impact on the relationship between PaO2 and CaO2. As such, changes in CBF in response to alterations in CaO2 are fairly linear, whereas the CBF response to PaO2 modulation is curvilinear. Under hypoxic conditions, increases in CBF can occur through cerebral vessel dilation within the branches of the extracranial (Lewis et al., 2014), intracranial (Wilson et al., 2011; Imray et al., 2014; Willie et al., 2014a) and pial vessels (Wolff et al., 1930). The magnitude of this increase in CBF is generally sufficient to maintain CDO2 across a range of hypoxic severities (Figure 2-1). 9 Figure 2-1. CBF and CDO2 reactivity to reductions in CaO2 Volumetric measures of cerebral blood flow (CBF) and cerebral oxygen delivery (CDO2) reactivity in response to reductions in arterial oxygen content (CaO2) reported across seven separate studies. Modified from (Hoiland et al., 2016). Hypoxic cerebrovascular dilation is believed to be primarily initiated through three erythrocyte mediated mechanisms (Figure 2-2), including: 1) The nitrite reductase pathway whereby circulating nitrite is reduced via deoxyhemoglobin to nitric oxide (Cosby et al., 2003; Doyle et al., 1981); 2) The S-nitrosohemoglobin (SNO-HB) pathway in which s-nitrosothiols (SNO) bind with circulating nitric oxide via covalent bonding in the lung to form SNO-HB (Jia et al., 1996), which can then release vasoactive SNO in the cerebrovasculature when the erythrocyte becomes deoxygenated (Lima et al., 2010; Stamler et al., 1997), and; 3) Adenosine triphosphate (ATP) 10 release from deoxyhemoglobin (Bergfeld & Forrester, 1992; Ellsworth et al., 1995, 2009). These three erythrocyte mediated pathways, regardless of their relative contribution to hypoxic cerebral vasodilation, lead to nitric oxide mediated increases in potassium channel conductance directly or through increases in guanylate cyclase (cGMP) activity (Pearce et al., 1990). Figure 2-2. Erythrocyte mediated pathways of hypoxic cerebral vasodilation ATP; adenosine triphosphate, AA: adenosine A2A receptor, Ca+; calcium channel. cAMP: cyclic adenosine monophosphate, cGMP; cyclic guanosine monophosphate, COX; cyclooxygenase, Deoxy Hb; deoxyhemoglobin, ENOS; endothelial nitric oxide, EET’s; epoxyeicosatrienoic acid, K+; potassium channel, MLCK; myosin light chain kinase, NO; nitric oxide, NO2; nitrate, PG; prostaglandins, PKA; cAMP-dependent protein kinase, PKG; cGMP-dependent protein kinase, SNO; s-nitrosohemoglobin, 5N; 5-neucleotide. 11 As evidence of the highly redundant control of CBF in humans, numerous downstream signaling molecules have also been implicated in contributing to cerebrovascular dilation in response to hypoxia, including ATP, nitric oxide, adenosine, K+ channels, and prostaglandins. 2.1.1.1 Adenosine triphosphate Adenosine triphosphate (ATP) is released from deoxyhemoglobin (Bergfeld & Forrester, 1992b; Ellsworth et al., 1995; Sprague et al., 1998; Dietrich et al., 2000; Kalsi & González-Alonso, 2012) and elicits vasodilation through three separate pathways; 1) ATP binds to P2Y2 purinoreceptors located on the endothelium stimulating endothelial nitric oxide production; 2) ATP breaks down into circulating adenosine and elicits vasodilation (Forrester et al., 1979; You et al., 1997; Dietrich et al., 2000) through the cyclicoxygenase (COX) pathway (You et al., 1997); 3) ATP binds to the P2X7 receptor on the erythrocyte, stimulating epoxyeicosatrienoic acid (EET) release (Jiang et al., 2007). This production of ATP from the erythrocyte is highly dependent on temperature (Kalsi & González-Alonso, 2012) and shear stress (Sprague et al., 1998); therefore, concomitant changes in core temperature and CO2 may alter cerebral hypoxic dilation. 2.1.1.2 Adenosine Adenosine is released from the breakdown of ATP by 5-nucleotide and binds to adenosine A2A receptors (Kalaria & Harik, 1988; Hein et al., 2013) which increase cAMP signaling (Sattin & Rall, 1970; Nordström et al., 1977) to increase K+ channel conductance (Hein et al., 2013) and cause vasodilation. Adenosine has been shown to elicit dilation in animal studies and adenosine antagonism or receptor blockade has been shown to cause reduced resting flow under both normoxic and hypoxic conditions (Wechsler R. L. et al., 1950; Gottstein & Paulson, 1972; Magnussen & Høedt‐Rasmussen, 1977). Importantly, however, the CBF reactivity to hypoxia is unaltered following adenosine administration (Bowton et al., 1988; Nishimura et al., 1993). Indeed, recent work in humans also show that theophylline - an adenosine antagonist - does not attenuate the CBF response to hypoxic stimuli at low altitude or high altitude (Hoiland et al., 2017), implying that it is not obligatory for hypoxic cerebrovascular vasodilation, despite a number of animal models suggesting otherwise (Mascarenhas et al., 2024). 12 2.1.1.3 Nitric oxide Nitric oxide elicits cerebral vasodilation through direct increases in potassium channel conductance (Bolotina et al., 1994) and via increasing cyclic guanosine monophosphate (cGMP) (Pearce et al., 1990). Evidence in humans using non-selective nitric oxide synthase blockade showed that hypoxic cerebral reactivity is decreased ~24% following L-NMMA infusion when assessed with duplex ultrasound (Hoiland et al., 2023). Similar findings have been observed with MRI based assessments with L-NMMA infusion whereby the hypoxic reactivity response is abolished (Van Mil et al., 2002). However, the contribution of each pathway and isoform of nitric oxide is still unclear and has been historically debated between the nitrite pathway and R-SNO pathway (Doctor & Stamler, 2011; Gladwin et al., 2006; Gladwin & Kim-Shapiro, 2008; Gladwin & Schechter, 2004; Premont et al., 2020). 2.1.1.4 Prostaglandins Prostaglandins are produced through the COX pathway after ATP binds to P2Y2 purinoreceptors. Prostaglandins then bind to IP and EP receptors (Davis et al., 2004) to increases cAMP signaling (Narumiya et al., 1999), and increasing k+ channel conductance. The ultimate contribution of prostaglandins on cerebral hypoxic vasodilation is unclear however. One study utilizing Indomethacin, which inhibits COX activity, showed that CBF reactivity to hypoxia in the anterior circulation was unchanged, while reactivity in the posterior circulation was reduced (Hoiland et al., 2015). Other studies have shown that apart from regulating CBF under resting normoxic conditions (Barnes et al., 2012; Peltonen et al., 2015; Mikhail Kellawan et al., 2020), COX does not regulated CBF under hypoxic conditions in an obligatory manner (Mikhail Kellawan et al., 2020). However combined COX inhibition using Indomethacin and ascorbic acid (to inhibit reactive oxygen species activation of the COX pathway) did appear to abolish the cerebral hypoxic reactivity, as assessed using transcranial doppler ultrasound (Harrell et al., 2019). 13 2.1.1.5 Potassium channels Adenosine triphosphate-sensitive potassium (KATP) channels are present in vascular smooth muscle cells and neurons, and elicit cerebral vasodilation by reducing cell membrane potential and inhibiting voltage gated calcium channel activity (Nelson et al., 1990; Faraci & Sobey, 1998). Work in humans using the KATP channel blocker glibenclamide has been shown to selectively reduce CBF hypoxic reactivity in the anterior circulation with no impact observed in the posterior circulation or in resting flow patterns (Rocha et al., 2020). This work suggests that greater redundancy - or differential mechanisms of action - may be present within the posterior cerebrovascular. 2.1.2 Carbon dioxide Regulation of CBF under normoxic conditions, is primarily regulated by alterations in the partial pressure of arterial carbon dioxide (PaCO2). Alterations in PaCO2 cause changes in blood flow by altering perivascular pH, and not necessarily by changes in arterial PaCO2 or arterial pH per se (Lambertsen et al., 1961; Harper & Glass, 1965; Kontos et al., 1977b, 1977a). These changes in perivascular pH - by way of altering arterial CO2 - illicit bi-directional changes (vasodilation with hypercapnia; vasoconstriction with hypocapnia) in cerebral tone and thus blood flow (Kety & Schmidt, 1948; Sato et al., 2012; Smith et al., 2014; Willie et al., 2015). Directional sensitivity is also observed in the cerebral vascular response to CO2 whereby higher reactivity is observed with increases (3-6% / mmHg PaCO2) vs decreases (1-3% / mmHg PaCO2) in PaCO2 (Sato et al., 2012; Willie et al., 2012). This higher sensitivity to hypercapnia is likely due to the associated effect of shear stress and the pressor response that occurs with increases in PaCO2. Indeed, increases in blood velocity causes increased shear stress between the erythrocyte and the endothelium, stimulating endothelial nitric oxide synthase expression from the forces on the endothelial wall (Fujii et al., 1991, 1992; Green et al., 2017), and from the erythrocyte (erythrocyte shear stress increases ATP release as described previously). Conversely, the lower CBF reactivity associated with hypocapnia may be resultant of ischemic mediated pathways 14 becoming activated following reductions in CBF and thus reduced local oxygen delivery (Harper & Glass, 1965; Ide et al., 2003; Tominaga et al., 1976; Wasserman & Patterson, 1961). 2.1.3 Blood pressure and autoregulation The brain was first reported to be a relatively pressure passive organ in studies completed by (Roy & Sherrington, 1890) and (Bayliss et al., 1895) examining changes in cerebral volume with alterations in arterial pressure using animal models. In 1959 Lassen published his landmark review based on 11 separate studies (primarily in clinical patients) that described the brain as possessing a large capacity to autoregulate flow under a wide range of blood pressure (~100 mmHg range) whereby CBF is maintained regardless of the underlying pressure (Lassen, 1959). Today it is better appreciated that this plateau of autoregulation is much smaller than originally proposed (~20 mmHg range) and that rapid deviations in blood pressure outside of the small autoregulatory window elicit respective increases and decreases in CBF [reviewed in:(Brassard et al., 2021)] as illustrated in Figure 2-3. 15 Figure 2-3. The contemporary autoregulatory plateau The relationship between relative changes in mean arterial pressure (MAP) from baseline (Δ%MAP) and concomitant relative changes in cerebral blood flow (Δ%CBF) when MAP decreases using non-pharmacological manipulation of blood pressure. Modified from (Brassard et al., 2021). Cerebral autoregulation is mediated through a combination of intrinsic myogenic control (Kontos et al., 1978), responses to changes in local cerebral metabolism (Paulson et al., 1990), and neurogenic components (Zhang et al., 2002; Hamner et al., 2010, 2012; Ogoh et al., 2010). When decreases in MAP are sufficient to reduce cerebral perfusion pressure (the difference between mean arterial blood pressure and intracranial pressure) there is a subsequent cerebral vasodilatory response that reduces vascular resistance to drive increases/maintenance of CBF. This vasodilatory response occurs in response to rapid localized tissue O2 and CO2 changes that occur with drops in perfusion pressure (ie. insufficient O2 delivery and CO2 washout) in addition to the neurogenic regulatory component. Conversely, with increases in blood pressure, vasoconstriction occurs, primarily mediated through stretch and pressure sensing within the vascular smooth muscle cells (Knot & Nelson, 1998) and through sympathetic mediated control [reviewed in: (Ainslie & Brassard, 2014; Brassard et al., 2017)]. 16 2.1.4 Metabolism Regulation of CBF by metabolism can be thought of as the overall metabolic demand of the brain governing changes in flow, and the transient, rapid localized changes in metabolism and blood flow in response to neural stimuli. Under resting conditions cerebral metabolic demand is almost entirely met by oxidative glucose metabolism as indicated by the high respiratory quotient (0.97) (Nybo et al., 2003; Dalsgaard et al., 2004a), with a small (5-10%) contribution of energy supplied through aerobic glycolysis (Dalsgaard et al., 2004b; Van Hall et al., 2009). While oxygen moves freely across the blood brain-barrier, glucose uptake is reliant on sodium-independent bi-directional facilitative transporters (GLUT1-8) [reviewed in: (Joost & Thorens, 2001; Carruthers et al., 2009; Mueckler & Thorens, 2013)] and sodium dependent glucose transporters (SGLTs) [reviewed in: (Wright et al., 2011) as depicted in Figure 2-4. The primary transporter associated with glucose uptake across the blood brain barrier is GLUT-1. Once taken up into the brain, glucose in the extracellular space is rapidly taken up into astrocytes, microglia and neurons through various GLUT and SGLT transporters (Figure 2-4) Glucose uptake in astrocytes is primarily mediated through GLUT-1, while GLUT-3 is the primary transporter for glucose uptake into neurons. Finally, GLUT-5 acts as the primary transporter of microglia which facilitates fructose uptake from the extracellular fluid. 17 Figure 2-4. Cerebral glucose transporters Movement of glucose molecules across the neurovascular unit at the blood-brain barrier. Under normal physiological conditions, glucose molecules (black dots) move from a higher concentration in the blood flow across endothelial cells and the basal membrane to a lower concentration in the brain extracellular fluid and then into astrocytes, neurons and microglia. Facilitative glucose transporters (GLUTs, ovals) and sodium-dependent glucose transporters (SGLTs, oblongs) are responsible for transporting glucose across membranes as shown. The most abundant glucose transporter is GLUT-1 (red), which is present in red blood cells and endothelial cells, and in astrocytes, neurons and microglia. In neurons, GLUT-3 (pink) is the major glucose transporter. In microglia, GLUT-5 (green) is the major GLUT transporter, which transports fructose and only has a low affinity for glucose. Other glucose transporters have been detected on cells at the neurovascular unit of the blood-brain barrier as indicated: GLUT-2 (purple), GLUT-4 (blue), GLUT-6 (gold), GLUT-7 (pale green), GLUT-8 (cyan), SGLT-1 (yellow), SGLT-2 (orange). Modified from (Patching, 2017). 2.1.5 Autonomic activity The cerebrovasculature is innervated by adrenergic and cholinergic fibers with the principal neurotransmitter for sympathetic innervation being noradrenaline. In the brain there is non-uniform, region specific distribution of α-1, α-2, β-1, and β-2 receptors, whereby extracranial conduit arteries are primarily populated by α-1 receptors with a progressive transition of distribution down to the parenchymal arterioles in which β receptors are most densely populated 18 [reviewed in: (Koep et al., 2022)]. Vascular responses to sympathetic stimulation are dependent on a number of factors including the density and distribution of receptors within each vessel; the concentration of neurotransmitter release, and; the presence of vasoactive and vasoconstrictive compounds. Quantification of the role of sympathetic and parasympathetic activity on CBF regulation is difficult in humans for a number of reasons including; 1) alterations in sympathetic and parasympathetic nerve activity imparting an indirect influence on CBF via alterations in cerebral perfusion pressure; 2) the presence of redundant mechanisms (Gross et al., 1983; Busija & Leffer, 1987); 3) heterogeneous distribution of sympathetic innervation and receptors; 4) species differences (Wagerle et al., 1986, 1990), and; 4) due to the presence of some form of cerebral sympatholysis resulting in a blunting a sensitivity to sympathetic activity (Remensnyder et al., 1962; Brassard et al., 2010). 2.2 Cerebral blood flow regulation in children and adolescents From birth through puberty, adulthood, and into old age, CBF during conditions of rest undergoes multiple alterations in blood flow patterns based on changes in metabolic requirement and alterations in physical activity (Talbot et al., 2023) and exercise, sensitivity to vasoactive factors (such as CO2), and the development and progression of disease [reviewed in: (Hoiland et al., 2019a). The pattern of CBF regulation during early childhood (Figure 2-5) is characterized by a progressive increase in CBF from infancy that is coupled to increases in cerebral metabolic demand, peaking around puberty (Goyal et al., 2014; Paniukov et al., 2020). These changes in flow patterns mirror that of neural development whereby children undergo increases in white matter volume (Deoni et al., 2012), cortical surface expansion (Remer et al., 2017), and alterations in functional connectivity (Long et al., 2017). As children progress through puberty and adolescence, a progressive decline in CBF is observed (Giedd et al., 1999; Satterthwaite et al., 2014; Tamnes et al., 2017) due to neuronal network alterations such as synaptic pruning (Huttenlocher, 1979) and increases in axon myelination (Kwon et al., 2020). 19 Figure 2-5. Changes in CBF across the lifespan Cross sectional global cerebral blood flow in children and adolescent (n=52) and adults (n=30) assessed using MR imaging. Modified from (Wu et al., 2016). While it is generally assumed the regulatory mechanisms underlying control of CBF are the same in children and adults, key differences likely lie within the impact of sex hormones on CBF, which is still poorly understood in adults (Barnes, 2017; Ruediger et al., 2021; Skinner et al., 2021), and the higher underlying metabolic requirement prior to puberty. Additionally, findings regarding sex differences have been very mixed in that most studies do not explicitly test sex differences. Among those that do, one study has reported higher brain perfusion in the parietal cortex in females than in males aged 5–18 years old (Taki et al., 2011), while another study reported that CBF decreases in males across adolescence, but a u-shaped trajectory of decreases then increases in flow occurs in female adolescents (Satterthwaite et al., 2014). 2.3 High-altitude High-altitude environments are generally classified as terrestrial regions with an altitude >1500m. However, when considering high-altitude in the context of its impact on altering physiological 20 processes, most consider 2000-2500m as the threshold for “high-altitude”. In addition to the lower barometric pressure, and thus a reduction in the partial pressure of inspired oxygen, high-altitude regions typically present with colder, drier climates, with an increased occurrence of food and water scarcity in remote locations whereby movement within the regions is generally more difficult (Moore, 2017). Despite these challenges, 219 million people reside at altitudes ≥2,000m, 25.2 million at altitudes ≥3,000m, 6.4 million at altitudes ≥4,000m, and 310,000 thousand at altitudes ≥5,000m (Tremblay & Ainslie, 2021). The relatively large population residing at >5000m is particularly fascinating as the partial pressure of inspired oxygen is only ~50% of sea-level values (PIO2= ~140mmHg vs ~78mmHg) (Figure 2-6) (Tremblay & Ainslie, 2021). 21 Figure 2-6. Population estimates from high-altitude regions Panel (A) Global map illustrating areas with altitudes of 1,500 m to 2,500 m, 2,500 m to 3,500 m, and ≥3,500 m (Antarctica is excluded). The 10 countries with the greatest total (B–D) and relative (E–G) populations residing at altitudes ≥1,500 m, ≥2,500 m, and ≥3,500 m are presented. Modified from (Tremblay & Ainslie, 2021). 22 In addition to the millions of people that live at high-altitude, these regions are commonly visited by military personnel (Muza, 2007) and individuals traveling for recreational trekking and climbing, religious pilgrimage (Basnyat, 2014), and sport purposes (Stray-Gundersen & Levine, 2008). In these individuals, upon arrival to high-altitude a number of cardiorespiratory, hematological, and cerebrovascular adjustments act in an attempt to compensate for the underlying systemic hypoxemia and maintain CDO2 and systemic function. 2.3.1 Acclimatization to high-altitude Acclimatization to high-altitude describes the various inter-related physiological processes (Figure 2-7) that occur in order to reduce the magnitude of systemic hypoxemia. Herein, the specific mechanisms and processes related to systems that ultimately affect CBF regulation (cardiorespiratory and hematological) will be discussed. Figure 2-7. Phases of respiratory, and hematological acclimatization at high-altitude Visualization of relative changes in ventilation, hematocrit (Hct), plasma volume, partial pressure of arterial carbon dioxide (PaCO2), bicarbonate (HCO3-), and partial pressure of arterial oxygen (PaO2) associated with ventilatory and metabolic compensation during acclimatization to high-altitude in lowland natives. Vertical grey bars represent changes in the pH of arterial blood and cerebral spinal fluid from sea-level. This figure has been adapted from (Hoiland et al., 2018) and represents general trends based on data collected in individuals at altitude ~5000m. 23 Early acclimatization through physiological changes related to the respiratory system account for some of the most immediate changes observed at high-altitude in an attempt to mitigate systemic hypoxemia. The acute physiological changes relating to respiratory function and oxygen carrying capacity are primarily affected by changes in ventilatory and metabolic compensation, and ventilatory sensitivity. Hypoxia mediated stimulation of the peripheral chemoreceptors results in marked increases in ventilation, primarily driven through increased breathing frequency (Chiodi, 1957). The degree to which this occurs is highly dependant on an individual’s ventilatory sensitivity to both O2 and CO2, and the severity of altitude exposure. Indeed, increases in ventilation act as one of the most noticeable and immediate compensatory responses experienced at high-altitude (West, 2006). The increase in ventilation serves to partially mitigate the reduction in PaO2 and SaO2; however, particularly at extreme altitudes, respiratory compensation is not sufficient to negate hypoxemia. As a result, initial exposure to high-altitude (>3000m) results in reductions in PaO2 and SaO2 and subsequent reductions in arterial oxygen content (CaO2) as illustrated by the equation: CaO2 (mL⋅ dL−1) = 1.34 × [Hb] (g ⋅ dL−1) × SaO2(%) + 0.003 × PaO2 (mmHg) Because of the increase in ventilation, the partial pressure of arterial carbon dioxide (PaCO2) decreases in accordance with the increase in ventilation and altitude of exposure. This hypocapnia results in a marked increase in arterial pH as shown by the Henderson-Hasselbalch equation, resulting in state of respiratory alkalosis. pH = 6.1 + log [ HCO3- ] ( 0.03 ⋅ PCO2 ) This increase in arterial pH is partially mitigated by stimulation of bicarbonate exertion through the kidneys in a diuretic response (Gledhill et al., 1975). Although this metabolic compensation is often not sufficient to restore pH to normative sea-level values, particularly at higher altitudes, (>4000m) and in the early stages of acclimatization (Tymko et al., 2022; Steele et al., 2022). 24 The initial increase in ventilation and subsequent reduction in PaCO2 is followed by a modest ventilatory depression through a reduced CO2 mediated drive to breath, detected at the level of the peripheral and central chemoreceptors (Sato et al., 1994; Powell et al., 1998). Despite this depression, however, ventilation remains markedly elevated compared to sea-level. Over days to weeks at altitude, ventilation begins to increase further as the hypoxic ventilatory response and hypercapnic ventilatory response are increased following high-altitude exposure >24 hrs (White et al., 1987; Sato et al., 1992; Bhaumik et al., 2003; Donoghue et al., 2005; Rupp et al., 2014) due to sensitizing of the carotid bodies (Dempsey & Morgan, 2015) and changes in pH. This augmented HVR appears to remain sensitized following both descent to sea level (5-days post) (Sato et al., 1992), and with subsequent re-ascent to high-altitude (Subudhi et al., 2014) At high-altitude, increases in [Hb] (and hematocrit) occur, leading to increases in CaO2. It was initially reported that the early haemoconcentration response with acute hypoxic exposure was as a consequence of reductions in total body water resultant of increased diuresis related to acid-base status compensation (Swenson et al., 1995; Hildebrandt et al., 2000; Loeppky et al., 2005). However, more recent work has challenged this original theory utilizing a large scale hypobaric chamber over a 4-day exposure, permitting the investigators to control for physical activity levels, and water and sodium consumption (Schlittler et al., 2021; Roche et al., 2022). These studies showed that 24-hour urine output was not changed, but plasma volume was still reduced as previously reported. These observations strongly suggest that plasma volume contraction, whereby fluid moves out of the intravascular space into the extra vascular space, is contributing to the early increase in [Hb] at high-altitude [reviewed in:(Siebenmann et al., 2023). Indeed, plasma volume contraction occurs within the first 24-hours of hypoxic exposure (Beidleman et al., 2017). With prolonged hypoxic exposure (>2 weeks), increases in red cell volume are observed, leading to further increases in [Hb] and CaO2 (Figure 2-7). This occurs through upregulation of the hypoxia-inducible factor-2 (HIF-2) pathway, leading to increases in erythropoietin (EPO) production. Upregulation of EPO is stimulated by local hypoxia at the level of the kidneys that is not compensated for by increases in renal blood flow (Olsen et al., 1993). The magnitude by which 25 red cell volume increases is highly altitude dependant whereby at lower altitudes (<3000m) the minimum duration of stay required to increase [Hb] is longer and a lower absolute increase in total red cell volume is observed (Rasmussen et al., 2013). Figure 2-8. Changes in blood volume at high-altitude Changes in intravascular volumes of whole blood, plasma, and red cells, (panel A) and hematocrit and hemoglobin concentration ([Hb] (panel B) in nine lowlanders across 4-weeks at high altitude (3454m) and following 14 days of de-acclimatization at sea-level. Modified from (Siebenmann et al., 2017). 2.3.2 Cerebral blood flow regulation at high-altitude During the acute stages of high-altitude exposure, the reduced barometric pressure, results in reductions in PaO2 and arterial oxygen saturation SaO2, leading to overall reductions in arterial 26 oxygen content CaO2. This necessitates a compensatory increase in CBF. However, across multiple studies using both rapid vehicular and gradual ambulatory ascent methodologies, CDO2 appears to be largely preserved across the varied hypoxic stimuli experienced at high-altitude (Figure 2-9). As mentioned, in the acute stages of high-altitude exposure, increases in alveolar ventilation result in a marked reduction in PaCO2. Under normoxic conditions, such reductions in PaCO2 would result in constriction of the cerebrovasculature and reductions in CBF (as described previously). The cerebrovasculature is thus undergoing both stimulation and inhibition to increases in CBF under these conditions. Despite this, however, multiple studies have shown that CBF at high-altitude appears to be modulated in a manner such that CDO2 is maintained, overcoming the countering influence of hypocapnia, with CaO2 being the principal determinant of CBF as shown in Figure 2-9. 27 0 1000 2000 3000 4000 5000 6000-1001020304050Altitude (meters)% CBF1233347777878999101011111191331414115-25-20-15-10-50510% CaO281313111111117777141421-15-10-505101520% CDO277778131311111114142122121211882288122110 2 4 6 8 10 12 14 16Days at altitude99999999886662222212124115555Ascent Acclimatization Figure 2-9. Cerebral blood flow, oxygen content and oxygen delivery during ascent and acclimatization at high-altitude in lowlanders Cerebral blood flow (bottom – green), arterial oxygen content (middle – red), and cerebral oxygen delivery (top – blue) reported from previous studies during ascent (left panels) and during acclimatization (right panels), across varying severities of altitude, rate of ascent, and duration of acclimatization. Individual studies are presented as numbered open squares with dotted connecting line (Studies: 1-(Severinghaus et al., 1966b); 2-(Huang et al., 1987)l; 3-(Otis et al., 1989); 4-(Jensen et al., 1990); 5-(Baumgartner et al., 1994); 6-(Lucas et al., 2011); 7-(Wilson et al., 2011); 8 (Subudhi et al., 2014); 9-(Willie et al., 2014a); 10-(Feddersen et al., 2015); 11-(Hoiland, et al., 2019b); 12-(Howe et al., 2019); 13-(Lafave et al., 2019); 14-(Lefferts et al., 2020). Mean responses are presented as a solid line indicating the line of best fit through the amalgamated data. Data during ascent and acclimatization are presented as percent change from sea-level or near sea-level values as reported in each respective study against altitude (ascent) or duration of stay (acclimatization). Arterial oxygen content was determined directly from arterial blood sampling or indirectly through measurement of peripheral oxygen saturation and hemoglobin or hematocrit. Cerebral oxygen delivery was determined by multiplying the calculated oxygen content by the respective measure of cerebral blood flow utilized in each respective study (i.e duplex ultrasound, transcranial doppler ultrasound, or Fick method). 28 As CaO2 increases following initial exposure at altitude through continued reductions in plasma volume (Figure 2-8), increases in PaO2 through upregulation of ventilatory sensitivity (Figure 2-7), and chronically through erythropoiesis mediated increases in red cell volume (Figure 2-8), CBF decreases toward baseline values. Again, the degree to which this occurs appears to be driven linearly by changes in CaO2 primarily through hemoconcentration, with increases in PaO2 and SaO2 contributing to a lesser degree (Howe et al., 2019). Throughout this process, however, CDO2 appears to be fairly well maintained despite the continued hypoxemia as illustrated in Figure 2-9. 2.3.3 Indigenous high-altitude populations Populations indigenous to high-altitude regions have developed specific, divergent adaptations over millennia spent living under hypoxic conditions (Beall, 2006, 2007). These populations are the Tibetans residing in the Qinghai-Tibetan Plateau, the Andeans residing in the Andean Altiplano, and the Ethiopians residing in the Semien Plateau (Figure 2-10). 29 Figure 2-10. Estimations of paths and dates of human migration to high-altitude Historical records indicate that the Amhara of Ethiopia tribe has been living at altitude for up to 70,000 years. Humans are thought to have occupied the Tibetan plateau for ~30,000–40,000 years and eventually crossed a land bridge that once connected present day Russia and Alaska, which facilitated North and South American settlement and eventual migration to the Andes 7000–10,000 years ago. Modified from (Tymko et al., 2019b) Andean highlanders are believed to have resided at high-altitude for the shortest period of time of the three populations (~7000-10,000 years), which generally aligns with contemporary theories of human migration into South America (Steffen, 2024). Hallmark systemic adaptations within Andeans include an exaggerated erythropoietic response leading to remarkably high hemoglobin concentration (Beall et al., 2002; Hancco et al., 2020; Oberholzer et al., 2020), low plasma volume (Stembridge et al., 2019), and a blunted hypoxic ventilatory response (Chiodi, 1957; Severinghaus et al., 1966a; Sorensen & Severinghaus, 1968a, 1968b; Lahiri et al., 1969; Moore, 2000; Brutsaert et al., 2005). Further, Andeans appear to be particularly susceptible to developing chronic mountain sickness, particularly at very high-altitudes (>5000m) (Hancco et al., 2020; Champigneulle et al., 2022, 2023). Tibetans (and Sherpa) have resided at high-altitude for ~30,000-40,000 years (Aldenderfer, 2003), and are perhaps the most widely known population due to their climbing and mountaineering feats. Thus, it is not entirely surprising that we generally consider Tibetans to be better adapted to high-30 altitude compared to Andeans (Lahiri & Milledge, 1965; Beall, 2000, 2007). Hallmark adaptations in Tibetans include large blood volumes with relatively normal hemoglobin concentrations (Stembridge et al., 2019), a “normal” hypoxic ventilatory response compared to lowlanders (Zhuang et al., 1993; Beall et al., 1997; Moore, 2000), and high exhaled and circulating nitric oxide (Beall et al., 2012). The high-altitude groups in Ethiopia (the Oromo and Amhara) are believed to have resided at high-altitude up to 70,000 years. Very little research work has been completed in these groups however it is believed that the Amhara have remarkably high oxygen saturation due to adaptations related to oxygen affinity (Cheong et al., 2017). 2.3.4 Cerebral blood flow regulation in indigenous high-altitude populations Based on published data, Andeans appear to present with lower CBF compared to lowlanders at high-altitude (Appenzeller et al., 2004, 2006; Claydon et al., 2005; Jansen & Basnyat, 2011). This lower CBF pattern may be due to Andeans presenting with 1) elevated arterial oxygen content via increased [Hb] (Beall et al., 2002; Hancco et al., 2020; Oberholzer et al., 2020); 2) lower cerebral glucose metabolism (Hochachka et al., 1994); and 3) markedly elevated blood viscosity (Tymko et al., 2019a; Tremblay et al., 2019; Stauffer et al., 2020). However, it is important to acknowledge that the majority of these CBF measurements have been obtained using transcranial doppler ultrasound, a technique that measures the velocity of blood as an index of flow. Thus, differences in intra cranial-vessel diameter can cause errors in quantifying CBF particularly when evaluating between group differences [reviewed in: (Hoiland & Ainslie, 2016)]. Despite early work evaluating cerebral blood velocity, which indicated that Sherpa presented with elevated CBF compared to both Andeans and lowlanders (Jansen & Basnyat, 2011), more recent work suggests an altered model of cerebrovascular adaptation. Indeed, both CBF and CDO2 appear to be reduced in Sherpa during ambulatory ascent and prolonged inhabitancy at high-altitude with comparable levels of CaO2 and PaCO2 (Hoiland et al., 2019b). Similar findings have been observed in pre-adolescent Sherpa children whereby global CBF was ~30% lower compared to age matched children residing at sea-level (Flück et al., 2017; Rieger et al., 2019), and in Tibetan high-altitude 31 natives whom displayed reduced CDO2 compared to Han Chinese living at high-altitude (Liu et al., 2016). Despite these findings, however, the mechanisms by which CBF and CDO2 are reduced have yet to be elucidated in these populations. Indeed, CO2 reactivity does not appear to differ compared to lowlanders (Jansen et al., 1999), and evaluations of cerebral autoregulation have produced mixed results. Indeed, no differences in autoregulation were observed between lowlanders and Sherpa at 5050m during spontaneous or driven changes in blood pressure (Smirl et al., 2014), whereas preserved dynamic cerebral autoregulation has been observed during ambulatory ascent to 3440 and 5050m when compared to lowlanders (Tymko et al., 2020). It is possible that, similar to the peripheral circulation (Gilbert-Kawai et al., 2017a; Davies et al., 2018), Sherpa present with greater cerebral capillary density (Xu & Lamanna, 2006) or possibly reductions in cerebral metabolic demand (Hoiland et al., 2019b). The latter mechanism has been investigated in Sherpa under normoxic conditions with no differences observed when compared to controls at sea-level (Hochachka et al., 1996); however, it may be possible that a reduction in cerebral metabolism is elucidated when exposed to hypoxic environments, as is the case in a number of hypoxia tolerant vertebrates (Nilsson & Lutz, 2004), permitting a greater reserve to increase flow to maintain CDO2. This mechanism(s) of adaptation would also likely reduce the occurrence of vasogenic oedema and/or intracranial hypertension particularly at extreme altitude (Lawley et al., 2015; Sagoo et al., 2016). This theory is supported by findings in elite climbing Sherpa whom present with reduced cortical atrophy and hyperintensity lesions compared to experience lowlander mountaineers (Garrido et al., 1993, 1996). 32 -50-40-30-20-1001020% CBFTibetan Andean13672554891110131213-15-10-5051015% CaO2Tibetan Andean735 5181312101113-35-30-25-20-15-10-505101520% CDO2Tibetan Andean1375581211101313 Figure 2-11. Cerebral blood flow, oxygen content and oxygen delivery in Andeans and Sherpa at high-altitude Studies incorporating Andean and Tibetan highlanders are presented as numbered closed squares with solid lines indicating the mean percent difference compared to lowlanders across studies. Studies: 1-(Huang et al., 1992); 2-(Jansen et al., 1999); 3-(Jansen et al., 2000); 4-(Jansen et al., 2007); 5-(Liu et al., 2016); 6-(Flück et al., 2017); 7-(Hoiland et al., 2019b); 8-(Sorensen et al., 1974a); 9-(Marc-Vergnes, 1974); 10-(Appenzeller et al., 2004); 11-(Claydon et al., 2005); 12-(Appenzeller et al., 2006) 13-(Stacey et al., 2023). Data from highlander studies are presented as percent difference compared to acclimatized lowlanders either reported within the study or compared to data collected in lowlanders at a similar altitude following >1 week stay at altitude. Lowlander data for these altitude comparisons was obtained from (Liu et al., 2016) (3675m – Duplex ultrasound and TCD – studies #1, 5); (Severinghaus et al., 1966b) (3800m – Fick method – studies #8, 9); (Villien et al., 2013) and Tymko et al., Unpublished (4300m – TCD – studies #2, 3, 4, 10, 11, 12). 2.3.4.1 Cerebral blood flow regulation in children and adolescents at high-altitude Studies investigating cerebrovascular function in children and adolescents of lowland descent and of high-altitude indigenous populations are quite limited. In lowlander children whom ascended to 3500m over five days, TCD measurements of cerebral blood velocity showed increases in blood velocity within the anterior cerebral arteries (ACA) and left middle cerebral artery (MCA), but unchanged velocity within the right middle and both posterior cerebral arteries compared to sea-level (Gavlak et al., 2013). These findings differ from those in adults whereby MCAv has been shown to be unchanged on day three at 3500m while ACAv was reduced (Lefferts et al., 2020). 33 Further, in adults following a similar, albeit slightly faster ascent profile (Hoiland et al., 2019b), regional and global CBF as assessed by duplex ultrasound was unchanged compared to low altitude. The discrepancy between findings in children and adults in these studies are difficult to tease apart due to the lack of assessment of vessel diameter, differences in baseline velocity between hemispheres, and lack of measures of CaO2 in children. In lowlander children brought to high-altitude by car (3800m) and compared directly against their parents using duplex ultrasound measures, relative increases in CBF have been shown to be similar to those of adults, albeit with higher flows across baseline and at high-altitude in children (Rieger et al., 2022). These data would indicate that although bulk CBF is different between children and adults, as discussed previously, the mechanisms regulating CBF under high-altitude hypoxia are likely similar within the respective population. Work in high-altitude children and adolescents has shown that Sherpa living at ~3800m elevation present with lower CBF compared to children living at low altitude (Flück et al., 2017). This pattern of CBF matches what has been shown in adult Sherpa from the same region (Hoiland et al., 2019b), and provides some evidence that the cerebrovascular phenotype in Sherpa is observable across the lifespan. In Andeans, no volumetric measures of CBF have been reported to date. One study utilizing velocity estimates of flow has reported that Andeans living at 3700m have lower basilar artery blood velocity compared to sea-level counterparts (Hogan et al., 2010). However, this is difficult to interpret without a measure of CaO2 or some indication of vessel diameter. Indeed, it may be that Andean children present with larger vessel diameters and thus similar CBF to sea-level groups, or that high CaO2 as is observed in adults (Figure 2-11), results in maintained CDO2. 2.4 Hypothermia Hypothermia refers to the generalized condition within the body whereby a reduction in core body temperature has occurred. Although marked intra-and inter-individual differences exist, the classification of hypothermia is commonly categorized into four levels based on the degree of thermal loss (Durrer et al., 2003; Dow et al., 2019). These respective classifications (shown in table 2-1) are accompanied by distinctive physiological changes and clinical signs and symptoms 34 that are primarily determined by the presence of shivering, level of consciousness, and functional capacity. Table 2-1. Categorization of hypothermia Stage Clinical Symptoms Core temperature (ºC) Cold Stress Normal mental status with shivering. Functioning normally. Able to care for self >35ºC Mild Hypothermia Alert, but mental status may be altered. Shivering present. Not functioning normally. Not able to care for self. 32-35ºC Moderate Hypothermia Decreased level of consciousness. Conscious or unconscious, with or without shivering 28-32ºC Severe/Profound Hypothermia Unconscious. Not shivering. <28ºC Table adapted from (Dow et al., 2019) Of note, these categorizations have been altered and revised various times throughout the 1900s and early 2000s (Tveita & Sieck, 2022), and vary between clinical organizations and research bodies. Body temperature and the rate of heat loss in a cold environment is determined by the products of the heat balance equation: Whereby metabolic heat production equals all summated metabolic activity, (+Work) which represents energy leaving (positive for concentric muscular contraction) or entering (negative for eccentric muscular contraction) the body as a product of external work; heat 35 exchange between the body and environment occurring through convective (direct transfer of heat to convective currents of air or water); conductive (direct transfer of heat to an adjacent, cooler object); evaporative (vaporization of water), and; radiative (emission of infrared electromagnetic energy) heat transfer. In this context, therefore, heat loss can occur via a number of different pathways that are influenced via metabolism and the external environment. When an individual is exposed to temperatures below the ambient temperature while lightly clothed or nude, heat loss occurs slowly though convective and radiative heat loss. When an individual is immersed in water, which has a markedly higher thermal conductivity compared to air (~25x) (Ramires et al., 1995) the rate of heat loss is ~2-5x greater compared to the same temperature in air. This is due to the greater rate of convective and conductive heat transfer whereby while air temperatures of 26⁰C is considered thermoneutral (Kingma et al., 2014), in water thermoneutral temperature is ~34-35 ⁰C. 2.4.1 Systemic physiological responses to cold stress and hypothermia With cold stress and mild hypothermia a number of systemic responses are activated in an attempt to maintain normothermia by reducing heat loss, and increasing heat production. The primary response which aims to limit heat loss is peripheral vasoconstriction, while shivering and non-shivering thermogenesis act to increase heat production. These two thermal mediated responses also elicit a number of physiological changes that don’t contribute to protection from heat loss, but have a great influence on altering CBF, including changes in blood pressure, hemoconcentration, ventilation, and metabolism. Herein, the pathways by which these responses are mediated will be described, and their individual impacts on regulation of CBF as they relate to the topics in Chapter 2.1 will be discussed. 2.4.1.1 Peripheral vasoconstriction Peripheral vasoconstriction acts as the first line of defense to protect against cold induced heat loss. This mechanism is highly efficient as it requires minimal energy to preserve core temperature 36 as opposed to the metabolic cost required to increase heat production. Further, as a consequence of peripheral vasoconstriction, increases in blood pressure and [Hct] are also observed, both of which may impact CBF regulation under conditions of cold stress and mild hypothermia. The mechanisms by which these responses arise are discussed herein. Reductions in environmental temperature sufficient to decrease skin temperature to <35ºC (Veicsteinas et al., 1982a) are detected by TPRM8 thermal receptors (McKemy et al., 2002; Peier et al., 2002) within the cutaneous tissue (Figure 2-12). Here, this afferent stimulation travels to the preoptic area of the hypothalamus where the signal is integrated with feedback from temperature receptors located in the spinal cord and brain that respond to changes in core temperature (Boulant, 2000). Efferent signals are produced which travel to the periphery in the form of skin sympathetic nervous activity and result in noradrenaline and Neuropeptide Y (NPY) release from postganglionic neurons. Binding of noradrenaline to A2C receptors (Charkoudian, 2010) and NPY to Y1 receptors (Stephens et al., 2001, 2004) within the cutaneous vasculature elicits vasoconstriction. This reflex vasoconstrictive response is supported by local cooling mechanisms (Alvarez et al., 2006) via increases in reactive oxygen species mediated stimulation of RHO Kinase and subsequent inhibition of myosin light chain phosphatase (Thompson-Torgerson et al., 2007). Rho kinase activation also elicits translocation of A2C receptors to the plasma membrane, increasing the sensitivity of receptors to norepinephrine (Chotani et al., 2000, 2004; Johnson, 2007). Finally, ENOS signaling is also reduced with local cooling, resulting in an increase in reflex vasoconstriction (Noma et al., 2006). 37 Figure 2-12. Mechanisms of reflex and local cold induced vasoconstriction DRG; dorsal root ganglia, RMR; rostral medullary raphe, MLCK; myosin light chain kinase, MLCP; myosin light chain phosphatase, NE; norepinephrine, NPY; neuropeptide Y, POA; preoptic area. Modified from Greaney et al., 2015 With greater cold stimuli and a greater area of cooling, increases in vascular resistance result in MSNA mediated elevations in skeletal muscle and visceral vascular tone, in addition to the skin sympathetic nerve activity responses innervating cutaneous vascular beds (Fagius & Kay, 1991; Wilson et al., 2007). The peripheral vasoconstrictive response becomes maximal at skin temperatures <31ºC (Veicsteinas et al., 1982b; Castellani & Young, 2016). Ultimately, this magnitude dependent peripheral vasoconstrictive response occurs in order to reduced convective heat loss and is one of the primary mechanisms responsible for protection of thermal balance. As a consequence of the systemic increase in vascular tone, regardless of the medium of external cold stress, MAP is markedly increased (average ~10-20mmHg) (Wilson et al., 2007; Mugele et al., 2023). As discussed in Section 2.1.3, increases in blood pressure can elicit increases in CBF if the magnitude of the increase in perfusion pressure is outside of the relatively small autoregulatory plateau. However, under conditions of cold stress and mild-hypothermia, other physiological 38 responses may outweigh the impact of increased MAP on CBF, as flow is typically reduced with mild-hypothermia (See section 2.4.2). 2.4.1.2 Shivering and non-shivering thermoregulation Barring prevention of heat loss is inefficient to maintain core temperature, or if the reduction in skin temperature is great enough, increases in metabolic heat production through shivering and non-shivering thermogenesis are initiated. Similar to peripheral vasoconstriction, while shivering and non-shivering thermogenesis does not directly impact CBF, a number of consequent responses related to ventilation, acid base status, fuel utilization, and metabolism may alter resting CBF patterns during cold stress and mild hypothermia. The cold induced shivering response represents a centrally controlled mechanism by which biomechanically inefficient muscle contractions are generated to increase metabolic heat production. Shivering usually begins in the torso muscles, and spreads distally to the limbs (Bell et al., 1992). The magnitude of this shivering response is dependent on the severity of the cold stimuli, and can be augmented by environmental factors such as wind and the medium interfacing with the skin (ie. cold water vs cold air). As shivering intensity and muscle recruitment increases (maximal shivering intensity is reached between 34-35 ºC core body temperature) (Castellani et al., 2006), whole-body metabolic rate increases, reaching ~200–250 W with exposure cold air exposure and ~350 W during cold water immersion. This elevation in metabolism elicits changes in ventilation and acid base status similar to that of exercise. The shivering response can create vast amounts of thermal energy, and thus requires a large increase in systemic metabolic demand despite cell level reductions in metabolism. It is estimated that shivering can contribute 24-32% of whole-body heat production (Blondin & Haman, 2018). The pathway mediating the shivering response, as interpolated based on rodent models, is initiated by cold sensitive cutaneous receptors (as previously described) which activate local inhibitory neurons in the median preoptic nucleus, decreasing the activity of the inhibitory projections located in the median POA (Boulant, 2000). This activation leads to disinhibition of neurons within the dorsomedial hypothalamus and subsequent activation of somatic premotor neurons in the rostral 39 medullary raphe. Premotor neurons, once activated, excite spinal motor outputs which drive shivering (Morrison, 2016). The generalized patterns of shivering can be classified into two groups based on their EMG signature (Israel & Pozos, 1989; Meigal, 2002); 1) Continuous low intensity shivering characterized by low-threshold type 1 muscle fiber activity and 2) bursts of high-intensity shivering characterized by high-threshold type 2 muscle fiber activity. Thus, patterns and respective shivering intensities can vary drastically between individuals based on body composition and morphology (primarily due to muscle fiber type distribution) and by differences in glucose and lipid availability (Bell et al., 1992; Haman et al., 2004; Blondin et al., 2017b). From a metabolic standpoint, the capacity for an individual to maintain shivering is quite flexible, due to an ability to alter substrate utilization between glycogen/glucose and lipid metabolism based on substrate availability (Martineau & Jacobs, 1989, 1991; Haman et al., 2004; Blondin et al., 2014; McInnis et al., 2020). Shivering substrate metabolism is not exclusive to glycogen with both lipids and protein contributing to shivering thermogenesis and the magnitude of their contribution dependent on substrate availability whereby under conditions of glycogen depletion, lipid oxidation can be upregulated without altering heat production. Non-shivering thermogenesis describes the various metabolic processes that produce heat without any form of external muscular work. Under cold conditions these metabolic pathways that are altered our activated to increase heat production are primarily brown adipose tissue (BAT), mitochondrial proton leak, and calcium channel cycling within resting skeletal muscle (Blondin & Haman, 2018). In humans, BAT is predominately located in the supraclavicular regions and produces heat through catabolizing circulating blood glucose and intracellular triacylglycerol, although the magnitude of its contribution to thermogenesis and changes in metabolic demand with cold stress are rather small (Dulloo, 2013; Blondin et al., 2017a; McInnis et al., 2020). Increases in oxidative phosphorylation uncoupling in skeletal muscle (mitochondrial proton leak) and calcium cycling contribute the greatest to non-shivering thermogenesis (Harper et al., 2002; Wijers et al., 2011). Increases in mitochondrial proton leak with cold stress and hypothermia are 40 thought to occur through increases in ROS sensitizing uncoupling proteins within skeletal muscle (Harper et al., 2002; Mailloux et al., 2011), although this is not entirely clear. It is estimated that 8-16% of heat production during mild cold stress is from skeletal muscle mitochondrial proton leak (Blondin & Haman, 2018). 2.4.1.3 Metabolism Beyond the impact of cold stress and hypothermia on energy and fuel utilization associated with shivering and non-shivering thermogenesis, decreases in body temperature also elicit reductions in metabolism at a cellular level. This reduction in metabolism in physiology is referred to as the Q10 principal which describes the reduction in metabolism of a cell, tissue, or organ associated with a decrease in temperature of 10⁰C (Mundim et al., 2020). The principle classically follows the Arrhenius model and is calculated as where T is the temperature in degrees Celsius or Kelvin, and k is the rate constant. In the human body, the Q10 is typically between 2 and 3 (Sakoh & Gjedde, 2003), with a value of ~2.2 - 2.4 generally associated with the Q10 of the brain (Russell-Buckland & Tachtsidis, 2020a). By way of this principle, a non-linear reduction in metabolism is associated with progressive hypothermia. For example, a reduction in core temperature from 38 to 34.4⁰C is predicted to reduced metabolism by ~25%, with a ~50% reduction at 29.4⁰C (Sakoh & Gjedde, 2003). Because the decrease in metabolism is exponential, smaller reductions in core temperature which can be safely examined in healthy humans (37-35⁰C) may not elicit as marked of a suppression of metabolism, particularly in a non-anesthetized or paralytic state. 2.4.1.4 Hemoconcentration While not occurring as a thermal protective response, alteration in [Hb] and blood properties occur with cold stress and mild hypothermia due to the peripheral vasoconstrictive response and due to 41 direct temperature-mediated properties. As these factors impact both shear stress and CaO2 they have a significant impact on CBF regulation. Herein, the mechanisms by which cold induced hemoconcentration occur are described and the ultimate impact it may have on CBF under cold stress and mild hypothermia are discussed. Cold stress and hypothermia elicit changes in hemorheological properties, via direct effects of temperature on blood viscosity (Rand et al., 1964; Snyder, 1971) and through eliciting a hemoconcentration response (Rostomily et al., 2020). Decreases in blood temperature cause increases in viscosity due to the individual molecules having less thermal energy to overcome the attractive forces between them (Peleg, 2018). The physiological basis for cold induced hemoconcentration have shifted over time. Originally, sympathetic mediated splenic contraction was believed to contribute to the rapid increase in hemoglobin concentration based on anesthetized animal studies (Gentile et al., 1995). However, this is unlikely to meaningfully contribute to the magnitude that [Hb] increases, both from a mathematical perspective and based on the available literature. Indeed, the increases in [HB] and Hct observed with cold stress are far too large for the spleen to provide. For example, breath hold studies in humans have reported splenic contractions of ~50-100 ml (Schagatay et al., 2020), which would be associated in an increase in Hct of ~0.5-1%. Previous work in splenected canines (Chen & Chien, 1977) and in neonates where their spleens do not possess a reservoir function (Chen et al., 1980) both show that hemoconcentration still occurs with consequent reductions in plasma volume. Further, hemoconcentration has been observed with very mild skin cooling which are likely too minimal to elicit increases in MSNA required to cause splenic contraction. It is likely that the hemoconcentration response is almost entirely mediated by plasma volume shift secondary to increases in MAP and venoconstriction (Young et al., 1987). This haemoconcentration response results in significant increases in CaO2 and thus has a profound impact on control of blood flow (see section: Cerebrovascular responses to cold stress and hypothermia). 42 2.4.1.5 Ventilation As a consequence of the increase in metabolism from shivering and increases in autonomic activity, particularly during cold water immersion, marked increases in ventilation occur with cold stress. Changes in respiratory pattern with hypothermia are dependent on the temperature of the cold stimuli and how that stimuli interfaces with the cutaneous cold receptors (air vs water), and the underlying stage/severity of hypothermia. Cold stress and mild-moderate hypothermia elicit marked increases in tidal volume and breathing frequency both from increases in metabolic activity (hyperpnea response) and increases in sympathetic activity (hyperventilatory response). This distinction between metabolic hyperpnea and sympathetic hyperventilation is best visualized by the cold shock ventilatory response which occurs immediately upon immersion or dousing with cold water. The cold shock respiratory pattern is characterized by an initial inspiratory gasp response (Burke & Mekjavic, 1991) and subsequent hyperventilation (Goode et al., 1975) whereby ventilation can increase up to 600-1000% of normothermic baseline (Cooper et al., 1976a; Tipton, 1989) resulting in profound hypocapnia (Keatinge et al., 1964; Cooper et al., 1976b; Golden & Tipton, 1988; Tipton, 1989). This increase in ventilation occurs prior to any reduction in core temperature and prior to development of shivering. 2.4.2 Cerebrovascular responses to cold stress and hypothermia As eluded to previously, CBF is primarily modulated by changes in arterial blood gases, perfusion pressure, metabolism, and autonomic activity. With cold stress, marked alterations in each of these systems occurs either directly from the cold stimuli (reduced local tissue metabolism) or as a consequence of the physiological responses activated to prevent against heat loss (shivering, peripheral vasoconstriction, hyperopia and hyperventilation, and increased SSNA and MSNA) With cold stress and mild hypothermia in humans, CBF is reduced compared to normothermia. This finding is reported across various animal models (Rosomoff & Holaday, 1954), in clinical populations in which induced hypothermia is utilized as a treatment modality (Marion et al., 1993), and in healthy spontaneously breathing individuals (Gibbons et al., 2020); (Figure 2-13). Importantly, however, the factors contributing to reductions in CBF between healthy and 43 anesthetized patients likely differ slightly. Indeed, as previously described, cold stress and mild hypothermia, which can be tested and observed in healthy individuals, elicits reductions in PaCO2, increases in CaO2 and sympathetic activity, and presumably a reduction in cerebral metabolism. In patient groups, particularly when deep hypothermia is utilized (core temp <30⁰C) the primary mechanism responsible for reductions in CBF are likely directly due to temperature mediated reductions in cerebral metabolism as; 1) patients are generally ventilated and given a paralytic drug, reducing the incidence for increases in ventilation and subsequent hypocapnia; 2) Blood pressure is pharmacologically managed, and fluid volume monitored or purposely altered, reducing the impact of haemoconcentration. However, the effect of anesthetic and paralytic drugs on reducing cerebral metabolism in these groups should not be ignored. 44 Figure 2-13. Reductions in CBF reported in a classic animal model, a clinical patient population, and in healthy humans. Panel A: Near linear reductions in cerebral blood flow (CBF) observed during core cooling from 35⁰C to 25⁰C in anesthetized canines (Rosomoff & Holaday, 1954). Panel B: Patients treated with hypothermia vs normothermia following sustaining closed head injury present with lower CBF during the first 24 hours with core temperature management (Marion et al., 1993). Panel C: Healthy, spontaneously breathing participants exhibit a modest reduction in global CBF following a ~1⁰C reduction in core temperature via cold water immersion (Gibbons et al., 2020). 45 2.4.3 Avalanche burial The earliest reported fatality from avalanche burial in Canada is believed to have occurred during the Winter of 1781-1782 in Nain, Newfoundland. Wallace J. McLean, a historian from Labrador discovered the account in the postscript of a letter dated 1782 from the missionaries in the town of Nain to the missionaries at Moravian. The letter read: “A Lamentable Circumstance has happened this last winter about twelve miles from us, upon the edge of a hill under which was an Esquimaux winter hauss where 31 Esquimaux lived, there gatherd a monstrous body of snow which shot all at once down and pressed the winter hauss even with the ground, with all the people in it excepting one man who was buried in the snow without. Out of 31 only 9 got out alive.” Since this first historical report, 919 avalanche related fatalities have been reported in Canada, with 225 individuals having survived within those same avalanches. The majority of these having occurred in British Columbia (~65%). Between January 1, 2011 and April 30, 2022, there were 77 avalanche-related deaths in B.C., equating to an average of 7 deaths each year (BC Coroners Service, 2022). In Europe, between 1970 and 2015 the mean fatality rate from avalanches within the alps was ~103 deaths per year (Techel et al., 2016). As described by Falk et al., in 1994 using avalanche reports from Switzerland, the calculated probability of survival during avalanche burial decreases drastically from 92% at 15 minutes, down to only 30% at 35 minutes. This rapid decrease in survivability is ascribed to acute asphyxiation of victims without an air pocket and free airways. After 35 minutes, survivability decreases to 27% at 90 minutes of burial, and only 3% at 130 minutes of burial. The blunting of the decrease in survivability is due to the presence of an air pocket being required for avalanche victims to survive being buried for these durations. Ultimately, however, death following burial durations >35min are generally resultant of progressive hypoxia and/or hypothermia. Death following burial durations <15minutes are likely resultant of blunt trauma or a non-patent airway from inhalation of avalanche debris. 46 These survival probability data were updated with more recent data from Switzerland in addition to data from Austria and Canada (Strapazzon et al 2024). The updated survival curves (Figure 2-14) show much of the same trajectory; however a steeper decrease in survival during the early stages of burial is observed within the Canadian data. This has been postulated to be due to differences in climate and snow properties (Haegeli et al., 2011; Strapazzon et al., 2021c). Figure 2-14. Avalanche burial survival curves Survival curve for completely buried victims in Austria (solid black line; 333 victims between 2005 and 2013), Canada (solid grey line; 301 victims between 1981and 2005) and Switzerland (dashed grey line; 422 victims between 1981 and 1991; dashed black line; 300 victims between 2005 and 2013). Modified from (Strapazzon et al., 2024). 2.4.3.1 The physiology of avalanche burial During avalanche burial, barring individuals have not succumbed to blunt force trauma or rapid asphyxia as described previously, progressive hypoxia and hypercapnia will develop (Wik et al., 2022). The rate that this occurs is determined by the presence and size of an air pocket (Brugger 47 et al., 2003a; Procter et al., 2016a), and the properties of the snow/debris field (Procter et al., 2016a; Strapazzon et al., 2017). Indeed, despite compacted snow comprising of 40-60% air, as CO2 accumulates in the air pocket through the continuation of metabolic processes and ventilatory gas exchange, displacement of O2 within the air pocket occurs (displacement asphyxia), resulting in a reduction in inspired O2 and increases in inspired CO2, leading to progressively more severe hypercapnic-hypoxia. This effect can be compounded by the formation of an ice mask, whereby moisture in expired air freezes to the wall of the air pocket, creating a barrier that reduces the movement of gases between the snow and air pocket. 48 Figure 2-15. Causes of death as they relate to duration of burial The cause of death in individuals who die rapidly following avalanche burial is generally attributable to blunt force trauma from striking an object while being carried by the avalanche or by acute asphyxia due to a blocked airway. As duration of burial increases due to the presence of an air pocket, individuals will progressively become hypercapnic, hypoxic, and hypothermic. 49 Hypercapnic-hypoxia elicits activation of peripheral and central chemoreceptors leading to a marked, stimulus dependent increase in ventilation. Because of the enclosed space during avalanche burial, any elevations in ventilation fails to rectify the PaO2 and PaCO2 levels to normative values. Rather, the rate of O2 desaturation and arterial CO2 accumulation becomes magnified. While minimal work has been performed investigating CBF during conditions of avalanche burial, we can draw some comparisons to prolonged breath hold studies. Previous work in elite free divers undergoing both maximal static dry apneas and simulated maximal apneas utilizing a dynamic end-tidal forcing system (minimum SaO2= ~60%; maximum PaCO2= ~53 mmHg), shows that pronounced hypercapnic-hypoxia elicits marked elevations in CBF that maintain CDO2 despite profound reductions in CaO2. Further, suppression of CMRO2 is observed in these populations due to the hypercapnic stimuli (Bain et al., 2016a, 2017) rather than hypoxemia. Indeed, measurements of CMRO2 under solely hypoxic conditions have shown that cerebral metabolism is maintained (Ainslie et al., 2014). Subsequent work examining the role of hypercapnia on CMRO2 has confirmed the findings observed in apnea divers whereby changes in PaCO2 are responsible for reducing cerebral metabolism as shown in Figure 2-16 (Caldwell et al., 2024). 50 Figure 2-16. Relationship between PaCO2 and CMRO2 Cerebral metabolic rate of oxygen (CMRO2) versus arterial partial pressure of carbon dioxide (PaCO2) in N=41. Blue dots represent male participants and red triangles female participants. Modified from Caldwell et al., 2024 The specific mechanism for this hypometabolism response is poorly understood, however it may be resultant of 1) by reductions in phosphofructokinase activity and thus glycolytic flux (Folbergrová et al., 1972; Miller et al., 1975; Borgström et al., 1976); 2) CO2-mediated extracellular acidosis, increasing adenosine concentrations and glutamatergic signaling, whereby cortical excitability is reduced (Thesen et al., 2012); 3) Increases in sympathetic activity via chemoreflex activation (Steinback et al., 2009, 2010a, 2010b), with hypercapnia-mediated elevations in circulating catecholamines (SECHZER et al., 1960; Metz, 1969; Davidson et al., 1986; Brofman et al., 1990), increasing the contribution of non-oxidative carbohydrate uptake. As duration of burial increases in concert with hypercapnia and hypoxia, hypothermia progressively develops. The rate of core cooling in avalanche burial is highly variable between incidents and individuals, with reported cooling rates from clinical case studies ranging from <1 to 9 ºC per hour (Pasquier et al., 2015; Ströhle et al., 2015); and even a 14 ºC reduction in core 51 temperature per hour in one report (Rauch et al., 2023). Factors such as air temperature, wind, snow temperature, clothing, body composition, perspiration prior to burial, and activity can impact this rate of cooling. The combination of hypercapnia, hypoxia, and hypothermia experienced during avalanche burial, otherwise referred to as “Triple H Syndrome” (Brugger et al., 2003a), is sparsely studied outside of animal models. To date, only two porcine studies have investigated this condition (Paal et al., 2013; Strapazzon et al., 2021a). (Strapazzon et al., 2021a) showed that combined severe hypothermia (28 ºC), hypercapnia, and hypoxia resulted in cardiac instability and a reduction in cerebral perfusion and tissue oxygenation. Notably, as with almost all animal models of avalanche burial, this study was performed in sedated animals whereby hypoxia and hypercapnia were modulated by use of a ventilator in a manner not necessarily representative of the progression of avalanche burial. 52 Chapter 3: Study 1 – Cerebral blood flow and oxygen delivery in Sherpa, Andeans, and lowlanders at 5000m Abstract Populations indigenous to high-altitude environments such as the Sherpa of the Tibetan plateau and the Andeans of the Peruvian altiplano display unique and divergent phenotypical adaptations under similar environmental constraints. In lowlanders, the patterns and mechanisms of cerebral blood flow (CBF) at high-altitude have been well investigated; however, whether divergent pathways of cerebrovascular control exist between these populations under similar hypoxic conditions is unknown. The purpose of this study was to compare resting patterns of CBF, arterial oxygen content (CaO2) and cerebral oxygen delivery (CDO2) between Sherpa, Andeans, and acclimatized lowlanders, all residing at ~5000m. We examined the hypothesis that Sherpa would present with lower CBF and CDO2 compared to Andeans and acclimatized lowlanders. We tested male lowlanders (n=14; 5050m), Sherpa (n=14; 5050m), and Andeans (n=14; 5100m). Duplex ultrasound was used to measure internal carotid (ICA) and vertebral artery (VA) blood flow to determine global CBF. Radial artery blood samples were obtained to measure arterial blood gases and calculate CDO2. Covariate analysis of CBF for severity of hypoxemia (CaO2)) revealed that Sherpa exhibited markedly lower ICA (~41% and 43%) and global CBF (~31% and 45%) compared to both lowlanders and Andeans, respectively. No differences in CBF or CDO2 were observed between Andeans and lowlanders, however both ICA and VA diameter were larger and blood velocity proportionally lower in Andeans compared to both lowlanders and Sherpa. These data highlight that CDO2 attenuation at high-altitude is an adaptive trait unique to Sherpa, and divergent from Andeans residing at the same altitude. 3.1 Introduction While the brain has a poor ability to function when its oxygen supply is interrupted (Lipton, 1999; Del Zoppo et al., 2011), it has a remarkable ability to function and maintain cerebral oxygen delivery (CDO2) when ambient oxygen availability is reduced. For example, upon initial arrival to altitude when hypoxemia is most profound, cerebral blood flow (CBF) increases in order to compensate for the reductions in arterial oxygen content (CaO2), and maintain CDO2 [reviewed in:(Hoiland et al., 2018)]. In lowlanders, as the duration of residence at altitude increases, 53 ventilatory and hematological acclimatization typically increase CaO2, which enables CBF to decrease toward sea-level values while maintaining CDO2. This response has been well characterized (reviewed in: Hoiland et al., 2018) and appears to be primarily a result of increases in hemoglobin concentration ([Hb)] with lesser contributions from ventilatory compensation (Howe et al., 2019). Populations indigenous to high-altitude, including Andeans, Tibetans (Sherpa), and Ethiopians display unique adaptations permitting what most consider to be enhanced physiological function under hypoxic conditions compared to lowlander counterparts (West, 2006). While each of these populations has adapted in favor of survival under hypoxic environments, divergent physiological adaptations appear between these high-altitude populations (Beall, 2006, 2007). Hallmark adaptations associated with high altitude in Andeans include larger lung volumes (Brutsaert et al., 1999), a blunted hypoxic ventilatory response (Moore, 2000), and elevated magnitudes of erythrocytosis, leading to high hematocrit and [Hb] in the presence of lower plasma volume (Stembridge et al., 2019). Conversely Tibetans and Sherpa present with enhanced muscle capillary density (Beall, 2007; Erzurum et al., 2007), high mitochondrial efficiency (Horscroft et al., 2017), unremarkable ventilatory responses to hypoxia [reviewed in: (Gilbert-Kawai et al., 2014)] and elevated plasma volume and total blood volume masking a relatively larger red cell volume when compared to lowlanders (Stembridge et al., 2019). These adaptations coincide with unique upstream gene expression signaling patterns (including hypoxia inducible factor pathway genes), that are divergent from gene activity in Andeans at high altitude (Beall, 2000; Bigham & Lee, 2014; Simonson, 2015). Understanding how differing populations regulate CBF at high-altitude remains an important question to better understand mechanisms contributing to maintenance of CBF and CDO2 under conditions of chronic hypoxemia. As regulation of CBF in hypoxia is mediated through erythrocyte associated pathways (Hoiland et al., 2023), differing adaptations relating to hematology (ie., marked erythropoietic response in Andeans compared to Sherpa) likely impact CBF regulation at high altitude across populations. In Sherpa and Tibetans, early research using intra-cranial velocity estimates of CBF suggested elevations compared to lowlanders (Huang et 54 al., 1992). In contrast, more recent volumetric measures of CBF and CDO2 have shown Sherpa and Tibetans to possess lower CBF and CDO2 compared to lowlanders (Liu et al., 2016; Hoiland et al., 2019b). Conversely, intra- and extra-cranial blood velocity estimates of CBF in Andeans indicate it is lower in Andeans compared to lowlanders (Appenzeller et al., 2004, 2006; Claydon et al., 2005). Classic work using invasive measurements of the cerebral arterial-venous difference of oxygen (used to index CBF) also suggested that CBF was lower in Andeans living at 4380m (Milledge & Sorensen, 1972) and 3800m (Sorensen et al., 1974a) compared to lowlanders during the early stages of acclimatization (3-5 days at 3800m). However, recent volumetric comparisons conducted at 4380m report no marked differences in CBF between nine acclimatizing lowlanders (14 days at 4380m) and nine Andeans (Stacey et al., 2023). Indeed, it is important to acknowledge and query comparisons of CBF across populations using intracranial velocity measurements as differences in vessel diameter may mask or negate similarities and differences in CBF (Ainslie & Hoiland, 2014) and to consider the duration of acclimatization for lowlander participants. While a number of studies have been completed examining CBF regulation in these high-altitude populations, differing assessment techniques (primarily intracranial velocity measurements), equipment and altitudes of study can make direct comparisons between populations difficult. Indeed, to date no studies have directly compared CBF between high-altitude populations using matched CBF techniques (assessing flow) at the same terrestrial altitude either retrospectively or in an a-priori study. Thus, the purpose of this investigation is to compare regional and global CBF and CDO2 between Andeans, Sherpa, and acclimatized lowlanders at a closely matched altitude of ~5000m. It was hypothesized that 1) Sherpa would present with lower CBF and CDO2 compared to lowlanders at matched altitudes, suggestive of a unique protective physiological adaptation. 2) Andeans and lowlanders will present with similar CBF and CDO2. 3.2 Methods Ethical Approval Studies completed in Lowlanders and Sherpa participants were obtained as part of the UBC 2016 Nepal expedition and were approved by the Clinical Research Ethics Board of the University of British Columbia (H16-01028) and the Nepal Health Research Council. Details on this expedition 55 and the overall research frame work have been published elsewhere (Willie et al., 2018). Studies completed in Andeans were approved by the International Committee of Ethics in Research of the Institute of Tropical Medicine "Daniel Alcides Carrion" of the National University of San Marcos (CIEI-2019-002) and completed as part of the Expedition 5300 high altitude research expedition completed in 2020. Participants Participant characteristics including age, height, weight, years spent at altitude and altitude of birth/childhood are presented in Table 3-1. Lowlander participants comprised of expedition team members and researchers within the UBC 2016 Nepal expedition framework (Willie et al., 2018). All lowlander participants were born and resided at altitudes <1500m within Canada, the UK and New Zealand. Sherpa participants were also recruited within the UBC 2016 Nepal expedition framework and worked as trekking and climbing guides in the Khumbu region of Nepal. All Sherpa participants were born and resided at altitudes >2000m. Andean participants whom were recruited resided in La Rinconada, Peru (5100 m) and reported to a temporary medical research laboratory set-up within the city of La Rinconada. The Andean participants in this study were selected from a larger data set of Andean participants with and without chronic mountain sickness (CMS). Based on previously published data and discussions (Hancco et al., 2020; Oberholzer et al., 2020; Champigneulle et al., 2022, 2023), the classical threshold for excessive erythrocytosis of 21 g/dL in males is likely inappropriate for this population given their residing altitude of 5100m, and would disproportionately select participants with relatively low [Hb] within the population. Therefore, Andean participants were included in this study if arterial [Hb] values at time of testing were ≤24.0 g/dL and if participants did not present with CMS symptoms scoring ≥6 based on the Qinghai CMS scoring system (considering the adjusted excessive erythrocytosis threshold used in this study). 56 Table 3-1. Participant characteristics Lowlanders Sherpa Andeans n 14 male 14 male 14 male Age (years) 29 ± 7 25 ± 7 40 ± 3 * † Height (cm) 179 ± 6 168 ± 7 * 164 ± 5 * Body Mass (kg) 74.1 ± 7.0 61.4 ± 8.0 * 66.1 ± 6.5 * BMI 23.2 ± 2.1 21.7 ± 3.0 24.6 ± 1.6 Years at altitude 0 21 ± 9 * 37 ± 6 * † * Signifies a significant difference compared to lowlanders, P<0.05. † Signifies a significant difference compared to Sherpa, P<0.05. BMI, body mass index. Experimental measures Measurements of CBF, arterial blood gases and blood pressure were obtained in all participants following ~10 minutes of supine rest. Data in Lowlanders and Sherpa was obtained following 2-3 weeks spent at 5050m (~3-4 weeks spent >2400m). All Andean participants had resided at 5100m for >3 months prior to testing. Volumetric measures of cerebral blood flow in the right internal carotid artery (ICA), and left vertebral artery (VA) was assessed using a 10 MHz multi‐frequency linear array duplex ultrasound (uSmart 3300, Teratech, Burlington, MA, USA) following published technical recommendations (Thomas et al., 2015). Vessel diameter was assessed using B-mode imaging while pulse-wave mode was used to simultaneously measure peak envelope blood velocity. The ICA diameter and velocity were measured at least 1.5 cm distal to the common carotid bifurcation to eliminate recordings of turbulent and retrograde flow, while VA diameter and velocity were measured between C4 and C5, and C5 and C6, or proximal to entry into the vertebral column. The specific location in which the vessels were insonated was determined on an individual bases to ensure optimal image quality in each participant. Ultrasound scans were completed by (C.W.) in lowlanders and Sherpa in Nepal, and by (C.A.H) in Andeans in Peru. A minimum duration of one-minute videos were recorded and stored as video files for offline analysis using automated edge detection software (Woodman et al., 2001) following study completion. 57 Arterial blood samples were obtained using single radial arterial punctures with participants resting in the supine position. Measured arterial blood variables included the partial pressure of arterial oxygen (PaO2), partial pressure of arterial carbon dioxide (PaCO2), arterial oxygen saturation (SaO2), bicarbonate (HCO3-), [Hb], and pH. In lowlanders and Sherpa, blood samples were analyzed using an ABL90 blood gas analyzer. In Andeans, blood samples were analyzed using an I-STAT blood gas analyzer. All blood gas data were analyzed at an assumed core body temperature of 37.0°C. Mean arterial blood pressure (MAP) was assessed in duplicate with participants resting in the supine position using an automated sphygmomanometer (Lowlanders and Sherpa: UA-767FAM, Life Source, Mississauga, Canada); (Andeans: HEM-7321-E, Omron, Kyoto, Japan). Calculations Volumetric blood flow within the ICA and VA was calculated using the formula: Q (mL/min) = (0.5 × Peak envelope velocity) × (π × (0.5 × Diameter)2 ) × 60s. The global CBF (gCBF) was estimated as twice the sum of the unilateral ICA and VA flow measurements. The gCBF conductance (CVC) was calculated using the formula: gCBF CVC (ml/min/mmHg) = gCBF / MAP. The CaO2 was calculated using the formula : CaO2 (ml/dl) = (1.34 × [Hb] × SaO2) + (0.003 × PaO2). The CDO2 was calculated using the formula: CDO2 (ml/min) = gCBF × CaO2. Statistical analysis Statistical analysis was performed using IBM SPSS software. Data are presented as means ± SD with statistical significance set at P<0.05. One-way ANOVA’s were used to detect differences between groups with post-hoc comparisons made using Bonferroni multiple comparison tests when applicable. As metrics which are known to affect CBF regulation differed between groups (age, CaO2, and PaCO2; see Results), an ANCOVA analysis was also performed on metrics of CBF (ICA flow, VA flow, and gCBF). Following evaluation of the ANCOVA model assumptions, only CaO2 was included as a covariate alongside population as a fixed effect. The variable selected was included as it has been shown to influence CBF in humans (Willie et al., 2014b) and improved 58 the model fit, as determined by the −2 log likelihood indicating their acceptability in the model. For transparency, CBF results are presented with and without CaO2 as a covariate included in the model. 3.3 Results Arterial blood gases and blood pressure Arterial blood gas and blood pressure data is presented in Table 3-2. Hemoglobin concentration was markedly higher in Andeans compared to both Sherpa (P<0.001) and Lowlanders (P<0.001). Both PaCO2 and pH were similarly higher in Andeans (P<0.001; and P=0.002) and Sherpa (P=0.020; and P=0.044) compared to lowlanders, while HCO3- was similar between groups. Andeans presented with lower SaO2 compared to both Sherpa (P=0.004) and lowlanders (P<0.001), but PaO2 in Andeans was only lower compared to lowlanders (P=0.004). The CaO2 was higher in Andeans when compared to both Sherpa (P=0.003) and lowlanders (P=0.027). Across all three groups, there were no differences in systolic blood pressure, diastolic blood pressure, or MAP. 59 Table 3-2. Arterial blood gas and blood pressure Lowlanders Sherpa Andeans Hb (g/dl) 15.6 ± 1.0 15.9 ± 1.2 20.4 ± 1.4 * † pH 7.446 ± 0.023 7.420 ± 0.027 * 7.407 ± 0.031 * PaCO2 (mmHg) 29.2 ± 2.4 32.1 ± 3.3 * 33.3 ± 2.3 * PaO2 (mmHg) 44.4 ± 6.9 42.1 ± 5.7 37.1 ± 3.9 * SaO2 (%) 82.7 ± 4.4 78.4 ± 5.6 71.4 ± 6.2 * † SpO2 (%) 88 ± 2 86 ± 4 79 ± 3 * † HCO3- (mmol/l) 20.1 ± 1.4 20.8 ± 2.0 21.1 ± 2.1 MAP (mmHg) 93 ± 8 86 ± 9 85 ± 13 CaO2 (ml/dl) 17.4 ± 1.8 16.8 ± 2.3 19.6± 2.1 * † * Signifies a significant difference compared to lowlanders, P<0.05. † Signifies a significant difference compared to Sherpa, P<0.05. Hb, hemoglobin; PaCO2, partial pressure of arterial carbon dioxide; PaO2, partial pressure of arterial oxygen; SaO2, arterial oxygen saturation; SpO2, peripheral oxygen saturation; HCO3-, bicarbonate; MAP, mean arterial pressure; CaO2, arterial oxygen content. Cerebral blood flow Metrics of CBF are presented in Table 3-3, Figure 3-1 and Figure 3-2 without covariate analysis. No differences in ICA flow, VA flow, or gCBF were observed between groups. However, in both the ICA and VA, Andeans presented with larger vessel diameter compared to Sherpa (ICA and VA; P<0.001) and lowlanders (ICA; P<0.001, VA; P=0.023) with concomitantly lower ICA and VA velocity compared to Sherpa (ICA; P=0.004, VA; P<0.001) and lowlanders (ICA; P=0.001, VA; P<0.001). Additionally, no differences in gCBF CVC were observed. While CDO2 was not different between lowlanders and Andeans, both groups presented with elevated CDO2 compared to Sherpa (P<0.001). 60 Table 3-3. Regional and global cerebral blood flow Lowlanders Sherpa Andeans ICA Diameter (cm) 0.524 ± 0.054 0.475 ± 0.037 0.631 ± 0.097 * † ICA Velocity (cm/s) 39.02 ± 6.92 38.04 ± 8.75 27.98 ± 7.26 * † ICA Flow (ml/min) 252.29 ± 56.90 205.03 ± 67.52 261.76 ± 73.88 VA Diameter (cm) 0.372 ± 0.037 0.338 ± 0.044 0.428 ± 0.071 * † VA Velocity (cm/s) 21.63 ± 5.19 21.23 ± 5.71 13.24 ± 3.10 * † VA Flow (ml/min) 71.15 ± 20.81 60.24 ± 28.72 59.50 ± 23.37 gCBF (ml/min) 646.89 ± 113.64 530.53 ± 178.98 642.52 ± 141.56 gCBF CVC (ml/min/mmHg) 7.00 ± 1.40 6.19 ± 2.12 7.70 ± 2.16 CDO2 (ml/min) 111.94 ± 18.48 87.70 ± 27.02 * 124.59 ± 24.57 † * Signifies a significant difference compared to lowlanders, P<0.05. † Signifies a significant difference compared to Sherpa, P<0.05. ICA, internal carotid artery; VA, vertebral artery; gCBF, global cerebral blood flow; CVC, conductance; CDO2, cerebral oxygen delivery. 61 020406080ICA velocity (cm/s)P=0.004P=0.0010.00.20.40.60.81.0ICA diameter (cm)P<0.001P<0.001Lowlanders Sherpa Andeans0100200300400500ICA Flow (ml/min)0.00.10.20.30.40.50.6VA diameter (cm)P<0.001P=0.023010203040VA velocity (cm/s)P<0.001P<0.001Lowlanders Sherpa Andeans050100150VA Flow (ml/min) Figure 3-1. Regional cerebral blood flow Internal carotid artery (ICA) and vertebral artery (VA) vessel diameter, blood velocity and blood flow in Lowlanders (n=14), Sherpa (n=14), and Andeans (n=14). Data are presented as individual data points (open circles) with mean and standard deviations underlaid. Connecting bars above data with accompanying P-values indicate a significant difference between groups. Significance for all comparisons is set at P<0.05. 62 0102030CaO2(ml/dl)P=0.003P=0.027Lowlanders Sherpa Andeans020040060080010001200gCBF (ml/min)Lowlanders Sherpa Andeans050100150200CDO2 (ml/min)P<0.001P=0.030 Figure 3-2. Oxygen content, global cerebral blood flow, and oxygen delivery Arterial oxygen content (CaO2), global cerebral blood flow (gCBF), and cerebral oxygen delivery (CDO2) in Lowlanders (n=14), Sherpa (n=14), and Andeans (n=14). Data are presented as individual data points (open circles) with mean and standard deviations underlaid. Connecting bars above data with accompanying P-values indicate a significant difference between groups. Significance for all comparisons is set at P<0.05. Covariate analysis An ANCOVA was used to investigate the effect of population type on resting CBF within the three groups by controlling for the severity of hypoxemia (CaO2), which is known to impact CBF regulation and resting CBF patterns. Metrics of CBF with co-variate analysis are presented in Table 3-4 and Figure 3-3. With covariate analysis, ICA flow was higher in Andeans and Lowlanders compared to Sherpa (P=0.002 and P=0.002, respectively), while VA flow was not different across groups. Global CBF was similar between Andeans and Lowlanders, but was elevated in both populations compared to Sherpa (P=0.005 and P=0.003, respectively). 63 Table 3-4. Regional and global cerebral blood flow with covariate analysis Lowlanders Sherpa Andeans ICA Flow (ml/min) 244.20 ± 15.68 186.20 ± 16.37 * 288.67 ± 17.22 † VA Flow (ml/min) 72.01 ± 6.64 62.24 ± 6.93 56.64 ± 7.28 gCBF (ml/min) 632.43 ± 36.75 496.88 ± 38.36 * 690.63 ± 40.34 † * Signifies a significant difference compared to lowlanders, P<0.05. † Signifies a significant difference compared to Sherpa, P<0.05. ICA, internal carotid artery; VA, vertebral artery; gCBF, global cerebral blood flow. Data reported as the estimated marginal means ± SE. 64 0100200300400500ICA Flow (ml/min)P=0.038P<0.001020406080100120VA Flow (ml/min)Lowlanders Sherpa Andeans02004006008001000gCBF (ml/min)P=0.039P=0.0070100200300400500ICA Flow (ml/min)020406080100120VA Flow (ml/min)Lowlanders Sherpa Andeans02004006008001000gCBF (ml/min)With covariatesWithout covariates Figure 3-3. Cerebral blood flow with and without covariate analysis Internal carotid artery (ICA) and vertebral artery (VA), and global cerebral (gCBF) blood flow in Lowlanders (n=14), Sherpa (n=14), and Andeans (n=14) presented without (left figures) and with (right figures) covariate analysis (covariate; CaO2). Data are presented as means with standard deviations. Connecting bars above data with accompanying P-values indicate a significant difference between groups. Significance for all comparisons is set at P<0.05. Data without covariate analysis are presented as means ± SD. Data with covariate analysis are presented as estimated marginal means ± SE. 65 3.4 Discussion This is the first study to compare CBF, CDO2 and the systemic determinants that influence CBF and CDO2 using volumetric measures of flow and arterial blood gases in acclimatized lowlanders, Sherpa, and Andeans residing at ~5000m elevation. The results of this study demonstrate that 1) Analysis of flow without covariance did not reveal any differences in CBF between populations within the ICA, VA, or globally, although CDO2 was lower in Sherpa compared to lowlanders and Andeans. However, when CaO2 was included as a covariate, differences in ICA flow and gCBF were observed whereby CBF was higher in both lowlanders and Andeans compared to Sherpa. 2) Andeans present with markedly elevated cerebral vessel diameters yet consequently lower blood velocity in both the ICA and VA compared to Lowlanders and Sherpa. These findings are in agreement with our hypothesis that patterns of CBF would be lower in Sherpa compared to lowlanders but would not differ between Andeans and lowlanders. That CDO2 and CBF (with covariance analysis) are attenuated in the Sherpa - the most adapted of all three groups (Beall, 2006) - potentially reflects an adaptive trait to this environment. Differential pattern of CBF While initial analysis did not reveal any differences in regional or global CBF between groups, this appears to be due to differences in CaO2 between groups. With covariate analysis, both ICA flow and gCBF were higher in lowlanders and Andeans compared to Sherpa. Higher CBF and CDO2 in lowlanders compared to Sherpa aligns with previous reports from our group obtained during ascent to 5050m (Hoiland et al., 2019b). Data obtained at 4300m also has shown no difference in CBF between Lowlanders and Andeans (Stacey et al., 2023). Similar to CBF, CDO2 seems to follow a similar pattern within each population, whereby Sherpa present with 22% and 33% lower CDO2 compared to lowlanders and Andeans; with no differences between the latter. As measurements of variables that contribute to CBF regulation were either not different (MAP) or were accounted for within the covariate analysis, differences in other physiological variables must account for these differences in resting CBF and CDO2. We have previously postulated that Sherpa may exhibit a reduction in CMRO2 under hypoxic conditions (Hoiland et al., 2019b) and we continue to suspect that alterations in metabolism may contribute to this unique pattern of CBF and CDO2. A reduction of cerebral metabolism in a population residing at high-altitude that 66 frequents even higher altitudes (such as during mountaineering) may provide a greater buffer to increase CBF in the presence of greater severity hypoxia. This is also evidenced by lower cortical atrophy and white matter hyperintensities post ascent of 8000m peaks in Sherpa compared to lowlanders suggesting greater defense against cerebral hypoxic insults (Garrido et al., 1996). Together, we interpret these findings in Sherpa to be neurologically beneficial from an oxygen conservation perspective and, as such, reflective of an adaptative trait in view of their greater evolutionary history in this environment. In Andeans, one could argue that CDO2 is disproportionately elevated given the markedly elevated CaO2 yet relatively similar CBF compared to lowlanders. This finding is relatively surprising as previous work has shown that cerebrovascular hypoxic reactivity is inversely related to [Hb] whereby lowlanders with lower [Hb] and Andeans following hemodilution exhibit greater hypoxic reactivity (Hoiland et al., 2023). However, it may be that the relatively small plasma volume in Andean participants may limit the total plasma glucose pool, necessitating higher CBF to maintain a sufficient volume of glucose delivery for brain. This theory has been postulated to explain the reported lower cerebral metabolic rate observed in Andean highlanders compared to lowlanders when tested under normoxic conditions at sea-level (Hochachka et al., 1996). Cerebrovascular function and structure A specific phenotype observed within the peripheral vasculature of Andeans with elevated [Hb] (Tremblay et al., 2019) CMS (Rimoldi et al., 2012) is markedly elevated arterial vessel diameters with low blood velocity. This specific pattern of peripheral blood flow has been suggested to be a consequence of elevated blood viscosity, and thus vessel wall shear stress, known as the “shear stress normalization hypothesis” (Kamiya & Togawa, 1980; Girerd et al., 1996). This pattern of vascular remodeling appears to be present within the cerebrovasculature of this Andean high-altitude population whereby both ICA and VA diameters are markedly larger compared to Sherpa and lowlanders. Future studies are needed to determine if the elevated vessel diameters may indeed be a response to chronically elevated vessel shear stress, a result of greater erythrocyte mediated vasodilation under chronic hypoxic conditions, or both. 67 Vessel diameters within the Sherpa appear to be similar to that of lowlanders, but Sherpa still present with lower ICA flow and gCBF. From a structural cerebrovascular perspective this could indicate greater capillarization downstream within the cerebrovasculature. High capillarization in the peripheral tissue is a hallmark adaptation in Sherpa (Gilbert-Kawai et al., 2017b; Horscroft et al., 2017) therefore, if altered cerebral metabolism is not responsible for the lower CBF and CDO2 compared to lowlanders, differences in the microstructure of the cerebrovasculature that optimize the efficiency of oxygen utilization (Gayeski et al., 1988) is the next most tenable mechanism responsible for lower CBF and CDO2 observed in Sherpa. Limitations The data reported in this study were collected as part of two separate high-altitude field expeditions. Data collected in Sherpa and acclimatized lowlanders was obtained as part of the 2016 UBC Nepal expeditions in which a number of studies were completed comparing Sherpa and lowlanders as they ascended and stayed at high-altitude over multiple weeks. This work was able to take place due to relationships with specific local groups in Nepal that developed over multiple years and included careful consideration to both logistical and cultural considerations. Therefore, studying female Sherpa was not an option for this specific expedition. Similarly, data collected in Andeans was part of a 2020 field expedition in collaboration and support from the mining cooperative within La Rinconada. Thus, the first of these expeditions under the umbrella of Expedition 5300 have focused on studying miners working in the gold mines within and surrounding La Rinconada. Unfortunately, females are prohibited from this mining profession in La Rinconada (Enserink, 2019) and thus were not included in data collection for this study. Subsequent and upcoming expeditions in La Rinconada have a much greater focus on studying female and children Andean residents (Champigneulle et al., 2023). Although our approach using regional volumetric measurements of CBF circumvents many of the limitations of only measuring intra-cranial cerebral blood velocity (Ainslie & Hoiland, 2014) we acknowledge that these regional measures of blood flow into the brain are relatively obtuse compared to more advanced imaging modalities. Although not currently feasible at 5000m, future research that incorporates modern neuroimaging and invasive monitoring for analysis of localized 68 cerebral perfusion and molecular signatures of neuronal function would be needed to extend our current findings. The covariate analysis of CBF between groups attempted to control for the impact of differences in variables that are know to effect CBF. However, while variables such as age and PaCO2 were different between groups, they did not meet the required ANCOVA model assumptions and thus could not be included in the statistical analysis. However, differences in age and PaCO2 do not necessarily explain the findings observed, as older in age in Andeans would be expected to present as lower CBF compared to lowlanders and Sherpa, whereas higher PaCO2 in Andeans and Sherpa compared to lowlanders would elicit higher CBF in both groups. Thus the differences in CBF observed between groups are likely to be magnified if age and PaCO2 were matched between groups. 3.5 Conclusion In this study, covariate analysis showed that Sherpa present with lower gCBF compared to both lowlanders and Andeans and ultimately, lower CDO2. Andeans however had similar regional and global CBF compared to lowlanders, but presented with an altered vascular phenotype whereby both ICA and VA vessel diameters were markedly larger compared to Sherpa and lowlanders. As Sherpa are considered to be better adapted to high-altitude than Andeans and lowlanders, the cerebrovascular phenotype of lower oxygen delivery in Sherpa may be indicative of a beneficial adaptation, possibly allowing for greater reserve to increase oxygen delivery under more severe hypoxic conditions or may be present due to differences in cerebral oxygen handling or demand. 69 Chapter 4: Study 2 – Cerebral blood flow in Andean children and adolescents living above 5000m Abstract A number of indigenous populations have resided at high-altitude for generations, resulting in various phenotypical adaptations promoting successful high-altitude adaptation. While many of these adaptations have been investigated in adults, little is known regarding how children residing at high-altitudes adapt, particularly with regards to the cerebrovasculature. In this study, we evaluated regional and global cerebral blood flow (CBF) in Andean children and adolescents living in the highest city in the world at 5100m. Eighteen Andeans (ages 6-17years) living in La Rinconada, Peru (5100m) were compared to sex, age, size, and maturity matched high-altitude Sherpa (3400m) living in Nepal (n=18) and lowlanders (44m) living in Cardiff, Wales (n=18). Volumetric measurements of CBF were assessed using duplex ultrasound of the common carotid, internal carotid, and vertebral arteries to assess regional and global CBF. Despite Andean living under a more severe hypoxic environment, global CBF was similar between Andeans (687.01±138.49ml/min), Sherpa (711.27±110.27ml/min) and lowlanders (704.88±59.23ml/min). In contrast, vertebral artery blood flow was 24% lower in Andeans compared to lowlanders. The similar global CBF in Andean children might be achieved through elevated hemoglobin concentration. However, lower posterior perfusion in Andeans requires further investigation to determine whether it represents an adaptive or maladaptive response. 4.1 Introduction High-altitude environments present with a number of physiological stressors that challenge various homeostatic functions, such as cold stress, resource availability, and perhaps most notably, hypoxia. Upon exposure to normobaric or hypobaric hypoxia, in lowlanders, cerebral blood flow (CBF) is increased in order to maintain cerebral oxygen delivery (CDO2) (Hoiland et al., 2016). Indigenous high-altitude adults, such as Tibetan and Andean highlanders, appear to demonstrate lower CBF when compared to lowlander adults at the same altitude [reviewed in:(Jansen & Basnyat, 2011)]. Volumetric measures of CBF during ascent to high-altitude reveal that adult Tibetan’s (Sherpa) present with both a lower global CBF and CDO2 compared to adult lowlanders (Hoiland et al., 2019b). A lower global CBF was also reported in adult Tibetans when compared 70 to adult Han Chinese when assessed using volumetric flow measures, following 5-years acclimatization to high-altitude(Liu et al., 2016). In Andeans, markedly elevated hemoglobin concentration(Beall, 2006), reduced cerebral metabolism (Hochachka et al., 1994), and possibly elevated blood viscosity (Tymko et al., 2019a) are likely responsible for observations of reductions in CBF (Milledge & Sorensen, 1972; Marc-Vergnes, 1974; Sorensen et al., 1974b; Claydon et al., 2005; Appenzeller et al., 2006). The mechanisms mediating lower CBF - and consequently CDO2 - in adult Sherpa remain unclear; however, it has been postulated to be due to reductions in cerebral metabolism which may aid oxygen conservation at high altitude (Hoiland et al., 2019b). While mechanisms of CBF regulation in adult permanent altitude residents continue to be elucidated, there is a paucity of research in children and adolescent high-altitude residents. In lowlanders, CBF increases in proportion to neural development, myelination, and metabolic demand until approximately 6-10 years of age (Schöning & Hartig, 1996; Takahashi et al., 1999; Wu et al., 2016; Paniukov et al., 2019), before declining during adolescence (Satterthwaite et al., 2014; Vandekar et al., 2019; Talbot et al., 2023). A similar developmental trajectory in CBF has been noted in Bolivian children and adolescents living at 4000m in Bolivia (Virués-Ortega et al., 2011). In Sherpa children residing at 3800m, CBF is markedly reduced when compared to lowlander children assessed under normoxic conditions at sea-level (Flück et al., 2017). Previous comparative reports of CBF in Andean children at high-altitude are extremely limited, however, and solely dependent on velocity measurements (Hogan et al., 2010) which present with a number of caveats [reviewed in:(Ainslie & Hoiland, 2014) ]. One study has reported reduced basilar artery blood velocity - but similar middle cerebral artery blood velocity - in children residing at 3700m compared to sea-level residents (Hogan et al., 2010). One of the most prominent high-altitude settlements in terms of size and altitude severity is La Rinconada in Peru. Located at an altitude between 5100 and 5300m, La Rinconada operates as an artisanal gold mine with an approximated population of 50,000 Andean highlanders who primarily work in the mines or as shop keepers (Champigneulle et al., 2023). Little is known about how being born and/or raised at this highest permanent residence may influence CBF and other physiological systems. The adult La Rinconada population has a high prevalence of chronic 71 mountain sickness, characterized by remarkably high hemoglobin concentration ([Hb]), blood viscosity, and blood volumes (Hancco et al., 2020; Stauffer et al., 2020; Oberholzer et al., 2020; Champigneulle et al., 2022) which likely contributes to their CBF regulation. Hemoglobin has been shown to be a particularly potent regulator of CBF due to its role in nitric oxide signaling (Hoiland et al., 2023) and contribution to arterial oxygen content (CaO2) (Carr et al., 2023). In lowlander children, [Hb] increases with maturation(Perkins et al., 2022). Data is more limited in Andeans, with one study showing that [Hb] increases with age from 6-59 months (Ocas-Córdova et al., 2018). This response appears to be modulated by altitude whereby in the same data set, [Hb] is elevated with increasing altitude. Similar results have been reported in Andean children aged 1-16 years whereby [Hb] is elevated at higher altitudes (León-Velarde et al., 2004). Assuming this pattern of elevated [Hb] with altitude occurs in the population studied in La Rinconada, it could be postulated that despite living at extremely high altitude, elevated [Hb] and thus elevated CaO2, may not necessitate a compensatory blood flow response to maintain oxygen delivery for the given environmental hypoxic stimuli compared to other high-altitude dwelling populations. In addition to the lack of understanding regarding CBF regulation in children and adolescents at high-altitude, evaluation of individuals across this age range may provide a greater understanding of when in the life cycle that the phenotypical adaptations we see in adult high-altitude populations are occurring. Therefore, the purpose of this study was to quantify CBF in children and adolescents of Andean descent living at 5100m and compare them to age, sex, maturity and body mass-matched Sherpa children living at high-altitude (3800m) and lowlander children at sea-level. It was hypothesized that despite the higher altitude of residence, Andean children and adolescents living at 5100m would present with similar CBF compared to Sherpa and lowlander children and adolescents. 4.2 Methods Ethical Approval Data for this study were collected during two separate high-altitude expeditions in the Peruvian Andeans [Expedition 5300:(Champigneulle et al., 2023)], the Khumbu valley of Nepal (Willie et al., 2018) and from data collected at Cardiff Metropolitan University in Wales, UK. Prior to 72 commencement of each study, a detailed verbal and written explanation of the experimental measures was provided to the study participant and their parent/guardian. The participant’s parent/guardian and participants provided written, informed ascent or consent, respectively. Documents were provided to participants in their native language by; a medical student from the Universidad Nacional del Altiplano de Puno and a study investigator in Peru; a Nepali Physician and Sherpa language translator in Nepal; a member of the research team at Cardiff Metropolitan University in Wales. The studies were approved by the International Committee of Ethics in Research of the Institute of Tropical Medicine "Daniel Alcides Carrion" of the National University of San Marcos (CIEI-2019-002), the University of British Columbia Clinical Research Ethics Board (H16-01028) and the Nepal Health Medical Research Council (227-2016), and by the Cardiff Metropolitan University Natural Sciences Research Ethics Sub-committee (PGR-1339). All three studies conformed to the standards set by the Declaration of Helsinki (except registry in a database). Participants Eighteen Andean children between the ages of 6-17 years were recruited for this study in the city of La Rinconada, Peru. Data in 18 Sherpa and 18 lowlander children were obtained from two separate datasets matching as closely as possible for sex, age, height, weight, and maturity offset to the Andean participants (Table 1). Information was obtained from the mothers of the Andean participants regarding location of pregnancy, pregnancy duration, location of birth, and birth weight. This information is included as descriptive variables and is not intended to be compared across groups. All Sherpa participants were of Sherpa descent and were born in the Khumbu valley of Nepal, as confirmed by their parents. Experimental measures Volumetric measures of CBF within the common carotid artery (CCA), internal carotid artery (ICA), and vertebral artery (VA) was assessed using a 10 MHz multi‐frequency linear array duplex ultrasound (uSmart 3300, Teratech, Burlington, MA, USA) according to published technical recommendations(Thomas et al., 2015). Vessel diameter was measured using B-mode imaging while pulse-wave mode was used to simultaneously measure peak envelope blood velocity. The 73 ICA diameter and velocity were measured at least 1.5 cm distal to the common carotid bifurcation to eliminate recordings of turbulent and retrograde flow, while VA diameter and velocity were measured between C4 and C5, and C5 and C6, or proximal to entry into the vertebral column. The specific location in which the vessels were insonated was determined on an individual basis to ensure optimal image quality in each participant. One-minute videos were recorded and stored as video files for offline analysis using automated edge detection software (Woodman et al., 2001) following study completion. Volumetric blood flow within the CCA, ICA and VA was calculated using the following formula: Q = (0.5 × Peak envelope velocity) × (π × (0.5 × Diameter)2 ) × 60 The global CBF (gCBF) was estimated as twice the sum of the unilateral ICA and VA flow measurements. Body mass was measured using an electronic scale with subjects barefoot and dressed in light clothing. Sitting height was recorded with participants seated on a flat based chair and wall mounted measurement device. Anthropometrics, age and sex were used to calculate maturity-offset (Mirwald et al., 2002) whereby participants were classified as pre- or post-peak height velocity (PHV) if they were ± 1.0 year pre- or post-PHV respectively. In Andeans, the end-tidal partial pressure of oxygen (PETO2) and carbon dioxide (PETCO2) was assessed using an AGM100 gas exchange monitor (MediPines Corporation, Yorba Linda, USA) and heart rate and oxygen saturation were assessed with a finger tip pulse oximeter (Pulsox-3Si, Konica Minolta, Japan. In Sherpa, PETO2 and PETCO2 were measured using a metabolic cart (Oxycon Pro, Carefusion), heart rate was assessed using a 3-lead ECG (ADI BioAmp ML132), and oxygen saturation was assessed with a fingertip pulse oximeter (Rad-5, Masimo SET, Irvine, CA). In lowlanders, PETO2 and PETCO2 were measured using a calibrated gas analyser (ML206, AD Instruments Ltd) and heart rate was assessed via finger photoplethysmography (Finometer PRO, Finapres Medical Systems). Finally, hemoglobin concentration was acquired in Andean participants from a capillarized blood sample at either the ear lobe or finger tip and subsequently analyzed using a Hemoque blood 74 analyzer (HemoCue®Hb201+, HemoCue AB, Ängelholm, Sweden). In Andean participants, arterial oxygen content (CaO2) was estimated using the following equation CaO2 = (SpO2 x [Hb] x 1.34 + (PaO2 x 0.003) whereby capillary [Hb] is used in place of arterial [Hb], PETO2 is used in place of PaO2 and SpO2 is oxygen saturation. Cerebral oxygen delivery (CDO2) is presented for Andean participants using the calculated CaO2 value multiplied by the measured gCBF. Study Protocol Participants rested in the supine position for at least 10 minutes prior to measurement acquisition. Volumetric CBF scans were acquired in consecutive order (CCA, VA, and ICA) prior to collection of end-tidal gases, SpO2, heart rate and Hb data. CCA measurements were only recorded in Andean and Sherpa participants while Hb values were only recorded in Andean participants. While Hb values are not compared between groups, since this measure is not commonly reported in this specific population, it has been included in the manuscript as a descriptive assessment. Statistical analysis Statistical analysis was performed using IBM SPSS software (Version 23.0, Chicago, USA). Data are presented as means ± SD with statistical significance set at P<0.05. One-way ANOVA’s were used to detect differences between groups with post-hoc comparisons made using Bonferroni multiple comparison tests when applicable. A secondary analysis was performed to examine sex differences using a mixed-effects model, whereby group and sex were included as fixed factors within the model. 4.3 Results Demographics and resting cardiorespiratory data are presented in Table 4-1. As participants were matched for age, size, and maturity offset (5 females and 4 males pre-PHV; 3 females and 3 males at PHV; 2 females and 1 male post-PHV in all three groups), these variables were comparable between groups (all P>0.05). Heart rate was higher while PetCO2 was lower in Andeans compared 75 to lowlanders (P=0.005 and P<0.001), but not different compared to Sherpa. Oxygen saturation and PETO2 was lower in Andeans compared to Sherpa (P<0.001) and lowlanders (P<0.001). Table 4-1. Participant demographics and cardiorespiratory variables Andean Children Sherpa Children Lowlander Children n 18 (8 male) 18 (8 male) 18 (8 male) Age (yrs) 11.7 ± 3.2 11.6 ± 2.5 11.3 ± 2.8 Height (cm) 140.2 ± 14.5 143.5 ± 15.3 145.4 ± 12.7 Sitting Height (cm) 74.5 ± 7.5 73.8 ± 8.1 75.5 ± 6.5 Body Mass (kg) 38.1 ± 12.7 37.0 ± 13.7 38.2 ± 12.4 BMI (kg/m2) 18.8 ± 3.07 17.4 ± 3.4 17.6 ± 2.9 Maturity offset (yrs) -0.96 ± 2.40 -1.33 ± 2.09 -1.41 ± 2.10 PETCO2 (mmHg) 24.1 ± 2.7* 24.5 ± 2.5* 38.3 ± 3.8 PETO2 (mmHg) 50.1 ± 5.9*! 58.3 ± 4.3* 105.7 ± 4.9 SpO2 (%) 82 ± 4! 94 ± 2 - Heart rate (bpm) 86 ± 15* 86 ± 18 70 ± 9 Hemoglobin (g/dl) 16.6 ± 1.7 - - CaO2 (ml/dl) 18.3 ± 1.7 - - Diff compared to LL = * Diff compared to Sherpa = ! The CBF data are presented in Table 4-2 and Figure 4-1. Andeans had lower CCA velocity compared to Sherpa (P=0.004), however there were no differences in CCA diameter or flow. Although there were no differences in ICA velocity or flow observed between the three groups, ICA diameter was larger in Andeans compared to lowlanders (P=0.025), but not compared to Sherpa. Vertebral artery velocity in the Andean children was markedly lower compared to both Sherpa and lowlanders (P=0.033 and P<0.001); however, VA diameter was not different between groups. This resulted in 24% lower VA flow in Andeans compared to lowlanders (P=0.022). Notably, however, gCBF was not different between groups. 76 Table 4-2. Cerebral blood flow measures in Andean, Sherpa, and Lowlander Children and Adolescents Andean Children Sherpa Children Lowlander Children CCA Diameter (cm) 0.621 ± 0.053 0.595 ± 0.052 _ CCA Velocity (cm/s) 43.88 ± 10.40* 53.22 ± 8.78 _ CCA Flow (ml/min) 400.89 ± 66.01 463.06 ± 122.32 _ ICA Diameter (cm) 0.516 ± 0.042* 0.482 ± 0.050 0.472 ± 0.039 ICA Velocity (cm/s) 42.97 ± 7.95 50.02 ± 9.09 49.84 ± 9.33 ICA Flow (ml/min) 273.60 ± 55.79 275.02 ± 51.19 256.38 ± 20.33 VA Diameter (cm) 0.375 ± 0.054 0.356 ± 0.033 0.372 ± 0.019 VA Velocity (cm/s) 21.14 ± 7.15*! 26.54 ± 4.30 29.56 ± 5.96 VA Flow (ml/min) 72.93 ± 31.60* 80.62 ± 19.64 96.09 ± 19.23 gCBF (ml/min) 687.01 ± 138.49 711.27 ± 110.27 704.88 ± 59.23 CDO2 (ml/min) 126.5 ± 27.1 - - Diff compared to LL = * Diff compared to Sherpa = ! 77 Figure 4-1. Regional and global cerebral blood flow Regional and global cerebral vessel diameter, blood velocity and blood flow. Due to exclusion of ultrasound video files with insufficient image quality, metrics of ICA blood flow are reported for n=14 Andeans, VA blood flow metrics are reported for n=15 Andeans, and gCBF are reported for n=14 Andeans. Full data sets are presented for Lowlander and Sherpa participants. Andeans Sherpa Lowlanders02004006008001000gCBF Flow (ml/min)Andeans Sherpa Lowlanders0100200300400ICA Flow (ml/min)Andeans Sherpa Lowlanders050100150VA Flow (ml/min)P=0.0220.30.40.50.60.7ICA diameter (cm)P=0.005020406080ICA velocity (cm/s)0.20.30.40.50.6VA diameter (cm)01020304050VA velocity (cm/s)P=0.033P<0.00178 Individual pregnancy and birth information for Andean participants are included in Table 4-3. Briefly, 14 participants were carried at 5100m during pregnancy, while four were carried at 3850m. Thirteen participants were carried to term (37-38 weeks), four participants were born pre-term (30-36 weeks), and one participant was born post-term (41 weeks). Five of the participants were born `in La Rinconada at 5100m while the remainder were born between altitudes of 3400-3850m. Average birth weight was 3.04 ± 0.60 kg. Nine participants have resided in La Rinconada since birth.79 Table 4-3. Pregnancy and birth metrics for Andean participants M: Male; F: Female Sex differences Males presented with larger VA diameters compared to females across groups (main effect; P=0.004), however, there were no differences in any other demographic, cardiorespiratory, or cerebrovascular variable between sexes. Subject Altitude of pregnancy (meters) Pregnancy duration (weeks) Birth Weight (kg) Altitude of birth (meters) Age of arrival in La Rinconada (years) F01 5100 35 3.5 3840 0 F02 5100 38 3 3825 0 F03 5100 38 3.5 3825 7 F04 5100 36 2.03 3825 0 F05 5100 38 3.5 3825 4 F06 5100 38 2.8 3825 6 F07 5100 35 2.7 5100 0 F08 5100 38 3.2 5100 0 F09 3825 38 N/A 3825 9 F10 3825 38 3.6 3825 17 M01 5100 30 1.8 3825 6 M02 3878 38 3.2 3878 1 M03 5100 38 2.1 5100 0 M04 5100 41 3.8 5100 0 M05 5100 37 N/A 5100 0 M06 5100 37 3.5 3400 1 M07 3825 38 3.2 3825 10 M08 5100 38 3.2 3850 0 Average 4820 37 2.97 4260 2 SD 525 3 0.68 666 3 80 4.4 Discussion For the first time, we assessed CBF in Andean children and adolescents living above 5000m in the highest city in the world, La Rinconada, Peru. As expected, although end-tidal PCO2 was comparable, Andean children presented with greater hypoxemia compared to both lowlanders and Sherpa children residing at lower altitude. Despite this marked hypoxemia, subtle regional differences in CBF were observed whereby ICA diameter was larger and VA flow lower in the Andeans when compared to lowlanders. Notably, however, global CBF was not different between the three populations. Comparable global CBF between populations: While similar resting CBF values may seem surprising in this population comparison, it does align with recent reports in adult Andean residents at 4300m whereby CBF has been shown to not differ compared to lowlander counterparts (Stacey et al., 2023). However, elevations in [Hb] and hence oxygen content likely help to maintain oxygen delivery; therefore, CBF does not need to be elevated (see methodological considerations). This observation is consistent with the notion that CBF is regulated, at least in part, via CaO2 – rather than PaO2 per se - to maintain CDO2 (Hoiland et al., 2023). It is also important to note that haematocrit (and therefore viscosity) is also likely to be higher in the Andean group. The limited data available suggest that the inverse relationship between blood haematocrit and CBF (Metry et al., 1999) is also a function of oxygen delivery rather than viscosity per se (Todd et al., 1994; Tomiyama et al., 1999). Reductions in posterior blood flow: Although no differences in either anterior or posterior flow were observed between Andeans and Sherpa, posterior blood flow - as measured through the VA - was 24% lower in Andeans compared to lowlanders. Similar findings of lower VA flow have previously been observed in both adult and adolescent Sherpa compared to lowlander counterparts (Flück et al., 2017; Hoiland et al., 2019b). The significance of these findings is related to the anatomical link from the VA to areas of the brainstem that subserve critical homeostatic function. Observations of a lower VA flow – and potentially flow to the brainstem - contrast with findings in adults at sea level which have reported that blood flow to the brainstem increases more than that to the middle and anterior regions for a given severity of isocapnic hypoxia (Willie et al., 81 2012; Ogoh et al., 2013). Consistent with these observations, positron emission tomography scan data also indicate that phylogenetically older regions of the brain (including the brainstem) tended to show larger vascular responses to hypoxia than evolutionary younger regions (Binks et al., 2008). This may differ in children however, as previous research has shown that girls present with a smaller relative VA blood flow response under poikilocapnic hypoxia compared to their mothers (Morris et al., 2017). Regardless, the implications of the chronic reductions in posterior CBF on developmental cognition and other aspects of homeostatic function require future research. Indeed, it may be that reductions in posterior CBF underlie the reported increased risk of neurodevelopmental problems during childhood at high altitude (Wehby, 2013) . Acid-base balance: As expected, both SpO2 and PETCO2 were markedly reduced in Andeans compared to lowlanders; however, resting PETCO2 was similar between Andeans and Sherpa. Given the fundamental gas equation linking alveolar PCO2, metabolism and alveolar ventilation (PACO2 = VCO2/VA), this observation suggests that Andeans are hypoventilating relative to their Sherpa counterparts at lower altitudes. As arterial PCO2 is a potent stimulant for CBF [reviewed in: (Hoiland et al., 2019a)], Andeans presenting with similar PETCO2 and a greater level of hypoxemia would be expected to elicit higher CBF compared to Sherpa, however this was not the case. Although changes in metabolic demand or acid-base balance should be considered as possibly contributing to differences in CBF, it seems more likely that changes in CaO2 may explain the maintenance of CBF across groups. Methodological considerations: Hemoglobin: Unfortunately, we were unable to obtain Hb data in our Sherpa and lowlander data sets. Values presented in Andeans appear to be higher than normative values expected in sea-level children populations. Indeed, Perkins et al., 2022 recently reported Hb data from a population of youths in a similar age range and from the same institution/region as the present study (Cardiff, Wales). Values for [Hb] in boys and girls reported for both pre- and post-peak height velocity were 13.3-14.2 g/dl and 12.5-13.2 g/dl, respectively. Mean [Hb] values also have been reported in Tibetans children and adolescents residing at 3800m whereby in males aged 6-17 years, mean [Hb] was 14.0-16.4 g/dL and in females aged 6-17 years, mean [Hb] was 14.1-15.1 g/dL (Garruto et al., 82 2003) . These reported values appear to be marginally higher than those reported in the lowlanders by Perkins et al but slightly lower than those reported in the Andean data set here-in. This aligns with the adaptations observed in Andean adults whereby Hb concentration and volume appears to be markedly elevated compared to lowlanders and other high-altitude populations (Beall et al., 2002; Beall, 2006, 2007; Stembridge et al., 2019). Timeline of adaptations: Due to the relatively small sample size in each group, we were unable to examine whether there are any differences in CBF across the reported age range between groups. Further whether the pattern of CBF responses in Andeans are occurring via the same mechanisms observed in adults is yet to be seen. Recently, Rieger et al., 2022 demonstrated that during automobile ascent to 3800m, both lowlander adults and children demonstrated the same pattern of CBF change following 4 days at 3800m. This would suggest that the responses to hypoxia and/or adaptations within a population are similar across age and may not differ between adults and children; therefore, the phenotypical differences observed between these high-altitude groups are occurring prior to adulthood, but the timeline of this has yet to be examined. Sex differences: While not an a prori research question, sex differences were investigated between populations in this study as part of a secondary analysis. Apart from VA diameter, which was observed to be larger in males compared to females, no other sex differences were observed in any other cardiovascular or cerebrovascular parameter. Previous work has shown that despite no discernible disparities in CBF between male and female children during early development (ages 2-7 years) (Paniukov et al., 2019) there are age and maturation-dependent sex-specific discrepancies during puberty (Satterthwaite et al., 2014; Vandekar et al., 2019).. At mid-pubertal and post-pubertal stages, females exhibit higher CBF compared to males, as demonstrated by magnetic resonance imaging in large cohorts of subjects. Studies utilizing duplex ultrasound of extracranial CBF do not necessarily show the same sex differences in CBF, however they do suggest that differences in vessel diameter and velocity characteristics may differ. Indeed, Talbot et al recently showed using duplex ultrasound that vessel diameter was larger in males and velocity higher in females in 7 to 18 year olds, while ICA and VA flow is not different between males and females between these ages (Talbot et al., 2023). Ultimately, to evaluate whether there are any sex 83 differences in CBF regulation within children and adolescence at high-altitude, a considerably larger dataset with an extensive distribution across various age groups and stages of maturation will be necessary. This is because CBF regulation is likely to be strongly influenced by the developmental stage, and a small sample size may overlook such differences when conducting in-depth sub-analyses of specific maturation stages. 4.5 Conclusion This study investigated global and regional CBF in Andean children and adolescents living above 5000m in La Rinconada, Peru. Global CBF was similar in sex, age, size, and maturity matched Andeans, Sherpa living at 3800m in Nepal, and lowlanders living near sea-level in Wales, despite the greater severity of hypoxemia in the Andean children. However, there was lower VA flow in the Andean children. Whether lower posterior CBF – or even similar anterior CBF - in the presence of hypoxemia are adaptations resulting from altered cerebral metabolic demand or are associated with negative physiological or neural developmental outcomes requires further investigation. 84 Chapter 5: Study 3 – Cerebral blood flow and metabolism under conditions of simulated avalanche burial in humans: the triple H syndrome Abstract Avalanche burial exposes individuals to a combination of hypothermia, hypercapnia, and hypoxia. While these combined stressors have been explored in pre-clinical models, they have yet to be studied in healthy humans. The purpose of this study was to investigate the impact of mild hypothermia on cerebrovascular function during room air breathing and progressive hypercapnia and hypoxia, simulating avalanche burial. In 14 participants (6 female), the radial artery and internal jugular bulb were catheterized to measure blood gases, intravascular pressure, biomarkers of neurovascular damage, and quantify cerebral oxygen delivery (CDO2) and metabolic rate of oxygen (CMRO2). Measurements were assessed before and during mild hypothermia (-1.8±0.6°C; esophageal temperature) induced via cold water (7⁰C) immersion. Progressive hypercapnic-hypoxia was imposed during normothermia and hypothermia using dynamic end-tidal forcing in 2-minute stages (-5mmHg PaO2; +2mmHg PaCO2) to a maximal stimulus of 40mmHg PaO2 and +20mmHg PaCO2, or until volitional tolerance. Duplex ultrasound was used to quantify global cerebral blood flow (gCBF), CDO2 and CMRO2. Biomarkers of brain injury and blood brain barrier permeability, tau, neurofilament light (Nf-L), and glial fibrillary acidic protein (GFAP) were quantified. During room air breathing, hypothermia increased CaO2 (+2.5mL/dL), MAP (+23mmHg) and reduced PaCO2 (-4.7mmHg). Consequently, gCBF was decreased by ~17% (-144mL/min), while CDO2 and CMRO2 were maintained. During hypercapnic-hypoxia, hypothermia reduced CBF and CDO2, but CMRO2 was still not different. Arterial tau, Nf-L, and GFAP were reduce with hypothermia. These results suggest that mild-hypothermia may be neuroprotective, as indexed by reduced biomarkers of neurovascular damage that are not due to changes in cerebral metabolism. 5.1 Introduction Under conditions of avalanche burial, the likelihood of survival is reduced from 92% following 15-minutes to 30% following 35-minutes of burial (Falk et al., 1994b). The probability of survival 85 is drastically reduced past this 35-minute time point. However, the presence of an open or closed air pocket surrounding the burial victim may prolong the duration an individual can survive (Procter et al., 2016b). Regardless of the presence of an air pocket, as burial time increases the partial pressure of inspired oxygen progressively declines while inspired carbon dioxide increases, resulting in progressive hypercapnic-hypoxia (Grissom et al., 2000; Brugger et al., 2003b). This hypercapnic-hypoxic environment may also be accompanied by hypothermia during prolonged snow burial, resulting in hypothermic-hypercapnic-hypoxia (triple H syndrome) (Brugger et al., 2003a). Although these three stressors have been investigated in isolation in countless studies [reviewed in; (Strapazzon et al. 2024)], the impact of this trifecta of environmental stimuli on cerebrovascular function is relatively unknown. This information is important to determine as loss of consciousness and death are ultimately caused by insufficient oxygen delivery to the brain. Furthermore, during many real-world scenarios, (e.g., suffocation, loss of circulation, ischemia and encapsulation), hypercapnia and hypoxia co-exist and the net strain on the brain is dictated by the interaction of these two stressors. Countless case reports of individuals that suffocate in cold water or snow indicate that moderate-to-severe hypothermia may delay neurological damage otherwise caused by progressive hypercapnic-hypoxia [reviewed in; (Strapazzon et al. 2024)]. Hypoxia and hypercapnia in isolation cause increases in both ventilation and cerebral blood flow (CBF). With hypoxia, CBF is increased in a compensatory magnitude to maintain cerebral oxygen delivery (CDO2). This elevation in CBF is primarily mediated by erythrocyte signaling pathways which act on various downstream vasodilatory pathways which have been implicated in human CBF regulation [reviewed in:(Hoiland et al., 2016; Carr et al., 2023)]. Hypercapnia produces a greater relative increase in CBF through CO2 mediated alterations in perivascular pH and resultant reductions in vessel tone [reviewed in:(Hoiland et al., 2019)]. These combined stimuli during experimental human models or in breath holding, results in a marked increase in blood pressure, CBF and maintained CDO2 despite the underlying hypoxia (Bain et al., 2016b, 2016a). However, in an animal model of avalanche burial, profound increases in the partial pressure of arterial carbon dioxide (PaCO2) with concomitant reductions in the partial pressure of arterial oxygen (PaO2), results in profound reductions in cardiac output that resulted in death (Paal et al., 86 2013). These changes pose a particular challenge in terms of survival as the brain has minimal energy storage capacity and requires a constant oxygen supply to maintain function. Although hypothermia is an additional ‘stressor’, it may prolong brain survival by reducing its oxygen demand via the Q10 principal (Erecinska et al., 2003). Potentially, this response may partially compensate for the reductions in CBF that result from cardiac instability. Although there is some evidence of direct linear Q10 effect on CMRO2 with heating in humans (Bain et al., 2020), it is unknown if the similar reductions also occur with cooling in healthy humans. Despite little evidence supporting a Q10 effect in the conscious cold-stressed human brain, therapeutic hypothermia is used clinically to protect the brain from hypoxic insults caused by pathology and acute injury. Hypothermia has been shown to cause a reduction in CBF in healthy humans due to a combination of hemoconcentration and hyperventilation-mediated reductions in PaCO2 (Gibbons et al., 2020); and based on pre-clinical and clinical data, a reduction in cerebral metabolism (Erecinska et al., 2003). Importantly however, the relative contribution of each of these stimuli on the reduction CBF has not been established. Further, the finding of reduced metabolism during cooling has only been shown in patient groups whom are confounded by various comorbidities and importantly, anesthesia protocols known to suppress neuronal activity (Erecinska et al., 2003). In a recent study, a porcine model was used to study the effect of triple H syndrome on cerebrovascular function (Strapazzon et al., 2021b). It was found that the combination of hypothermia, hypercapnia, and hypoxia resulted in a marked decrease in cerebral tissue oxygen tension, largely caused by reduced cerebral perfusion pressure secondary to severe cardiovascular instability (i.e. decreased cardiac output). It is important to note, however, that this study induced a severe level of hypothermia (28º C core body temperature) and induced hypoxia and hypercapnia sequentially which does not simulate the concurrent and progressive changes in arterial blood gases that occur during avalanche burial. For example, a mild hypoxic stimuli was first induced (FIO2=17%) for 20 minutes after which mechanical ventilation was reduced to 6 breaths per minute and CO2 was allowed to accumulate for a further 20 minutes (Strapazzon et al., 2021b). While PaCO2 increased markedly by the end of the test (+19.6mmHg), the severity of hypoxemia was 87 mild (PaO2=66.4mmHg; SaO2=90.2%). The use of a mechanical ventilator also eliminates the natural increase in ventilation (and thus the respiratory muscle pump) that is otherwise caused by each of hypercapnia, hypoxia and hypothermia, a response promotes increases stroke volume and cardiac output and stabilizes central circulation (Harms C. A. & Dempsey J. A, 1999). Thus, further work, particularly in spontaneously breathing humans, is still required to understand how CDO2 and cerebral metabolism is affected under conditions of hypothermic-hypercapnic-hypoxia. The purpose of this study was to determine the effects of mild hypothermia on CBF, CDO2, cerebral metabolism, and biomarkers of neurovascular injury during room-air breathing and during progressive combined hypercapnic-hypoxia, simulating avalanche burial. It was hypothesized that 1) during room-air breathing, hypothermia would elicit decreases in both CBF and CMRO2; 2) during progressive hypercapnic-hypoxia, CBF, CMRO2 and circulating blood-brain biomarkers would be lower compared to normothermic hypercapnic-hypoxia. 5.2 Methods Ethical approval This study was approved by the Clinical Research Ethics Board of the University of British Columbia (H21-00773) and adhered to the principles set by the Declaration of Helsinki except for registry in a database. All study participants provided written informed consent prior to participation. Participants Fourteen healthy participants (8 male / 6 female: 175 ± 10cm, 70.7 ± 10.3kg, 27 ± 4yrs) were recruited for this study. All participants were healthy and free from respiratory, cardiovascular, or cerebrovascular disease. Participants arrived to the laboratory having abstained from alcohol and caffeine for 12 h, and strenuous exercise for 24 h. Contraceptive use in female participants was recorded but not controlled for. Of the six female participants, two participants reported using an intra-uterine device, two reported using oral contraceptives, and two reported not using any contraceptives. Participants were instructed to consume a light snack two hours prior to arriving at the lab. 88 Experimental Protocol Participants were instrumented with a rectal and esophageal thermistor (RET-1; Physitemp Instruments, Clifton, NJ, USA) for continuous measurement of core body temperature. The rectal thermistor was self-inserted by the participant 15cm past the anal sphincter. The esophageal thermistor was placed by a research team member and advanced to the level of the right atria using established methodology (Mekjavic & Rempel, 1990). Participants were then instrumented with an internal jugular venous catheter and a radial arterial catheter placed using sterile technique. A 16 G central venous catheter (Arrow, Markham, ON) was advanced into the internal jugular vein under sterile conditions and local anesthesia (Lidocaine, 2.0%) with ultrasound guidance. The catheter was then advanced up to 15 cm cephalad. 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). A 20 G arterial catheter (Arrow, Markham, ON) was advanced into the radial artery under local anesthesia (Lidocaine, 2.0%) with ultrasound guidance. Baseline measurements of ventilation, blood pressure, core temperature, arterial and jugular venous blood gases, arterial and jugular venous blood brain biomarkers of neurovascular damage and cytokines, and volumetric CBF were obtained while participants lay in the supine position (see experimental measures). Following baseline, participants completed a progressive hypercapnic-hypoxic breathing protocol. Using a dynamic end-tidal forcing system, the partial pressure of end-tidal oxygen (PETO2) was reduced by 5mmHg (to a minimum absolute PETO2 of 40mmHg) and the partial pressure of end-tidal carbon dioxide (PETCO2) was increased by 2mmHg (to a maximum increase in PETCO2 of +20mmHg) simultaneously in 2-minute stages (Figure 5.1). This breathing protocol was performed until participants either completed 10 stages (20 minutes) of the breathing protocol, or until participants volitionally ended the breathing trial due to reaching maximal tolerance. Under conditions where a participant reached a minimum absolute PETO2 of 40mmHg prior to finishing stage 10, PETO2 was maintained at 40mmHg while PETCO2 continued to increase in 2-minute stages as described. 89 Figure 5-1. Hypercapnic-hypoxic breathing protocol. Changes in the partial pressure of arterial oxygen (PaO2) and arterial carbon dioxide (PaCO2) during the progressive hypercapnic-hypoxic breathing protocol presented in one representative participant. Following a period of recovery (~60 min), participants were transferred to a custom designed temperature controlled circulating water immersion tub (HHH Scientific Manufacturing, Kelowna, BC). Participants lay semi-supine in the immersion tub with their toes and hands supported out of the water. The water temperature in the tub upon entry was 21⁰C and at the level of the sternal notch. Once participants were laying in the tub, 60kg of crushed ice was rapidly added over 30-60s to reduce the water temperature to 7⁰C over ~10 minutes. This cooling protocol was adapted from previous reported cold water immersion studies (Cahill et al., 2011; Kulkarni et al., 2019) and was chosen as it minimized the ventilatory cold shock response and participant discomfort. Participants remained in the water immersion tub until either; 1) core temperature decreased by 2⁰C; 2) an immersion time of 1-hour had been completed; 3) participants reached volitional tolerance and requested to be removed from the cold water. 0 2 4 6 8 10 12 14 16 18 2002040608010040455055606570Time (minutes)PaO2(mmHg)PaCO2 (mmHg)90 Upon completion of the cooling protocol, participants were transferred back to the supine experimental bed, and after a 10-minute resting period, ventilation, blood pressure, core body temperature, arterial and jugular venous blood gases, arterial and jugular venous blood brain biomarkers of neurovascular damage and cytokines, and volumetric CBF were obtained during room air breathing and while completing the same hypercapnic-hypoxic breathing protocol. Experimental measures Cerebral blood flow Volumetric measures of CBF within the internal carotid artery (ICA), and vertebral artery (VA) was assessed using a 10 MHz multi‐frequency linear array duplex ultrasound (uSmart 3300, Teratech, Burlington, MA, USA) according to published technical recommendations (Thomas et al., 2015). Vessel diameter was measured using B-mode imaging while pulse-wave mode was used to simultaneously measure peak envelope blood velocity. The ICA diameter and velocity were measured at least 1.5 cm distal to the common carotid bifurcation to eliminate recordings of turbulent and retrograde flow, while VA diameter and velocity were measured between C4 and C5, and C5 and C6, or proximal to entry into the vertebral column. The specific location in which the vessels were insonated was determined on an individual bases to ensure optimal image quality in each participant. Ultrasound videos were recorded and stored as video files for offline analysis using automated edge detection software (Woodman et al., 2001). Volumetric blood flow within the ICA and VA was calculated using the following formula: Q = (0.5 × Peak envelope velocity) × (π × (0.5 × Diameter)2 ) The global CBF (gCBF) was estimated as twice the sum of the unilateral ICA and VA flow measurements assuming matched flow in contralateral arteries. Cardiorespiratory Cardiovascular and respiratory variables were sampled continuously throughout the protocol at 1 kHz via an analog-to-digital converter (Powerlab, 16/30, ADInstruments, Colorado Springs, CO, USA). Heart rate was be measured using a 2-lead electrocardiogram (ECG; ADI BioAmp ML132). The radial arterial and jugular venous catheters was connected to a waste-less sampling system 91 (VAMP, Edwards Life Sciences) and connected in series to a pressure monitor (BP amp, ADInstruments). Continuous beat-to-beat blood pressure was measured directly via the radial arterial catheter. Both PETO2 and PETCO2 were sampled at the mouth and recorded by a calibrated gas analyzer (ML206, ADInstruments, Colorado, CO), while respiratory flow, tidal volume, respiratory rate and minute ventilation were measured by a spirometer (FE141 ADInstruments, Colorado Springs, CO, USA) and pneumotach flow head (MTL1000L, ADInstruments, Colorado Springs, CO, USA) connected in series with the mouthpiece and a bacteriological filter. Hematological Arterial and jugular venous blood gases were assessed by drawing ∼1.0 mL samples into a pre-heparinized syringe (SafePICO, Radiometer, Copenhagen, Denmark) and analyzed immediately using a commercial blood gas analyzer (ABL90 Flex Plus, Radiometer). Post processing was completed to correct blood gas values to individual esophageal temperature values - which estimates arterial blood temperature - recorded simultaneously with the blood sample. Blood gas analysis included measurement of the partial pressure of oxygen (PaO2), partial pressure of carbon dioxide (PCO2), oxygen saturation (SO2), oxygen content, [Hb], Hct, glucose, lactate, potassium (K+), bilirubin (bil), bicarbonate (HCO3-), and hydrogen ions (H+). Blood-brain biomarkers and cytokines Arterial and jugular venous blood samples were obtained simultaneously using serum separator tubes. Samples were placed in a dark space for 10 minutes prior to being centrifuged at 600g for 10 minutes at 4 °C. Serum was aliquoted into cryovials and subsequently flash frozen in liquid nitrogen prior to storage in a -80°C freezer. Serum samples were analyzed for markers of neuronal and astroglial injury using the Quanterix platform and Simoa HD-1 analyzer (Billerica, MA) by a blinded investigator. The following assays were performed: GFAP discovery kit (102336), which reflects astroglial damage and BBB permeability; total tau advantage kit (101552) and NF-L advantage kit (103186), which reveal axonal damage. Inflammatory cytokines (interleukins 6 and 1-beta) were quantified using a cytokine 3-plex A advantage assay (101160). 92 Calculations Arterial and venous oxygen content (CaO2 and CvO2, respectively) were calculated as: O2 content (mL/dL) = [Hb × 1.34 × (SO2 ⁄ 100)] + (0.003 × PO2) Cerebral blood flow through the ICA and VA was calculated as: Flow (mL/min) = (peak envelope blood velocity ⁄ 2) × (π (0.5 × diameter)2) × 60 gCBF was calculated as: gCBF (mL/min) = 2 × (QICA + QVA) \ Cerebral oxygen delivery (CDO2) was calculated as: CDO2 (mL/min) = (gCBF ⁄ 100) × CaO2 Cerebral perfusion pressure (CPP) was calculated as: CPP (mmHg) = MAP - JV pressure Cerebral oxygen extraction fraction was calculated as: O2 extraction (%) = [(CaO2 – CvO2) ⁄ CaO2] × 100 Cerebral metabolic rate of oxygen (CMRO2) was calculated as: CMRO2 (mL/min) = (gCBF ⁄ 100) × (CaO2 – CvO2) The cerebral metabolic rate of glucose and lactate (CMRglucose and CMRlactate, respectively) were calculated by the trans-cerebral exchange of arterial and venous values: CMRGlucose (mmol/min) = (arterial glucose – venous glucose) × (gCBF ⁄1000) CMRLactate (mmol/min) = (arterial lactate – venous lactate) × (gCBF ⁄1000) 93 Oxidative glucose index was calculated as: OGI = (CaO2 – CvO2) ⁄ (arterial glucose – venous glucose) Oxidative carbohydrate index was calculated as: OCI = (CaO2 – CvO2) ⁄ [(arterial glucose – venous glucose) + [0.5(arterial lactate – venous lactate)]] Statistical Analysis: All statistical analyses were preformed using Statistical Package for the Social Sciences (SPSS, V24). All variables were assessed for normal distribution by using the Shapiro-Wilks test. Two apriori paired sample two tailed t-tests were performed between normothermia room air vs hypothermia room air and between the final stage of hypercapnic hypoxic breathing during normothermia vs hypothermia to address the two separate research hypothesis. Significance was set at P<0.05. A secondary time point comparison was performed in an attempt to delineate the impact of reductions in PaCO2 on CBF from hypothermia mediated reduction in CBF. For this, during the hypothermic condition, the hypercapnic hypoxic breathing stage with PaCO2 and PaO2 at or near the values obtain during normothermic room air baseline were compared to normothermic room air values (ie. normothermia vs hypothermia with PaCO2 and PaO2 matched) using the same statistical approach described previously. 5.3 Results Effect of cooling during room air breathing Thermoregulation Core body temperature was reduced from 37.3 ± 0.3ºC during normothermic room air breathing to 35.5 ± 0.6ºC (P<0.001) during hypothermic room air breathing as measured by the esophageal thermistor (Figure 5-2) with a mean cooling rate of 2.3⁰C/hr. As expected, a similar response was observed in the rectal thermistor measurement of core temperature whereby core temperature was 94 reduced from 37.6 ± 0.3ºC during normothermic room air breathing to 36.0 ± 0.6ºC (P<0.001) during hypothermic room air breathing. 343536373839Esophageal Temperature (°C)Normothermic Hypothermic* Figure 5-2. Core body temperature Core body temperature assessed with an esophageal thermistor, pre and post cooling. Data are presented as individual data points with mean values in grey (normothermia) and blue (mild-hypothermia) bars. * indicates a significant difference (P<0.05) Cardiorespiratory Cardiorespiratory variables are presented in Table 5-1. Following cooling, both HR and MAP were elevated compared to normothermia (+15bpm and +23.3mmHg, respectively). Since IJV pressure was not different, CPP was increased (+18.2 mmHg). Ventilation was increased (+30.3L/min), mediated by elevations in both tidal volume and respiratory rate (+0.95L and +9 breaths/min, respectively). Consequently, PETO2 was elevated (+12.5mmHg) and PETCO2 decreased (-5.3mmHg) with cooling. 95 Table 5-1. Cardiorespiratory variables during room air breathing and maximal hypercapnic-hypoxia under normothermic and hypothermic conditions Normothermic Hypothermic Room Air Hypercapnic Hypoxia Room Air Hypercapnic Hypoxia RA vs RA HH vs HH MAP (mmHg) 90.9 ± 6.4 114.2 ± 10.0 111.5 ± 10.2 118.4 ± 13.3 P=0.004 P=0.127 JV pressure (mmHg) 13.0 ± 2.4 16.7 ± 4.5 15.7 ±5.9 17.4 ± 6.8 P=0.072 P=0.781 CPP (mmHg) 79.0 ± 4.3 97.2 ± 11.7 97.0 ± 10.4 102.8 ± 17.3 P<0.001 0.203 HR (beats/min) 71 ± 12 112 ± 17 86 ± 22 100 ± 24 P<0.001 P=0.235 Respiratory rate (breaths/min) 13 ± 4 30 ± 9 22 ± 8 32 ± 9 P<0.001 P=0.564 Tidal volume (L) 1.146 ± 0.294 2.712 ± 0.527 2.091 ± 0.508 2.625 ± 0.264 P<0.001 P=0.364 Ventilation (L/min) 14.1 ± 4.0 80.2 ± 24.6 44.4 ± 12.7 83.2 ± 21.2 P<0.001 P=0.918 PETO2 (mmHg) 86.5 ± 7.1 51.5 ± 6.7 99.0 ± 5.0 61.2 ± 9.3 P<0.001 P=0.011 PETCO2 (mmHg) 40.4 ± 2.0 54.2 ± 3.6 35.1 ± 3.1 49.3 ± 6.3 P<0.001 P=0.028 MAP; mean arterial pressure, JV pressure; jugular venous pressure, CPP; cerebral perfusion pressure, HR; heart rate, PETO2; partial pressure of end-tidal oxygen, PETCO2; partial pressure of end-tidal carbon dioxide. Hematology and blood gases Hematology and blood gas data during room air breathing are presented in Table 5-2. Cooling elicited a hemoconcentration response whereby both arterial hemoglobin and hematocrit were elevated compared to normothermia (+1.6g/dL and +5%, respectively). Elevated ventilation elicited an increase in PaO2 (+14.0mmHg), and SaO2 (+1.2%), while PaCO2 was decreased (-4.7mmHg). Despite reductions in both PaCO2 and HCO3-, [H+] was increased (+5.8nmol/L) and pH reduced (-0.06) following cooling. The increase in ventilation and hemoconcentration resulted in a marked increase in CaO2 with cooling (+2.5mL/dL). Additionally, cooling elicited an increase in arterial and venous K+, Na+, glucose, and lactate, while bilirubin was only increased in arterial blood. Along with the increase in arterial bilirubin with core cooling, an increase in net cerebral bilirubin uptake was also observed. There was also a greater A-v PO2 and O2 content difference 96 with hypothermia, ie., increased O2 extraction. No other difference in arterial to venous gradients were observed. Table 5-2 Hematological and blood gas variables during room air breathing and maximal hypercapnic-hypoxia under normothermic and hypothermic conditions Normothermic Hypothermic Room Air Hypercapnic Hypoxia Room Air Hypercapnic Hypoxia RA vs RA HH vs HH Hb (g/dL) A 14.3 ± 1.0 14.6 ± 1.1 15.9 ± 1.3 15.2 ± 1.3 P<0.001 P=0.078 V 14.4 ± 1.0 14.7 ± 1.2 15.9 ± 1.4 15.4 ± 1.3 P<0.001 P=0.040 A-V -0.1 ± 0.1 -0.2 ± 0.4 0.0 ± 0.2 -0.2 ± 0.4 P=0.005 P=0.413 A 43.7 ± 3.2 44.7 ± 3.5 48.7 ±4.1 46.7 ± 4.0 P<0.001 P=0.078 Hct (%) V 44.1 ± 3.1 45.0 ± 3.7 48.6 ±4.2 47.1 ±4.0 P<0.001 P=0.031 A-V -0.4 ± 0.3 -0.4 ± 2.2 0.1 ± 0.5 -0.7 ±1.2 P=0.003 P=0.604 pH A 7.40 ± 0.01 7.30 ± 0.03 7.34 ± 0.03 7.28 ± 0.03 P<0.001 P=0.001 V 7.36 ± 0.01 7.29 ± 0.03 7.30 ±0.02 7.27 ± 0.04 P<0.001 P=0.018 A-V 0.04 ± 0.01 0.01 ± 0.01 0.04 ±0.01 0.01 ± 0.02 P=0.950 P=0.923 O2 A 19.1 ± 1.4 15.9 ± 2.5 21.6 ± 1.8 18.4 ±2.9 P<0.001 P=0.017 Content V 13.9 ± 1.6 13.7 ± 2.4 15.6 ± 1.8 15.5 ± 2.4 P=0.001 P=0.085 (mL/dL) A-V 5.2 ± 1.1 2.1 ± 0.9 6.0 ± 1.3 2.8 ± 1.2 P=0.036 P=0.035 PCO2 (mmHg) A 43.1 ± 1.7 57.0 ± 4.3 38.4 ± 2.9 51.8 ± 7.0 P<0.001 P=0.030 V 51.5 ± 2.4 59.6 ± 4.2 47.2 ± 2.7 56.5 ± 7.3 P<0.001 P=0.053 A-V -5.4 ± 11.7 -2.4 ± 2.1 -8.8 ± 3.0 -4.8 ± 3.4 P=0.374 P=0.315 A 84.4 ± 6.7 49.2 ± 10.0 98.4 ± 5.4 60.7 ± 16.8 P<0.001 P=0.047 PO2 V 38.0 ± 3.3 38.7 ± 4.6 37.1 ± 2.8 40.1 ± 3.7 P=0.321 P=0.761 (mmHg) A-V 46.4 ± 6.8 10.0 ± 7.4 61.4 ± 5.8 20.9 ± 17.0 P<0.001 P=0.070 A 96.8 ± 1.0 79.0 ± 10.3 98.0 ± 0.5 87.0 ± 9.1 P<0.001 P=0.057 SO2 V 69.9 ± 5.7 67.5 ± 9.7 71.4 ± 5.2 72.8 ± 7.4 P=0.359 P=0.324 (%) A-V 26.9 ± 5.5 11.0 ± 4.0 26.6 ± 5.1 14.1 ± 5.0 P=0.868 P=0.068 97 Normothermic Hypothermic Room Air Hypercapnic Hypoxia Room Air Hypercapnic Hypoxia RA vs RA HH vs HH K+ (mmol/L) A 3.9 ± 0.2 4.1 ± 0.4 4.5 ± 0.2 4.1 ± 0.2 P<0.001 P=1.000 V 3.9 ± 0.3 4.0 ± 0.3 4.5 ± 0.2 4.2 ± 0.3 P<0.001 P=0.430 A-V -0.1 ± 0.1 0.1 ± 0.2 0.0 ± 0.1 -0.1 ± 0.1 P=0.169 P=0.017 A 141 ± 2 142 ± 2 142 ± 2 142 ± 1 P=0.034 P=0.045 Na+ V 142 ± 1 142 ± 1 143 ± 2 142 ± 2 P=0.001 P=0.275 (mmol/L) A-V -1 ± 1 0 ± 1 -1 ± 0 0 ± 1 P=0.752 P=0.598 Glu (mmol/L) A 5.3 ± 0.4 5.8 ± 0.5 6.8 ± 1.0 7.0 ± 1.3 P<0.001 P=0.007 V 4.9 ± 0.4 5.5 ± 0.5 6.3 ± 1.0 6.9 ± 1.4 P<0.001 P=0.011 A-V 0.4 ±0.1 0.2 ±0.2 0.5 ± 0.2 0.2 ± 0.2 P=0.156 P=0.351 Lac (mmol/L) A 0.6 ± 0.3 0.6 ± 0.2 2.4 ± 1.3 1.1 ± 0.4 P<0.001 P=0.009 V 0.6 ± 0.2 0.6 ± 0.2 2.4 ±1.3 1.2 ± 0.4 P<0.001 P=0.015 A-V 0.0 ± 0.1 0.0 ± 0.1 0.0 ± 0.1 0.0 ± 0.2 P=0.089 P=0.351 Bil (µmol/L) A 6.8 ± 8.6 11.6 ± 9.9 15.3 ± 11.2 14.7 ± 9.5 P<0.001 P=0.021 V 7.4 ± 9.1 11.2 ± 10.2 11.3 ± 12.6 14.0 ± 9.8 P=0.052 P=0.122 A-V -0.6 ± 2.4 0.9 ± 1.7 4.0 ± 4.6 2.1 ± 3.2 P=0.013 P=0.219 HCO3- (mmol/L) A 25.1 ± 0.8 24.2 ± 1.1 21.5 ± 1.3 22.2 ± 1.1 P<0.001 P<0.001 V 25.6 ± 0.8 24.1 ± 1.3 21.9 ± 1.2 22.5 ± 1.0 P<0.001 P<0.001 A-V -0.4 ± 0.2 0.0 ± 0.4 -0.3 ± 0.3 -0.3 ± 0.4 P=0.244 P=0.347 H+ (nmol/L) A 40.3 ± 1.3 50.6 ± 3.6 46.1 ± 3.0 53.0 ± 4.1 P<0.001 P=0.001 V 43.8 ± 1.2 51.8 ± 3.5 50.1 ± 2.6 54.3 ± 4.6 P<0.001 P=0.044 A-V -3.6 ± 1.1 -1.2 ± 1.4 -4.0 ± 1.5 -1.3 ± 1.9 P=0.394 P=0.299 Hb; hemoglobin, Hct; hematocrit, PCO2; partial pressure of carbon dioxide, PO2; partial pressure of oxygen, SO2; oxygen saturation, K+; potassium, Na+; sodium, Glu; glucose, Lac; lactate, Bil; bilirubin, HCO3-; bicarbonate, H+; hydrogen ions. Cerebrovascular Cerebral blood flow and cerebral metabolism metrics are presented in Table 5-3. With cooling, both ICA and VA flow were reduced (-47.6mL/min; and -28.0mL/min; respectively) resulting in 98 a ~17% reduction in gCBF (-144mL/min) and a ~30% reduction in gCBF CVC (-2.8mL/min/100mmHg) post cooling. As CaO2 was elevated with cooling, CDO2 was not different during room air breathing. No differences in OEF, CMRO2, OGI or OCI were observed during room air breathing following cooling. Table 5-3. Cerebrovascular variables during room air breathing and maximal hypercapnic-hypoxia under normothermic and hypothermic conditions Normothermic Hypothermic Room Air Hypercapnic Hypoxia Room Air Hypercapnic Hypoxia RA vs RA HH vs HH ICAd (cm) 0.504 ± 0.072 0.531 ± 0.064 0.482 ± 0.119 0.513 ± 0.110 P=0.281 P=0.247 ICAv (cm/s) 51.4 ± 10.8 76.4 ± 14.1 48.9 ± 13.0 62.0 ± 14.0 P=0.366 P<0.001 ICA flow (mL/min) 302.8 ± 62.0 502.3 ± 97.9 255.2 ± 70.9 379.7 ± 144.4 P=0.015 P=0.003 VAd (cm) 0.408 ± 0.050 0.425 ± 0.047 0.368 ± 0.049 0.397 ± 0.046 P=0.013 P<0.001 VAv (cm/s) 28.9 ± 5.4 48.1 ± 9.4 27.3 ± 6.5 44.6 ± 11.2 P=0.185 P=0.294 VA flow (mL/min) 117.2 ± 35.1 237.2 ± 104.5 89.2 ± 29.1 172.0 ± 60.6 P=0.004 P=0.026 gCBF (mL/min) 840.0 ± 142.1 1494.5 ± 264.3 696.4 ± 173.8 1008.5 ± 180.8 P=0.002 P=0.002 CDO2 (mL/min) 160.2 ± 29.9 235.8 ± 52.0 149.7 ± 38.9 189.8 ± 35.2 P=0.189 P<0.001 CMRO2 (mL/min) 42.7 ± 10.3 31.2 ± 16.5 38.1 ± 7.9 25.9 ± 9.3 P=0.481 P=0.504 OEF (%) 27.4 ± 5.7 13.9 ± 5.9 27.8 ± 5.8 14.8 ± 5.0 P=0.804 P=0.094 CMRgluc (mmol/min) 3.6 ± 1.2 3.1 ± 1.8 3.4 ± 1.3 2.0 ± 2.4 P=0.434 P=0.076 CMRLac (mmol/min) -0.4 ± 0.4 0.1 ± 0.8 0.0 ± 0.4 -0.2 ± 1.0 P=0.082 P=0.185 OGI 6.0 ± 3.1 4.5 ± 3.5 5.7 ± 1.8 8.6 ± 4.9 P=0.741 P=0.112 OCI 6.3 ± 3.1 4.1 ± 2.7 5.9 ± 2.6 10.3 ± 9.9 P=0.669 P=0.145 gCBFCVC (mL/min/100mmHg) 9.3 ± 1.8 12.6 ± 2.1 6.5 ± 1.8 9.0 ± 1.8 P<0.001 P<0.001 ICA; internal carotid artery, VA; vertebral artery, gCBF; global cerebral blood flow, CDO2; cerebral oxygen delivery, CMRO2; cerebral metabolic rate of oxygen, OEF; oxygen extraction fraction, CMRgluc; cerebral metabolic rate of glucose, CMRLac; cerebral metabolic rate of lactate, OGI; oxidative glucose index, OCI; oxidative carbohydrate index, gCBFCVC; global cerebral blood flow conductance. 99 Figure 5-3. Regional and global cerebral blood flow Internal carotid artery (ICA), vertebral artery (VA), and global cerebral blood flow (gCBF) during room-air and maximal hypercapnic hypoxia during normothermia and hypothermia. Paired t-tests were performed between normothermic normoxia vs hypothermic normoxia and between normothermic hypercapnic-hypoxia (HH) vs hypothermic hypercapnic hypoxia. Data are presented as individual data points with mean values in grey (normothermia) and blue (mild-hypothermia) bars. * indicates a significant difference (P<0.05) 0200400600800ICA Flow (ml/min)0100200300400VA Flow (ml/min)500100015002000gCBF (mL/min)Normothermic NormoxiaNormothermic HHHypothermic NormoxiaHypothermic HH******100 100150200250300350CDO2 (ml/min)Normothermic NormoxiaNormothermic HHHypothermic NormoxiaHypothermic HH*020406080CMRO2 (ml/min)Normothermic NormoxiaNormothermic HHHypothermic NormoxiaHypothermic HH Figure 5-4. CDO2 and CMRO2 Cerebral oxygen delivery (CDO2) and cerebral metabolic rate of oxygen (CMRO2) during room-air and maximal hypercapnic hypoxia during normothermia and hypothermia. Paired t-tests were performed between normothermic normoxia vs hypothermic normoxia and between normothermic hypercapnic-hypoxia (HH) vs hypothermic hypercapnic hypoxia. Data are presented as individual data points with mean values in grey (normothermia) and blue (mild-hypothermia) bars. * indicates a significant difference (P<0.05) Blood-brain biomarkers Blood-brain biomarker and cytokine data are presented in Table 5-4. With cooling, arterial and venous GFAP were both decreased (-15.3pg/mL and -19.1pg/mL; respectively) with no differences in cerebral release or uptake. Although arterial Tau was also reduced (-0.44pg/mL), there was an increase in cerebral tau release (-0.51pg/mL) post-cooling. Both arterial and venous Nf-L were reduced (-1.8pg/mL and -2.2pg/mL; respectively). In addition, there was an apparent reduction in cerebral Nf-L release (+0.5pg/mL). Increases in circulating cytokines were also observed post-cooling whereby arterial and venous IL-6 was increased (+0.93pg/mL and +1.06pg/mL; respectively) and venous IL-1b was slightly increased (+0.14pg/mL). There were no a-v differences in these cytokines. 101 Table 5-4. Blood-brain biomarker and cytokine variables during room air breathing and maximal hypercapnic-hypoxia under normothermic and hypothermic conditions Normothermic Hypothermic Room Air Hypercapnic Hypoxia Room Air Hypercapnic Hypoxia RA vs RA HH vs HH GFAP (pg/mL) A 70.2 ± 24.4 102.4 ± 35.4 54.9 ± 21.0 68.9 ± 29.7 P<0.001 P=0.021 V 75.0 ± 30.1 108.9 ± 38.0 55.9 ± 22.1 76.3 ± 25.8 P<0.001 P=0.018 A-V -4.8 ± 9.1 -6.5 ± 5.4 -1.0 ± 4.3 -7.4 ± 12.7 P=0.110 P=0.778 Tau (pg/mL) A 1.20 ± 0.50 2.15 ± 1.03 0.76 ± 0.40 1.12 ± 0.69 P=0.004 P=0.009 V 1.35 ± 0.65 1.74 ± 1.06 1.41 ± 0.71 1.19 ± 0.89 P=0.777 P=0.162 A-V -0.15 ± 0.51 0.41 ± 1.40 -0.66 ± 0.65 -0.07 ± 0.97 P=0.035 P=0.120 Nf-L (pg/mL) A 6.8 ± 3.5 9.0 ± 5.3 5.0 ± 3.0 5.7 ± 3.6 P<0.001 P=0.002 V 7.5 ± 4.0 9.2 ± 5.2 5.3 ±3.5 6.2 ± 3.7 P<0.001 P<0.001 A-V -0.8 ± 0.8 -0.2 ± 0.5 -0.3 ± 0.5 -0.5 ± 0.5 P=0.022 P=0.643 IL-6 (pg/mL) A 0.61 ± 0.28 0.83 ± 0.38 1.54 ± 0.68 1.82 ± 0.60 P<0.001 P=0.003 V 0.66 ± 0.30 0.73 ± 0.28 1.72 ± 0.86 2.32 ± 0.98 P<0.001 P=0.001 A-V -0.05 ± 0.10 0.10 ± 0.27 -0.18 ± 0.73 -0.50 ± 0.93 P=0.471 P=0.076 IL-1b (pg/mL) A 0.51 ± 0.92 0.56 ± 0.98 0.57 ± 0.99 0.77 ± 1.18 P=0.202 P=0.151 V 0.53 ± 0.92 0.55 ± 1.01 0.67 ± 1.14 0.75 ± 1.12 P=0.040 P=0.524 A-V -0.02 ± 0.09 0.01 ± 0.08 -0.10 ± 0.17 0.03 ± 0.08 P=0.139 P=0.662 GFAP; glial fibrillary acidic protein, Nf-L; neurofilament light, IL-6; interleukin 6, IL-1b; interleukin 1b. 102 050100150200250Arterial GFAP**012345Jugular Venous Tau0510152025Jugular Venous NF-L**050100150200250Jugular Venous GFAP **-4-2024A-V TauNormothermic NormoxiaNormothermic HHHypothermic NormoxiaHypothermic HH*-4-3-2-101A-V NF-LNormothermic NormoxiaNormothermic HHHypothermic NormoxiaHypothermic HH*-30-20-1001020A-V GFAPNormothermic NormoxiaNormothermic HHHypothermic NormoxiaHypothermic HH012345Arterial Tau**0510152025Arterial NF-L** Figure 5-5. Neurovascular unit brain biomarkers Blood brain biomarkers of tau, neurofilament light (Nf-L), and glial fibrillary acidic protein (GFAP) in arterial (top row), jugular venous (middle row), and arterial-venous differences (bottom row) during room-air and maximal hypercapnic hypoxia under normothermic and hypothermic conditions. Paired t-tests were performed between normothermic normoxia vs hypothermic normoxia and between normothermic hypercapnic-hypoxia (HH) vs hypothermic hypercapnic hypoxia. Data are presented as individual data points with mean values in grey (normothermia) and blue (mild-hypothermia) bars. * indicates a significant difference (P<0.05)103 Effect of cooling during hypercapnic-hypoxic breathing Cardiorespiratory Cardiorespiratory variables during hypercapnic-hypoxic breathing are presented in Table 1. Following cooling, during maximal hypercapnic-hypoxia, PETO2 and PETCO2 were altered to a similar magnitude (less absolute severity of hypoxia and hypercapnia) as during room air breathing (+9.7mmHg and -4.9mmHg; respectively) as tolerance to hypercapnic-hypoxia was not different between temperature conditions (Stages completed: 7 ± 2 vs 7 ± 2; P=0.210). No other differences in any cardiorespiratory variables were observed. Hematology and blood gases Hematology and blood gas data during hypercapnic-hypoxic breathing are presented in Table 2. During maximal hypercapnic-hypoxia, PaO2 and CaO2 were both higher during the hypothermic condition compared to normothermia (+11.5mmHg and +2.5mL/dL; respectively). Conversely, PaCO2 was lower during the hypothermia trial compared to normothermia (-5.2mmHg), however [H+] was elevated (+2.4nmol/L) while both HCO3- and pH were lower (-2.0mmol/L and -0.02; respectively). Similar to the room air breathing stage, arterial and venous glucose and lactate were elevated during the hypothermic hypercapnic-hypoxia breathing trial (+1.2mmol/L and +0.6mmol/L; respectively) Cerebrovascular Cerebral blood flow and cerebral metabolism metrics during hypercapnic-hypoxic breathing are presented in Table 3. With hypothermia ICA flow (-122.6mL/min), VA flow (-67.0mL/min), gCBF (-486mL/min) and gCBF CVC (-3.6) were all lower during maximal hypercapnic-hypoxia compared to during normoxia. As a result of the lower CBF during the hypothermia trial, CDO2 was also lower (-46 mL/min). There were no apparent differences in CMRO2, OEF, CMRgluc, CMRlac, OGI, or OCI. Blood-brain biomarkers and cytokines Blood-brain biomarker and cytokine data during hypercapnic-hypoxic breathing are presented in Table 4. During the hypothermia stage, arterial and venous GFAP (-33.5pg/mL and -32.6pg/mL; 104 respectively) and Nf-L (-3.3pg/mL and -3.0pg/mL; respectively) were both lower compared to normothermia, although there were no differences in cerebral release. Arterial Tau was also reduced (-1.03pg/mL) while arterial and venous IL-6 were increased during the hypothermia trial (+0.99pg/mL and +1.59pg/mL). There were no a-v differences in any of these latter neurovascular biomarkers and cytokines. Effect of normalized CO2 on CBF during hypothermia To account for the impact of hyperventilation, i.e., hypocapnia, on CBF following hypothermia, the hypercapnic hypoxic breathing stage during the hypothermic trial that most closely matched the PaCO2 and PaO2 obtained during the normothermic normoxic baseline was compared (ie. normothermic normoxia vs hypothermia with PaCO2 and PaO2 matched). With this comparison, there were no differences in PaCO2 (43.1 ± 1.7 vs 42.3 ± 2.0mmHg; P=0.086) or PaO2 (84.4 ± 6.7 vs 87.1 ± 9.2mmHg; P=0.102) during normothermia and hypothermia and consequently ICA flow (302.8 ± 62.0 vs 289.4 ± 53.1mL/min; P=0.771), VA flow (117.2 ± 35.1 vs 112.5 ± 36.3mL/min; P=0.550) and gCBF (840.0 ± 142.1 vs 806.4 ± 97.5mL/min; P=0.303) were not different between normothermia and hypothermia. Both CDO2 (160.2 ± 29.9 vs 167.6 ± 25.6mL/dL; P=0.084) and CMRO2 (42.7 ± 10.3 vs 33.9 ± 8.1mL/min ;P=0.074) were also not different within this comparison; however, gCBF CVC (9.3 ± 1.8 vs 7.5 ± 1.0mL/min/100mmHg; P<0.001) was lower with hypothermia, due to the notable elevations in MAP (90.9 ± 6.4 vs 108.7 ± 8.1; P<0.001). Both IJV pressure (13.0 ± 2.4 vs 16.6 ± 6.2mmHg; P<0.001) and CPP pressure (79.0 ± 4.3 vs 92.4 ± 8.2mmHg; P=0.021) were increased with hypothermia. 5.4 Discussion This study evaluated the impact of mild hypothermia on CBF and cerebral metabolism during room air breathing and during maximal progressive hypercapnic-hypoxia simulating conditions of avalanche burial. Herein we showed that 1) regional and global CBF is lower with hypothermia during room air breathing and throughout hypercapnic hypoxia; 2) The reduction in CBF with hypothermia during room air breathing is primarily mediated via reductions in PaCO2; 3) Mild hypothermia did not reduce CMRO2 during room air breathing or during maximal hypercapnic-hypoxia compared to normothermic conditions; and 4) Blood brain biomarkers indicative of 105 neurovascular damage - tau, Nf-L and GFAP - were reduced systemically following hypothermia during room air breathing and during hypercapnic-hypoxia. These results indicate that mild-hypothermia may be neuroprotective, as indexed by reduced biomarkers of neurovascular damage that are not due to changes in cerebral metabolism. CBF during mild-hypothermia During room air breathing, hypothermia resulted in a reduction in gCBF through decreases in both ICA flow and VA flow. This is consistent with previous research, as reductions in CBF with mild-hypothermia has been shown across a number of pre-clinical models (Rosomoff & Holaday, 1954; Erecinska et al., 2003), in various patient populations (Marion et al., 1993; Erecinska et al., 2003), and in healthy humans (Gibbons et al., 2020). Indeed, duplex ultrasound measures in healthy humans have reported a reduction in gCBF by ~255 ml/min under room air conditions (Gibbons et al., 2020). In these previous studies the relative contribution of CaO2, PaCO2, and CMRO2 on the reduction in CBF are difficult to quantify. Herein, for the first time, we show that reductions in CMRO2 are not observed with mild hypothermia; therefore, reductions in cerebral metabolism do not seem to contribute to the decreases in CBF observed in this study. The reduction in CBF is primarily attributable to the decrease in PaCO2 induced by hyperventilation, and the increase in CaO2 caused by both hemoconcentration and hyperventilation. When participants end-tidal PO2 and PCO2 were returned to near normothermic baseline values during the earliest stages of the ramped hypercapnic-hypoxic breathing protocol, the reduction in CBF associated with hypothermia was almost completely abolished. Thus, it seems that hypocapnia is the primary driving factor behind the reduction in CBF in healthy humans during mild hypothermia. Further, as has been highlighted in previous work (Caldwell et al., 2021a, 2021b), hypocapnia mediated reductions in CBF appear to occur independently from changes in arterial pH, which was also reduced (ie., more acidotic) following cooling. Previous modeling work in pigs has suggested that the quantifiable reduction in CMRO2 with the magnitude of hypothermia in this study could be as low as ~2-4% (Russell-Buckland & Tachtsidis, 2020b). This estimate, however, can vary widely across different animal models (Erecinska et al., 2003). Moreover, this magnitude of change may be too small to be observed in this study using 106 our measurement techniques. Indeed, it is likely that mild hypothermia does have a hypometabolic effect on the brain as reductions in Brownian motion by way of decreasing temperature is a fundamental principal of physics; however, this relatively mild level of core cooling is likely inconsequential or undetectable in the conscious human. Of note, previous work utilizing the same measurement techniques for assessing CBF and CDO2 have shown increases in CMRO2 with passive heating of 2⁰C above resting conditions (Bain et al., 2020). This differential response between hyperthermia and hypothermia may be due to the countering effects of reductions in CO2 on cerebral metabolism (see section: effect of hypothermia and hypercapnic-hypoxia on CMRO2), the increased cerebral metabolic demand associated with shivering, or due to differences in temperature gradients between the core and cerebral tissue. Neurovascular biomarkers during mild hypothermia During both room air breathing and maximal hypercapnic hypoxia, a lower arterial concentration of GFAP, Tau, and Nf-L was observed during hypothermia compared to normothermia. Additionally, the a-v difference of Nf-L was reduced and a-v tau increased following hypothermia during room air breathing. For these biomarkers to be present in the blood they first must move across a leaky BBB or by cerebrospinal fluid –blood exchange and have a concentration gradient driving passive diffusion (Liebner et al., 2018). Thus, the reduction in GFAP, tau and Nf-L may be due of lower concentration of biomarkers within the perivascular space and/or a reduction in blood-brain barrier permeability. As GFAP - which is indicative of blood brain barrier permeability - was reduced, hypothermia-mediated reductions in blood brain barrier permeability (Karibe et al., 1994; Baumann et al., 2009) may be responsible for reducing diffusion of biomarkers into the circulation. While mild hypothermia may not be eliciting a neuroprotective response through reducing cerebral metabolism, there are other pathways by which hypothermia may be protective of the brain. In contrast, the increase in cerebral tau release with cooling is an interesting finding that may counter this point, considering all other biomarkers are reduced at the same timepoint. As tau is typically indicative of axonal damage (Zetterberg & Blennow, 2016), this could indicate an increase in some form of neurovascular unit damage associated with cooling. However, this difference in cerebral release may simply be a consequence of lower CBF and thus calculation of cerebral biomarker flux (the product of cerebral a-v differences and CBF), revealed 107 that only Nf-L flux was reduced (-0.62 ± 0.63 vs -0.23 ± 0.34 pg/ml/min; P=0.021) while tau, and GFAP were unchanged. The observation of lower systemic GFAP, Nf-L and tau with hypothermia during maximal hypercapnic hypoxia may indicate that hypothermia is reducing the degree of neuronal and astroglia “injury” associated with the hypercapnic-hypoxic breathing. Alternatively, however, this may be due to a lower magnitude of hypoxemia observed during the hypothermic trial due to the higher starting PaO2 caused cold-induced hyperventilation (see methodological considerations). Previous work in elite apnea divers exposed to a similar physiological stimulus (hypoxia, hypercapnia and elevated MAP), reported increases in arterial Nf-L, Tau and GFAP, that appear to be a similar magnitude as reported in this study (Bailey et al., 2022). However, in the divers, cerebral release of these blood-brain biomarkers is also observed, whereas we did not detect any trans-cerebral differences in our study during the combined hypercapnic-hypoxic stimuli (i.e., changes in a-v difference). Indeed, it seems that the increases in systemic blood brain biomarker concentrations that we observed are resultant of perhaps very small release from the brain on a magnitude that is too small to detect or is occurring at a timepoint that is being missed within our blood sampling protocol. Is mild hypothermia protective under conditions of avalanche burial? When we consider the “protective” effect of mild hypothermia during conditions of avalanche burial we have to consider the interplay of what is considered to be protective of the cerebrovasculature versus protective of the whole body. While all tissues fundamentally are affected by the Q10 principle, the shivering response elicited with mild hypothermia results in an increase in total body metabolism. This is evidenced in our study by ~4x increase in ventilation and marked increase in [H+] and reduction in pH. Under conditions of avalanche burial, this shivering response and marked increase in total oxygen consumption would likely outweigh the non-significant reductions in cerebral metabolism and hemoconcentration which would help protect cerebral oxygen supply matching. Further, the greater systemic oxygen requirement would result in a greater rate of CO2 production and O2 utilization. When breathing into an enclosed space, this would increase the rate at which displacement asphyxia occurs, amplifying the 108 magnitude and rate of hypercapnic-hypoxia (Strapazzon et al., 2024). Additionally, a similar number of stages were completed by participants during hypercapnic-hypoxic breathing in normothermia vs hypothermia. This observation would indicate that mild hypothermia does not necessarily provide any benefit in terms of tolerance to hypercapnic hypoxic breathing. Differences in tolerance to hypercapnic hypoxia between normothermia and hypothermia. Despite differences in the absolute magnitude of hypoxia due to hyperventilation following core cooling, and thus a higher starting PaO2, participants completed a similar number of stages (~7 stages) and thus similar absolute increase in PaCO2. Participants also reached a similar absolute ventilation at maximal hypercapnic-hypoxia. While not measured, maximal tolerable dyspnea/discomfort from the hypercapnic stimulus is likely the physiological mechanism responsible for cessation of the breathing protocol under these conditions. This claim is based on the matched terminal ventilation values, similar absolute increases in PaCO2 and researcher observations. It is important to highlight that maximal ventilatory tolerance was not reached in all participants, with one participant terminating the trial during the hypothermia hypercapnic-hypoxia test due to discomfort from the hypothermia stimuli and a second participant due to nausea. Effect of hypothermia and hypercapnic-hypoxia on CMRO2 Irrespective of core temperature, hypercapnic-hypoxia appears to reduce CMRO2. Previous work in elite apnea divers has highlighted the role of hypercapnia on inducing reductions in CMRO2 during maximal apnea (Bain et al., 2016b, 2016a, 2017). This modulation of CMRO2 has recently been shown to occur under solely hypercapnic and hypocapnic conditions (Caldwell et al., 2024), whereby increases and decreases in PaCO2 elicit decreases and increases in CMRO2, respectively. Therefore, in the context of the current findings, the impact of hypocapnia on the room air hypothermic measure of CMRO2 should also be considered. Indeed, although there was no observed decrease in CMRO2 between normothermic room-air and hypothermic room-air breathing conditions, PaCO2 was reduced by ~5 mmHg. Thus, based on previous reports (Bain et al., 2016a, 2017; Caldwell et al., 2024), a CO2 mediated increase in CMRO2 may be countering the temperature mediated reduction in CMRO2.(i.e., the Q10 effect). When the normothermic 109 room-air condition is compared to the hypothermic matched PaCO2 condition we still see that CMRO2 is unchanged; however, there is a statistical trend towards a decrease (42.7 vs 33.9 mL/min; P=0.074). Indeed, these ‘reductions’ in CMRO2 were observed in 11 out of 14 participants, and the relative difference is ~20%. These changes are similar to the relative increases in CMRO2 reported using identical measurement techniques during passive hyperthermia in healthy humans (Bain et al., 2020). Methodological considerations: Model of avalanche burial While this study attempted to emulate some of the physiological aspects of avalanche burial (hypothermia, hypercapnia, and hypoxia), there were necessary concessions based on what was ethically possible to perform in healthy humans, practical in a lab setting with invasive monitoring, and was repeatable pre and post cooling. First, reductions in core body temperature of only ~2⁰C were targeted due to the increased risk of arrythmia at core temperatures below 33-34⁰C. Cold water immersion was utilized for the hypothermic stimuli as it reduced core body temperature in a fairly rapid manner that limited the duration of participant discomfort (Kulkarni et al., 2019). As such, our experimental model might also be relevant to understanding some of the cerebrovascular changes with cold-water immersion per se; however, although not directly comparable to snow induced hypothermia, the decrease in core temperature was only slightly higher than mean rates of cooling under avalanche burial (Mittermair et al., 2021). Dynamic end-tidal forcing was utilized to alter arterial blood gases as it allowed us to tightly control end-tidal O2 and CO2 values during normothermia and hypothermia, as opposed to a snow pocket rebreathing protocol that may have more realistically simulated inspiratory gases of burial conditions on an individualized basis (Roubik et al., 2022). This approach also permitted us to acquire steady state conditions through the ramped breathing protocol, providing a more stable snapshot during our sampling time point which is important for reporting paired trans-cerebral blood measures and ultrasound CBF measurements. Difference in the magnitude of hypoxia between temperature conditions 110 As a result of hyperventilation during hypothermia, PaO2 was elevated by ~14mmHg during room-air breathing compared to normothermia. While a similar number of stages of hypercapnic-hypoxic breathing were completed between normothermia and hypothermia, because of this upshift of PaO2, the hypoxic stimuli during maximal hypercapnic hypoxia was less sever under hypothermic conditions. Thus, it is difficult to conclude how much of the decrease in CBF during hypothermic hypercapnic hypoxia is due to the differences in hypoxic stimuli, or are due to temperature specific mechanisms. Assumption of cerebral temperature An important consideration herein is that changes in core temperature are used with an assumption that the same magnitude of reductions in temperature are occurring within the brain. Systematic reviews on the topic seem to report divergent summaries depending on the clinical population studied and the site of core temperature measurement (McIlvoy, 2004; Childs & Lunn, 2013). Comparisons between rectal temperature and cerebral tend to show ~0.5⁰C positive bias for cerebral temperature (Tokutomi et al., 2007). It is important to consider that this bias appears across normothermia and hypothermia, so while it may be that we are underestimating the absolute cerebral temperature, the magnitude of cerebral temperature change is likely similar between the esophageal temperature and cerebral temperature. Non-cerebral sources of neurovascular unit brain biomarkers While alteration in cerebral release of GFAP, Tau, and Nf-L are likely primarily responsible for the differences in systemic circulating biomarker concentrations observed in the study, there are non-cerebral sources which should also be aknowledged. For example, GFAP can be produced within the peripheral nervous system along the peripheral nerve fiber track in non-myelinating Schwann cells and in the glia cells of the enteric nervous system. Similarly, Nf-L and tau can be produced at sites within the peripheral nervous system. . 5.5 Conclusion During room air breathing in healthy humans, mild hypothermia resulted in increases in CaO2 and reductions in PaCO2 that significantly contribute to the reduction in CBF with cooling. However, 111 mild hypothermia did not reduce CMRO2, and thus the decrease in CBF is likely not due to changes in cerebral metabolism. During maximal hypercapnic hypoxia, simulating avalanche burial conditions, CBF was lower during hypothermia vs normothermia, but again CMRO2 was not different and thus likely did not contribute to the differences in CBF. Across both room air and hypercapnic hypoxia, hypothermia reduced arterial tau, Nf-L, and GFAP with a reduction in cerebral release of Nf-L seen during room air conditions. These data indicate that mild hypothermia is likely not markedly protective of the cerebrovasculature from a metabolic standpoint during room air breathing or during progressive hypercapnic-hypoxia; however, it may contribute to maintaining neuronal and axonal health/function and blood brain barrier stability. 112 Chapter 6: Conclusion 6.1 Main findings The main findings of the studies within this thesis are summarized as follows: Study 1: Cerebral blood flow and oxygen delivery in Sherpa, Andeans, and lowlanders at 5000m. • Andean highlanders have similar global CBF compared to lowlanders; however, ICA and VA vessel diameters are markedly larger and blood velocity proportionally lower. • Sherpa highlanders present with lower global CBF and CDO2 compared to both Andeans and lowlanders at 5000m. Study 2: Cerebral blood flow in Andean children and adolescents living above 5000m • Despite Andean children and adolescents being more hypoxemic at 5100m, their global CBF was similar to Sherpa living at 3800m and lowlanders at sea-level. • Andeans had larger ICA vessel diameters and lower posterior CBF compared to lowlanders. Study 3: Cerebral blood flow and metabolism under conditions of simulated avalanche burial in humans: the triple H syndrome • Reductions in regional and global CBF during mild hypothermia are primarily mediated by decreases in PaCO2 and increases in CaO2. • Cerebral metabolism was not altered by mild hypothermia during room air breathing or during maximal hypercapnic hypoxia • Blood brain biomarkers indicative of blood brain barrier permeability and neuronal and axonal damage were reduced with hypothermia. 6.2 Contributions to the field Study 1 compared CBF and CDO2 between Sherpa, Andeans, and lowlanders with all three groups residing at the same altitude, thus standardizing the PIO2. In Andean highlanders, the majority of 113 previous reports have indicated that the overall pattern of CBF in this population is characterized by lower CBF (as indexed by intra-cranial velocity) compared to lowlanders (Appenzeller et al., 2004, 2006; Claydon et al., 2005). However, similar to a more recent study (Stacey et al., 2023) we showed that global CBF is similar to that of lowlanders. Further, in both the ICA and VA, vessel diameters are larger in Andeans compared to lowlanders which likely explains the differing findings from previous reports that have only acquired intra-cranial velocity measures that describe Andeans as having lower CBF. In Sherpa, in alignment with our previous study conducted during ascent to high-altitude (Hoiland et al., 2019b), our findings reveal that Sherpa exhibit both lower CBF and CDO2 compared to lowlanders and Andeans. This observation is suggestive that Sherpa possess an oxygen conservative cerebrovascular phenotype that may provide some form of protection against the high altitude environment. Study 2 provided the first volumetric measures of CBF and CDO2 in Andean children and adolescents living at high-altitude. Only one study to date has reported measures of intra-cranial blood velocity as an index of CBF (Hogan et al., 2010) in this population whereby blood velocity in the basilar artery was lower while MCA had similar velocity compared to lowlanders. As we report differences in ICA diameter within this population, previous reports utilizing solely velocity measures may underestimate true CBF if differences in diameter permeate through the intracranial vasculature. Indeed, the current understanding of high-altitude adaptation provides very little clarity regarding whether these adaptations are present at early stages of development or if they are only apparent in adulthood. In Study 1, adult Andeans had similar blood flow through the ICA and VA, yet markedly larger diameters compared to lowlanders. In the Andean children and adolescents, we see similarly larger ICA diameters with no differences in flow. However, similar findings were not observed within the posterior circulation between Andean children and adults. For example, in study 2, VA diameters were similar between Andeans and lowlanders, while VA flow was also lower in Andeans compared to lowlanders. This finding is intriguing as we consider the phylogenetically older regions of the (Binks et al., 2008), supplied by the posterior cerebral circulation to be more sensitive to hypoxia in order to preserve systemic function. Further, the comparisons between the adult high-altitude populations and children and adolescents within the same groups provides some incite into when in the lifespan the adaptations we associate with each group are occurring. In Andeans, as both adults and the children and adolescents present with 114 larger ICA diameters compared to Sherpa and lowlanders, this may indicate that differences in vessel caliber are beginning to occur before [Hb] and viscosity are markedly elevated in Andeans. In Study 3 we obtained the first measures of CMRO2 during mild hypothermia in healthy, spontaneously breathing individuals. While previous reports in clinical populations and in animal models generally report reductions in cerebral metabolism with hypothermia (Erecinska et al., 2003), we did not observe any differences in CMRO2 with mild hypothermia. This was also the case during progressive hypercapnic-hypoxia which simulated breathing conditions of avalanche burial. We also showed that CBF is reduced with mild hypothermia, as previously reported; however, as CMRO2 was not altered, the decrease in CBF with mild hypothermia can be attributed to reductions in PaCO2 and hemoconcentration; whereby despite the decrease in flow, CDO2 was preserved. These data may help provide some context to the recent reports that suggest that mild hypothermia does not improve survival outcomes compared to maintenance of normothermia in patient groups (Dankiewicz et al., 2021). 6.3 Weaknesses of the dissertation/methodological considerations Across all three studies included in this thesis, measurements of CBF were obtained using duplex ultrasound. While this technique provides a robust measure of flow, there are a number of caveats that should be acknowledged. The technique requires a highly skilled sonographer who has the ability to acquire consistent and repeatable scans, both within a participant and between participants. In all three studies, sperate sonographers were responsible for collected ultrasound images (Study 1: CAH and CKW; Study 2: CAH, DNF, and JT; Study 3: RLH, TDG, and MMT). While all of these individuals were highly trained in this technique and obtained blood flow measurements based on published guidelines (Thomas et al., 2015), minor differences in scanning experience, or site of scanning could lead to increased variability in blood flow measurements. Duplex ultrasound measures of the ICA and VA are also notoriously difficult during activities where the participants neck or upper body may be moving such as during exercise. In Study 3, participants were ventilating at a high rate and depth during hypercapnic-hypoxic breathing, in addition to shivering during the hypothermic stages. This introduces movement artifacts into the 115 ultrasound videos that have to be carefully removed during analysis or data has to be excluded in cases where movement artifacts are too severe Indeed under hypothermic-hypercapnic-hypoxia, gCBF data from three participants at maximal hypercapnic-hypoxia were not included due to image quality. Further, two participants were unable to complete the full hypothermia-hypercapnic-hypoxic trial due to discomfort related to the hypothermia. While studies 1 and 2 provide some of the first volumetric CBF data in Andeans residing at altitudes above 5000m, both studies are largely observational. Indeed, while it is important to first be able to classify the patterns of CBF in these high-altitude populations, further mechanistic work is needed to delineate regulatory mechanisms of CBF responsible for the phenotypical differences in resting CBF patterns in both Andeans and Sherpa. These include 1) assessments of CMRO2 at high-altitude to determine if differences in CDO2 in Sherpa are from altered cerebral metabolism; 2) MR imaging to evaluate if structural differences downstream of the extracranial vessels exist between lowlanders and highlanders, particularly in Andeans; 3) Perturbation and blockade studies to investigate the putative hypoxic mediated regulators of CBF using pharmacological interventions (eg. theophylline, L-NMMA, glibenclamide, etc.). The CBF response across aging also requires further investigation to determine whether the sigmoidal CBF pattern associated with childhood and adolescents is altered as individuals age. Indeed, much of our understanding of CBF changes and neural development are based on predominately western populations living under normoxic environments. Thus while we would assume to observe the same in high-altitude populations, whether systemic adaptations related to oxygen availability impact this CBF trajectory and ultimately the phenotypical pattern observed in adult high-altitude populations is unknown. In Study 3 we attempted to examine cerebrovascular responses under conditions of avalanche burial. We elected to utilize laboratory techniques that provided a higher level of control but were possibly not as applicable to avalanche burial as more applied techniques. First, a dynamic end-tidal forcing system was utilized to tightly and repeatably control arterial blood gases, however a protocol whereby participants breath into an enclosed snow structure or simulated snow structure using a substance such as Perlite may have elicited a more individualized response applicable to 116 avalanche burial conditions (Roubik et al., 2022). The effect of body and more specifically chest compression that occurs during snow burial was also not applied to limit participant discomfort and dyspnea as this was already a concern with the protocol as a whole. Along these lines, cold water immersion was used to induce hypothermia as opposed to snow burial due to the practicality of acquiring and using snow in a lab setting. This approach has the advantage of reducing core temperature as quickly as possible via conductive heat loss in order to limit participant discomfort, and to maintain access to the participant in case of emergency. However, the rate of both convective and conductive heat transfer is much greater in water vs snow. 6.4 Future directions and studies 6.4.1 Long term high-altitude adaptation in lowlanders Our understanding of how lowlanders respond to high-altitude remains limited to the period of acclimatization (<1 month at altitude) and we have fairly minimal understanding of how lowlanders adapt to high-altitude over a longer period of time. Research expeditions in lowlanders are generally limited to <1 month at high-altitude for various logistical issues. Notably cost, practicality, and psychological strain on participants. In 2019, in collaboration with a PhD student at the University of South Wales, we began mapping out a research expedition in an attempt to answer this question of long term adaptation in lowlanders at high-altitude. The expedition “lovingly” nicknamed the Lead Hut expedition was inspired by the Silver Hut expedition, led by Dr. Giffith Pugh and Sir Edmond Hillary in 1960-1961 (Milledge, 2010). That work would provide some of the first scientific understanding of how humans acclimatized to high-altitude over a longer period of time during a climbing expedition. The lead hut expedition aimed to take 20 lowlanders (10 male and 10 female) to Cerro de Pasco (4330m) to spend 6-months, during which serial measurements would be obtained across the entire period to track multisystem adaptations with high temporal resolution, including a period of deaclimatization following the 6-months stay. However, due to a combination of changes in visa duration allowances in Peru and the Covid-19 pandemic, this project ultimately could not be completed. Long term hypoxic adaptation continues to be examined through expedition work at the Concordia station in Antarctica (3233m) and in various regions in China where individuals migrate to high-altitude for military service or other 117 professions. These studies, however, are completed at an altitude that is on the lower end of the hypoxic stimuli threshold whereby long term adaptation is able to be observed. Particularly, for example, regarding CBF at high-altitude which generally requires a stimulus >3300m to be adequately perturbed (Figure 2-9). Across the few studies examining CBF in lowlanders to date it is difficult to classify resting CBF as “acclimatized” as the timepoints of assessment are generally too few and too short (Figure 2.9). Further, by assessing long term adaptation in lowlanders we may be able to determine whether the hematological and cerebrovascular responses seen in Andeans are truly a phenotypical response and not simply from chronic-hypoxic exposure in a human. Along these lines, studying high-altitude populations whom have descended to low altitude and reside under normoxic conditions may also permit us to investigate what adaptations are phenotypical and which may simply be a consequence of long term acclimatization. Future studies might also consider the effect of re-entry to high-altitude in groups of Sherpa originally born at altitude in Nepal, whom now reside in the USA or Canada. 6.4.2 Cerebrovascular phenotype of Ethiopian highlanders While we continue to make strides in understanding the phenotypical adaptations to hypoxia in Tibetans and Andean high-altitude populations, very little is understood regarding adaptations in Ethiopian highlanders. Indeed, if the more “efficient” adaptations to high-altitude in Tibetans may be resultant of their greater time spent as altitude and thus more generations for processes of natural selection to occur. In Ethiopians whom have resided at high-altitude for the longest period of time of any population (Simonson, 2015), we could assume that they have the most optimal adaptations under their environment. While our group has attempted to conduct research work in these populations, logistical and geo-political issues have hampered these attempts. Through work with researchers and students from the region we hope to be able to complete these measures in the near future to complete our understanding of how high-altitude populations adapt differently under the same hypoxic stimuli. 118 6.4.3 Longitudinal cerebrovascular measures in Andean children and adolescents While the work in children and adolescents included in this thesis helps provide some knowledge regarding how the cerebrovasculature of Andean children adapts to high-altitude, larger datasets will be required to assess how these patterns of CBF change across the lifespan and between sexes. Projects are currently ongoing evaluating CBF, as well as other systemic measures including echocardiography, blood volume, and hemorheology in Andean children living at sea-level, moderately high altitude in Cusco (3400m) and Puno (3800m), and in La Rinconada (5100m). This large dataset should provide us with a greater understanding of the effect of altitude severity, and age on CBF in Andean children. Additionally, these measures will provide us with the first volumetric measures of CBF that can be compared to data collected in Sherpa children living in Nepal at a matched altitude. These studies may also provide a greater understanding regarding the etiology of chronic mountain sickness. Indeed, the timeline by which CMS develops in Andeans is relatively unclear as longitudinal measures across age have yet to be completed. 6.4.4 Jugular bulb temperature measures in healthy humans As discussed in the limitations section, we are only able to assume that the cooling protocol we utilized in Study 3 to reduce core temperature also resulted in a comparable reduction in cerebral temperature. Future work should measure internal jugular bulb temperature (as a closer index of brain temperature) during similar cooling interventions to assess whether the intervention applied is affecting the end organ being measured. These studies will also permit us to assess a number of research questions including; 1) the effectiveness of various cooling and heating protocols on altering cerebral temperatures (eg. 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Thesis/Dissertation
eng
Kinesiology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Ainslie, Philip N., 1974-
Cerebrovascular function under extreme environmental hypoxia
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