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Cerebrovascular and ventilatory responses to acute normobaric hypoxia in girls and women Morris, Laura 2017

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CEREBROVASCULAR AND VENTILATORY RESPONSES TO ACUTE NORMOBARIC HYPOXIA IN GIRLS AND WOMEN  by  Laura Morris  B.A., Swansea University, 2015  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  THE COLLEGE OF GRADUATE STUDIES  (Interdisciplinary Studies)   THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan)  August 2017   © Laura Morris, 2017  ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled:   CEREBROVASCULAR AND VENTILATORY RESPONSES TO ACUTE NORMOBARIC HYPOXIA IN GIRLS AND WOMEN  submitted by  Laura Morris               in partial fulfillment of the requirements of   the degree of   Master of Health & Exercise Science     .    Dr. Ali McManus, School of Health & Exercise Science Supervisor Dr. Philip Ainslie, School of Health & Exercise Science Supervisory Committee Member Dr. Neil Eves, School of Health & Exercise Science Supervisory Committee Member Dr. Charlotte Jones, Southern School of Medicine  University Examiner                 iii Abstract  Physiological responses to hypoxia in children are incompletely understood. We aimed to characterize cerebrovascular and ventilatory responses to normobaric hypoxia in girls and women. Methods: Ten healthy girls (9.9 ± 1.7y; mean ± SD; Tanner stage 1 and 2) and their mothers (43.9 ± 3.5y) participated. Internal carotid (ICA) and vertebral artery (VA) velocity, diameter and flow (Duplex ultrasound) was recorded pre- and post-1h of hypoxic exposure (FIO2=0.126;~4000m) in a normobaric chamber. Ventilation (VE) and respiratory drive (VT/TI) expressed as delta change from baseline (∆%), and end-tidal carbon-dioxide (PETCO2) were collected at baseline (BL) and 5, 30 and 60 minutes of hypoxia (5/30/60 HYP). Heart rate (HR) and oxygen saturation (SpO2) were also collected at these time-points. Results: SpO2 declined similarly in girls (BL-97%; 60HYP-80%, P<0.05) and women (BL-97%; 60HYP-83%, P<0.05). Global cerebral blood flow (gCBF) increased in both girls (BL-687; 60HYP-912 ml.min-1, P<0.05) and women (BL-472; 60HYP-651 ml.min-1, P<0.01), though the ratio of ICA:VA (%) contribution to gCBF differed significantly (girls, 75:25%; women, 61:39%). The relative increase in VE peaked at 30HYP in both girls (27%, P<0.05) and women (19%, P<0.05), as did ∆%VT/TI (girls, 41%; women, 27%, P’s<0.05). Tidal volume (VT) increased in both girls and women at 5HYP, remaining elevated above baseline in girls at 30 and 60HYP, but declined back toward baseline in women. Conclusion: Girls elicit similar increases in gCBF and ventilatory parameters in response to acute hypoxia as women, though the pattern of these responses appear developmentally divergent.            iv Preface  Chapter 1: I wrote Chapter 1 with extensive feedback provided by Dr. McManus.  Chapter 2: A version of Chapter 2 has been published in Physiological Reports. The experiment was planned by Dr. McManus, Prof. Ainslie and myself, and completed at the University of British Columbia – Okanagan Campus. I conducted the data collection and analysis with the assistance of Daniela Flück. All listed authors contributed towards the interpretation of data. I wrote the manuscript with extensive feedback, input and critical review provided by Dr. McManus and Prof. Ainslie. All co-authors edited the manuscript. Ethical approval for this study was granted by the University of British Columbia Clinical Research Ethics Board (H16-00855). Dr. McManus was supported by a Natural Sciences and Engineering Research Council Discovery Grant (2015-03647), Prof. Ainslie was supported by a Natural Sciences and Engineering Research Council Discovery Grant (2015-0821-01) and a Canadian Research Chair in Cerebrovascular Physiology (950-230970), and Dr. Flück was supported by the Swiss National Science Foundation (P2ZHP3_158576). The authors disclose no perceived or potential conflicts of interest.   Chapter 3: I wrote Chapter 3 with extensive feedback provided by Dr. McManus.               v Table of Contents  Abstract ................................................................................................................................... iii	Preface ..................................................................................................................................... iv	Table of Contents ................................................................................................................... iv	List of Tables ........................................................................................................................ viii	List of Figures ......................................................................................................................... ix	List of Abbreviations ............................................................................................................. xi	Acknowledgements .............................................................................................................. xiii	Dedication ............................................................................................................................. xiv	Chapter 1 Literature Review  ................................................................................................ 1 1.1	 Introduction .............................................................................................................. 1	1.2	 The growing lung ..................................................................................................... 2	1.2.1	 Development of size, volume and capacity ........................................................... 3 1.2.2	 Ventilation ............................................................................................................. 5 1.2.3	 Ventilatory control ................................................................................................ 7 1.3	 The ventilatory response to hypoxia in children ..................................................... 9	1.3.1	 Hypoxic chemosensitivity in children and adults ................................................ 10 1.3.2	 Breathing patterns during hypoxia ..................................................................... 11 1.3.3	 Are respiratory responses influences by genetics? ............................................. 11 1.4	 Cerebrovascular physiology in healthy children .................................................. 13	1.4.1	 Basic development of the brain ........................................................................... 13 1.4.2	 Cerebrovascular anatomy ................................................................................... 15 1.4.3	 Measurement of CBF .......................................................................................... 16				Duplex ultrasound ........................................................................................ 18				Location and insonation of the ICA and VA ................................................ 18				Quantification of volumetric CBF ................................................................ 20				Analysis software .......................................................................................... 21				Methodological conciderations .................................................................... 22  vi 1.4.4	 CBF in children ................................................................................................... 22				Sex differences in CBF ................................................................................. 25 1.4.5	 Regulation of CBF .............................................................................................. 26			Regulation by arterial gasses ........................................................................ 27			Regulation by cerebral metabolism .............................................................. 28			Regulation by cerebral perfusion pressure ................................................... 29 1.5	 Cerebrovascular response to hypoxia in children ................................................ 30 1.6	 Aims and hypotheses .............................................................................................. 32 Chapter 2 Cerebrovascular and ventilatory responses to acute normobaric hypoxia in   girls and women .................................................................................................................... 33 2.1 Rationale ................................................................................................................ 33 2.2 Methods .................................................................................................................. 35 2.2.1	 Participants ......................................................................................................... 35 2.2.2	 Procedures .......................................................................................................... 35 2.2.3	 Primary measures ............................................................................................... 36 2.2.4	 Secondary measures ............................................................................................ 36 2.2.5	 Data and statistical analysis ............................................................................... 37 2.3	 Results .................................................................................................................... 37	2.3.1	 Ventilation ........................................................................................................... 39 2.3.2	 Drive to breathe .................................................................................................. 39 2.3.3	 Respiratory gas exchange ................................................................................... 40 2.3.4	 Cerebral blood flow ............................................................................................ 42 2.3.5	 Cerebral blood velocity and arterial diameter ................................................... 43 2.4	 Discussion .............................................................................................................. 44	2.4.1	 Ventilatory responses to hypoxia in children ...................................................... 44 2.4.2	 Cerebral hemodynamic responses to hypoxia .................................................... 46 2.4.3	 Limitations .......................................................................................................... 47 Chapter 3 Conclusion ........................................................................................................... 49 3.1	 The importance of studying the effect of hypoxia in children ............................. 49	3.2	 Methodological considerations .............................................................................. 50	3.2.1	 Laboratory versus field work studies .................................................................. 50  vii 3.2.2	 Mechanistic limitations ....................................................................................... 51 3.2.3	 Scaling for body size ........................................................................................... 51 3.3	 Futute studies ......................................................................................................... 53	3.3.1	 Determining the mechanisms behind regional CBF differences with age .......... 53 3.3.2	 Assessment of the acclimatization response to hypoxia in children ................... 55 References .............................................................................................................................. 57	Appendices ............................................................................................................................. 70 Appendix A: Parental assessment of Tanner staging ....................................................... 70	Appendix B: Lake Louise Sickness Score Questionnaire ................................................ 71	Appendix C: Cerebral Symptoms Questionnaire  ............................................................. 72	Appendix D: Certificate of Ethical Approval .................................................................... 73	                   viii List of Tables   Table 1    Descriptive characteristics. Data are mean (±SD) ………………………………...38 Table 2    Ventilatory responses at baseline (BL), and following 5 (5 HYP), 30 (30 HYP) and                  60 (60 HYP) minutes of hypoxic exposure in girls and women. Data are mean (±SD)…………………………………………………………...………………...41 Table 3    Internal carotid and vertebral artery diameter at baseline (BL) and following 60 minutes (60 HYP) of hypoxic exposure in girls and women. Data are mean (±SD) ……………………………………………………………………………..…….....44                                 ix List of Figures  Figure 1.0  Measurement of total lung capacity for 106 males (black circles) and 65 females (white circles)…………………………………………………………………….. 3 Figure 1.1  A volume-time spirometry trace of static lung capacities…………………………5 Figure 1.2  Relationship between mouth occlusion pressure (P0.1 cmH20) and respiratory drive normalized for body mass (VTBW/TI ml.kg-1.s-1) in 58 children (circles)…………7 Figure 1.3  Alterations in (A) end-tidal carbon dioxide (etCO2) and (B) peripheral oxygen saturation (SpO2) on ascent to altitude in healthy children and following acclimatization…………………………………………………………………....11 Figure 1.4  Anatomical course of the internal carotid and vertebral artery feed to the brain....16 Figure 1.5  TCD set up to analyze CBF velocity (a) and Duplex ultrasound analysis of extracranial blood flow (b)……………….……………………………………....18 Figure 1.6  Spectral Doppler trace differentiation between the ICA (A) and ECA (B)............19 Figure 1.7  B-mode image of the vertebral artery…….……………………………………....20 Figure 1.8  Glucose use of the human brain by age………………………………………......23 Figure 1.9  GlucoseRMR% and body-weight growth rate…………………………………..…..24 Figure 2.0  Developmental pattern of CBF change in males (blue) and females (pink) by age,     in multiple regions within the heteromodal association cortex………..…….…...26 Figure 2.1  Cerebral blood flow (CBF, black diamonds) and cerebrovascular reactivity to       CO2 (CVR, white squares) values in grey matter in participants separated by age……………………………………………………………………………......28 Figure 2.2  Stylized representation of the possible relationship between mean arterial       pressure and cerebral blood flow………...………………………………….…...30 Figure 2.3  Heart rate (panel a) and arterial oxygen saturation (panel b) at baseline (BL)           and following 5 (5 HYP), 30 (30 HYP) and 60 (60 HYP) minutes of hypoxia....38 Figure 2.4  Relative change from baseline (BL) in ventilation (∆%; panel a), tidal volume     (∆%; panel b), breathing frequency (∆%; panel c) and respiratory drive                (∆%; panel d) following 5 (5 HYP), 30 (30 HYP) and 60 (60 HYP) minutes of hypoxia………………………………………………………………………......40   x Figure 2.5  Relative change from baseline to 60 minutes of hypoxia in internal carotid          artery blood flow (∆%; panel a) and vertebral artery blood flow (∆%; panel b).…………………………………………………………………………..…....42 Figure 2.6  Global cerebral blood flow distribution at baseline and after 60 minutes of       hypoxia in girls (a) and women (b)……………..…………………..……….. ....43 Figure 2.7  Integrative changes in CBF, haematocrit (Hct), VE, cerebral capillary density and hypoxia-inducible factor-1 (HIF-1) during poililocapnic hypoxic exposure........55    xi List of Abbreviations                                              Abbreviation ACA AMS BA CBF CBFV CCA CMRO2 CO2 CPP CVR DER ECA ERV FIO2 fR FRC FVC gCBF  glucoseDER% glucoseRMR% HAPE HVR IC ICA ICP IJV IRV MAP Definition  Anterior cerebral artery Acute mountain sickness  Basilar artery  Cerebral blood flow Cerebral blood flow volume Common carotid artery  Cerebral metabolic rate for oxygen  Carbon dioxide Cerebral perfusion pressure  Cerebral vascular reactivity  Daily energy requirement  External carotid artery Expiratory reserve volume Fraction of inspired oxygen  Breathing frequency Functional residual capacity Forced vital capacity  Global cerebral blood flow  Glucose uptake as a percent of daily energy requirement Glucose uptake as a percent of resting metabolic rate  High altitude pulmonary edema  Hypoxic ventilatory response  Inspiratory capacity  Internal carotid artery Intracranial pressure  Internal jugular vein  Inspiratory reserve volume  Mean arterial pressure    xii  MCA MRI NIMH O2  OEF PaCO2 PaO2 PAP PCA PEF PETCO2 PO2 RMR ROI RV SaO2 SpO2 TCD TI TI/TTOT TLC VA VA VC VCO2 VD VE VE/VCO2 VE/VO2 VO2 VT VT/TI Middle cerebral artery Magnetic resonance imaging National Institute of Mental Health  Oxygen  Oxygen extraction fraction Arterial partial pressure of carbon dioxide Arterial partial pressure of oxygen  Pulmonary artery pressure  Posterior cerebral artery  Peak expiratory flow End-tidal carbon dioxide Partial pressure of oxygen  Resting metabolic rate Region of interest  Residual volume  Arterial oxygen saturation Oxygen saturation Transcranial Doppler Inspiratory time Respiratory timing Total lung capacity Vertebral artery Alveolar ventilation Vital capacity Carbon dioxide production Dead space Ventilation Ventilatory equivalent for carbon dioxide Ventilatory equivalent for oxygen Oxygen consumption Tidal volume  Respiratory drive  xiii Acknowledgements  I would like to begin by acknowledging my family and friends. Without their support and continued reassurance, I would never have possessed the courage (or funding!) needed to venture to the other side of the world and pursue this MSc. They have supported me throughout all my endeavours, and for that, I will always be grateful.   I would like to individually thank each of my lab mates for making me feel part of a fantastic team. I am honoured to have experienced this journey with them and have learnt a great deal in their company. I would also like to express my thanks to Dr. Phil Ainslie for his input, especially in helping me to better understand the important aspects of cerebral blood flow, and for allowing me to take over his environmental chamber during my testing.  Most importantly, it is with the utmost gratitude that my thanks are given to Ali McManus. Her capability as a supervisor, mentor, teacher and researcher are second to none. Without Ali’s passion and enthusiasm for research, nothing that is about to be presented would have been possible. Thank you for facilitating an incredible opportunity for me.                 xiv         For my family             1 Chapter 1 Literature Review   1.1 Introduction  Hypoxia is defined as an inadequate supply of oxygen (O2) to the bodily tissues, increasing in severity upon ascent as the environmental partial pressure of O2 (PO2) falls. In perspective, arterial oxygen saturation (SaO2) at sea level remains relatively constant around 96-99%, but ascending to 3000m and 5000m will reduce SaO2 to approximately 92% and 80%, respectively (Mazzeo, 2008). Oxygen is arguably the most integral component to our existence, and reductions in O2 availability greatly affect physiological function.  The ventilatory system has been described as the first interface between atmospheric O2 and the metabolic machinery of the body (Schoene, 2001). The lung is primarily designed to facilitate gas exchange, and whilst other roles include compound metabolism and filtration of unwanted materials, its cardinal function is to deliver inspired O2 from the air into the venous blood and to eliminate the bi-product carbon dioxide (CO2; West & Luks, 2012). In healthy individuals under normal atmospheric conditions (i.e. fraction of inspired oxygen, [FIO2] = 0.21), gas exchange across the alveolar-capillary membrane is exceptionally efficient and cellular respiration is rarely compromised. However, when PO2 falls, the arterial partial pressure of O2 (PaO2) is reduced and the work required to deliver and utilize the same amount of oxygen to respiring tissue becomes greater (Calbet & Lundby, 2009). A comprehensive understanding of the ventilatory responses to hypoxia in adults has been established for many years (Weil et al., 1970), though our knowledge of these responses in children remains surprisingly limited (Kohler et al., 2008). Of the few studies available, focus has revolved around studying the adaptations of those born and raised at high altitude (e.g. Bianba et al., 2014), and more recently, a growing interest in the low altitude dwellers response to periods of ≥1 days of hypoxia (Kriemler et al., 2015). Yet, data remains sparse and much of our understanding continues to be extrapolated from adult literature. This is problematic because of the documented developmental differences in ventilation (Gratas-Delamarche et al., 1993). Further study is needed to extend our knowledge of the ventilatory response to hypoxia in the child (Mohr, 2008).   2 Ventilatory induced changes in PaO2 and arterial partial pressure of CO2 (PaCO2) are tightly coupled with the regulation of cerebral blood flow (CBF; Ainslie & Ogoh, 2010). In adults, a reduction in PaO2 increases CBF to maintain adequate O2 delivery to the brain (Willie et al., 2012). This is crucial since the brain is the most oxygen demanding organ in the body relative to size – accounting for a mere 2% of body mass, but requiring at least 20% of the body’s total basal O2 consumption. Cerebral perfusion has similarly been shown to have distinct developmental patterns (Leung et al., 2016), which necessitates the need for child specific data in response to hypoxia. Unfortunately, this knowledge is limited to just one previous study to date. There are also many different methodological approaches available, such as the level of hypoxia, the length of exposure time and the type of cerebral vessels insonated (extracranial versus intracranial). As such, regional CBF changes in response to hypoxia in the heathy child are unknown.   It is becoming increasingly common for families to travel to regions of high altitude for ski or hiking excursions, with as many as ~150,000 children visiting the mountains every year in Colorado alone (Yaron & Niermeryer, 2008). Thus, it is surprising that the current recommendations given by practitioners to parents and their children are based upon little evidence, and are often extrapolated from adult studies (Samuels, 2004). This may be unrealistic or inappropriate advice if age and/or maturational differences in the ventilatory and cerebrovascular responses to hypoxia exist. The following review aims to address ventilatory and cerebrovascular function in children, both normatively, and in response to hypoxia where literature is available.    1.2     The growing lung      To contextualize what we know of the ventilatory response to hypoxia in children, it is important to first acknowledge the physiology of a healthy child under normal environmental conditions (i.e. FIO2 = 0.21). The development of size, volume and capacity of the lung as the child grows and matures, as well as an explanation of ventilation and ventilatory control will be discussed.     3 1.2.1      Development of size, volume and capacity  It is widely recognized that fundamental age and maturational differences exist in the way O2 consumption (VO2) and CO2 removal (VCO2) is mediated (Rowland, 2005). This difference has been attributed to developmental changes in the mechanical properties and metabolic capabilities which occur as the pulmonary system matures (e.g. Simon et al., 1972; Lanteri & Sly, 1993).  A key factor which contributes to pulmonary immaturity in children is lung size. The relationship between increasing lung volume (subdivisions including residual volume, functional residual capacity, vital capacity and total lung volume) and standing height is well established, with the earliest of reports documenting a correlation coefficient of .93 (Helliesen et al., 1958). As such, an increase in stature from 120 cm to 140 cm and then to 160 cm can be expected to align with an increase in total lung capacity (TLC) from 2L, to 3L, to 6L, respectively (Cook & Hamann, 1961; see Figure 1.0). Peak growth velocity of lung width and height closely correspond with one another and occur at approximately 12.2 years in girls and 13.8 years in boys, followed 6-8 months later by peak growth velocity of lung length, chest depth and thorax dimension (Simon et al., 1972; DeGroodt et al., 1988).    Figure 1.0     Measurement of total lung capacity for 106 males (black circles) and 65 females (white circles). There was a significant (p < .01) difference between the regression line for males (solid line) and that for females  4 (dotted line). The equations and lines are for geometric means. Adapted from Cook & Hamann, 1961 - ©permission received.   Likewise, alveolar surface area is closely correlated to height3. Alveoli are minute organs in the lung responsible for gas exchange (i.e. moving O2 and CO2 between the lungs and the blood), the number of which rapidly increases from ~24 to ~280 million between birth and 8 years of age (Dunnill, 1962). Thus, the surface area of these alveoli accumulates from 2.8 m2 postnatal to approximately 75 m2 once full stature is achieved (Dunnill, 1962). Astoundingly, the surface area of the complete lung increases ten-fold by the age of 18 (Davies & Reid, 1970). These structural changes are associated with reduced airway resistance and increased compliance into adulthood (Lanteri & Sly, 1993). This implies that there is a greater resistance to flow in the respiratory tract (i.e. it is more difficult to move air in and out of the lungs) and a lesser ability for the lung to stretch and expand during childhood. It is remarkable though that despite this immature pulmonary system, children can finely maintain PaO2 around the homeostatic region of 80-100 mmHg, and PaCO2 at approximately 38 mmHg (Marcus et al., 1994).  The determination of most static lung capacities are achieved using a spirometer, the results of which can be seen in Figure 1.1. Tidal volume (VT) is the amount of air moved in or out of the lungs during normal breathing which, in absolute form, increases with age in accordance with increased lung size. When accounting for body mass and surface area though, VT decreases over time. For example, Gaultier and colleagues (1981) observed a higher relative VT in children aged 6-8 (11.3 ml.kg-1) compared to children aged 8-16 (10.1 ml.kg-1). More recently, McMurray and Ondrak (2011) assessed seventy-three healthy 8-18 year olds and report a mean resting VT of 426 ml. Aside from values reported by smaller studies, pediatric norms for resting static lung volumes in healthy children are sparse. Following tidal inspiration, inspiratory reserve volume (IRV) can be measured by slowly inhaling to a maximum. Similarly, expiratory reserve volume (ERV) is measured following tidal expiration by slowly exhaling to a maximum. The sum of VT, IRV and ERV is representative of an individual’s vital capacity (VC). Following a slow maximal exhalation, any remaining air left in the lung beyond the ERV represents residual volume (RV). Along with functional residual capacity (FRC; which is a summation of ERV and RV), RV cannot be calculated with a simple spirometry maneuver. Alternative methods include gas dilution, whole  5 body plethysmography or radiography – for a detailed explanation of such approaches; readers are directed to Wagner et al. (2005). Total lung capacity is a summation of IRV, VT, ERV and RV, and must also therefore be obtained using alternative methods.    Figure 1.1     A volume-time spirometry trace of static lung capacities. IRV, inspiratory reserve volume; VT, tidal volume; ERV, expiratory reserve volume; RV, residual volume; FRV, functional residual capacity; IC, inspiratory capacity; VC, vital capacity; TLC, total lung capacity.  1.2.2     Ventilation  Basal metabolic rate is the minimum amount of energy needed to preserve the vital functions of waking life (Plowman & Smith, 2013). The basal metabolic rate of a child is approximately 25-35% higher than an adult (Bar-Or, 1983), which is largely attributed to the energy demands of growth, development and temperature regulation. Children sustain adequate ventilation (VE, total air movement between the environment and the lung) for basal metabolic function using a different pattern of breathing than adults. Children typically ventilate more (when normalized to body mass), breathing with a higher frequency (fR) and a larger relative VT. In a recent review by Fleming and colleagues (2011), fR of children aged ≤ 2 years was reported at approximately 44 breaths.min-1. From early childhood to 16 years of age, resting fR declines from ~26 breaths.min-1 to ~13-15 breaths.min-1. As noted in section 1.2.1 Development of size, volume and capacity, absolute VT increases with age and maturity whilst relative VT decreases.   6 Alveolar ventilation (VA) is the volume of effective gas exchange taking place in the alveoli (VA = [VT – dead space] x fR). VA is therefore smaller than pulmonary VE which is inclusive of both VA and physiological dead space (VD). Physiological VD can be segregated into anatomical VD (the volume of gas in the conducting airways) and alveolar VD (the volume of gas which reaches under- or un-perfused alveoli), considerably influencing the effectiveness of VE. VE is more frequently reported in pediatric literature due to the complexity and invasive nature of assessing VA, specifically, the VD portion of the VE equation. VD is calculated as follows;   𝑉𝐷 = 𝑉𝑇	x	 𝑃𝑎𝐶𝑂+ − 𝑃-.𝐶𝑂+𝑃𝑎𝐶𝑂+   Thus, PaCO2 can only be obtained by arterial sample which is unrealistic for healthy pediatric participants. In children aged 5 to 16 years old, non-invasive estimation of VD (using PETCO2 as a surrogate for PaCO2) accounts for approximately 33% of VT (Kerr, 1976), which is comparable to adult values on a per kg basis (2 ml.kg-1; Nunn & Hill, 1960). It is therefore unlikely that VD contributes to the VE differences between children and adults. Though VD is similar between ages, the comparably higher fR and VT in children results in an VA which is approximately twice as high as that of an adult (~100-150 ml.kg.min-1; Rupp et al.,1999).  The different breathing pattern in children results in greater ventilatory equivalents for O2 and CO2 (VE/VO2 and VE/VCO2, respectively) and implies that children must breathe more to obtain a given amount of O2, potentially increasing the energetic cost of breathing. Similarly, a higher VE/VCO2 implies breathing more to expel a given amount of CO2. To better understand the difference in ventilatory efficiency between children and adults, Kriemler and colleagues (2015) tested 20 father-child pairs to maximal exercise. Results indicate that VE/VO2 in prepubescent boys was greater than their genetically matched fathers (40.4 ± 5.9 and 35 ± 5, respectively). Similarly, VE/VCO2 was also greater in the boys (35.9 ± 5.3 vs 30.8 ± 4.3). As such, the higher metabolic cost for a child to sufficiently ventilate at a given metabolic demand can primarily be attributed to their tendency to breathe with a higher fR.    Respiratory drive and timing are important physiological markers of the breathing pattern, yet very little data exists on these components in children (Ondrak & McMurray, 2006). Respiratory drive  7 can be directly measured using mouth occlusion pressure, diaphragmatic EMG or phrenic EMG, though the ability to perform these measures in children is questionable and perhaps reason for the lack of data in this population. Instead, the ratio of VT and inspiratory time (TI; VT/TI) has been advocated as a surrogate of the drive to breathe in children, since VT/TI normalized to body mass correlates well with mouth occlusion pressure (r=0.63; Gaultier et al., 1981; see Figure 1.2). Gaultier and colleagues (1981) report inspiratory drive and respiratory timing (TI divided by a total respiratory cycle time; TI/TTOT) in 82 participants aged 4 to 32 years.  Resting VT/TI decreased with age whilst no change was observed in TI/TTOT across ages. This was later confirmed by Ondrak and McMurray (2006) who observed a ~31% reduction in VT/TI in 295 subjects aged 8 to 18 years. Although in contrast to Gaultier et al.’s (1981) findings, Ondrak and McMurray (2006) report age related attenuations in TI/TTOT, with a greater increase in TI (~42%) opposed to a decrease in relative VT (~11%) responsible for the overall attenuation of VT/TI with age.   Figure 1.2     Relationship between mouth occlusion pressure (P0.1 cmH20) and respiratory drive normalized for body mass (VTBW/TI ml.kg-1.s-1) in 58 children (circles). Mouth occlusion pressure was generated 0.1 seconds after an occlusion at the end-expiratory level. Triangles represent values in 20 adults which are not included in the calculation. Reproduced from Gaultier et al., 1981 - ©permission received.   1.2.3     Ventilatory control   Ventilatory control is tightly regulated by three fundamental components; sensors/receptors which  8 detect change, a central controller found in the pons and medulla of the brainstem which interprets and coordinates this information into impulses, and respiratory muscle effectors which respond to these impulses to induce a change in VE (West & Luks, 2012). This is largely an automatic process, but on occasion can be overridden by the cerebral cortex. For example, it is possible to half PaCO2 and increase arterial pH by approximately 0.2 units by voluntarily hyperventilating (West & Luks, 2012). Multiple sensors include lung receptors (stretch, irritant, J receptors, and bronchial C fibers), pain and/or temperature receptors, baroreceptors, receptors located in the nose, upper airway and muscle and chemoreceptors (central and peripheral). The close coupling of the chemoreceptor responsiveness to arterial blood with ventilation is key in the maintenance of a constant pH.   In normoxia, respiratory regulation is heavily reliant on central chemoreceptors; found in the retrotrapezoid nucleus region of the ventral surface of the medulla. These receptors are sensitive exclusively to changes in PaCO2, or more specifically, CO2 induced changes in H+ (and therefore pH) of the surrounding extracellular and neighboring cerebrospinal fluid – this response is known as the ‘reaction theory’ (Loeschcke, 1982). CO2 diffuses across the blood brain barrier into the cerebrospinal fluid as PaCO2 rises, which in turn liberates H+ ions and stimulates central chemoreceptors to increase VE (West & Luks, 2012). Children regulate PaCO2 (estimated using end-tidal CO2; PETCO2) at a lower set point compared to adults (38 ± 4 mmHg versus 40 ± 4 mmHg; Marcus et al., 1994). The reason for this earlier responsiveness to CO2 is poorly understood, but it is thought that greater sensitivity of the respiratory drive centers and/or a greater respiratory neural drive may play a role (Cooper et al., 1987; Gratas-Delamarche et al., 1993; Marcus et al., 1994). Furthermore, normal hemoglobin levels in healthy individuals increase with age from ≥110 g/L at 6 months to 4 years, to ≥115 g/L at 5 to 12 years, to ≥120 g/L in adult females/ ≥130 g/L in adult males (World Health Organisation, 2011). CO2 is transported dissolved in blood, carried by hemoglobin as a bicarbonate ion or bound to hemoglobin (Arthurs & Sudhakar, 2005). The blood is therefore a major storage site for CO2 and it is likely that younger children have a smaller storage capacity because of their lower hemoglobin levels. Potential differences in the storage capacity of muscle and/or fat tissue between children and adolescents are unknown (Cooper et al., 1987).    9 Peripheral chemoreceptors are situated in both the carotid bodies (at the bifurcation of the carotid artery) and aortic bodies (above and below the aortic arch) and respond primarily to changes in PaO2, but also increased PaCO2 and pH. Brady, Cotton and Tooley (1964) administered 12 newborn babies with hyperoxic gas (100% O2) and observed a 10% reduction in VE, suggesting that the peripheral chemoreceptor contribution to VE is active after just one hour of birth. When PaO2 is reduced the increased VE is believed to be mediated by the carotid body which drives stimulation of the brain stem respiratory center (Samuels, 2004). This response is regulated by the peripheral chemoreceptors since central chemoreception is unable to detect changes in PaO2. Unlike CO2 sensitivity, evidence to suggest that peripheral chemoreceptor sensitivity (and therefore the ventilatory response to hypoxia) is related to age is mixed, and will be discussed in more detail in 1.3.1 Hypoxic chemosensitivity in children and adults.   1.3    The ventilatory response to hypoxia in children  Increasing VE following hypoxic exposure acts to maintain O2 uptake, transport and delivery and has consequently been described as a key factor determining the effectiveness of acclimatization in adults (Bärtsch et al., 2002). These alterations to breathing patterns can be investigated both acutely, following an initial and/or short bout of hypoxia, or over the course of ventilatory acclimatization whereby hypoxic exposure is prolonged several hours, days or weeks etc. For more detail on ventilatory acclimatization to hypoxia, readers are directed to Ainslie, Lucas and Burgess (2013). Unlike adults, evidence of the ventilatory responses to hypoxia of any time-course in children is sparse (Kohler et al., 2008; Gavlak et al., 2013), with much of our understanding extrapolated from adult data. This is problematic because of documented, previously aforementioned differences in normoxic ventilation in the child. Moreover, it is prudent to note the specificities of terminology used to describe the interactions between VE and hypoxia. The term ‘hypoxic ventilatory response’ (HVR) is exclusively used to describe the increase in VE in response to isocapnic hypoxia (i.e. when CO2 levels are kept constant). The HVR is therefore solely mediated by changes in PaO2. Alternatively, the ‘ventilatory response to hypoxia’ will be used to describe the increase in VE in response to poikilocapnic hypoxia (i.e. when CO2 levels can vary naturally). This response is more indicative of real life situations whereby PaO2 and PaCO2 both influence the observed response. This is important because the initial increase in VE in  10 response to reduced PaO2 causes concomitant reductions in PaCO2 which actually attenuates the drive to breathe to preserve adequate levels of PaCO2 (Steinback & Poulin, 2007). It is therefore the interplay between VE, PaO2 and PaCO2 controlling the ventilatory response to hypoxia.   1.3.1     Hypoxic chemosensitivity in children and adults  Evidence that the ventilatory response to hypoxia is dependent on age is mixed. During isocapnic hypoxia, Honda and colleagues (1986) report no differences in the HVR of children, adolescents and adults. Using a progressive hypoxic technique participants began rebreathing into an 8-12L bag of room air set at 100 mmHg end-tidal PO2 (PETO2). Once stable, the O2 supply was switched off and a drop in PETO2 was initiated whilst maintaining PETCO2 at 5 mmHg above resting. A drop in PETO2 below 45 mmHg terminated the test. Of the 71 subjects who were allocated into 6 age-range categories (7-8, 9-10, 11-12, 13-14, 15-16 and 17-18 years), no significant difference in the magnitude of HVR was found. By contrast, in a comparison between 35 children (< 18 years) and 24 adults (18-49 years) using a similar rebreathing technique, Marcus and colleagues (1994) found that the HVR in children was significantly greater than in adults, and decreased with age. The authors concluded that children have a greater hypoxic chemosensitivity than adults; however, the relationship found was notably weak (r = 0.34). Kohler and colleagues (2008) more recently investigated VT/TI and VE between children and adults in the field (i.e. poikilocapnic, hypobaric hypoxia). Twenty pre-pubertal children and their biological fathers were recruited from lowland Switzerland to ascend by train to the Swiss Jungfrau-Joch research station (3450m) and remain there for 2 nights. Following day one at 3450m, VT/TI increased similarly in children (37%) and adults (45%) from baseline, as did VE (children, 42%; adults, 35%). This suggests that the ventilatory responses to hypobaric hypoxia are independent of age, aligning with the earlier report from Honda et al. (1986). Thus, of the few studies that are available documenting the ventilatory responses to hypoxia in children, a clear understanding is hindered by contrasting findings and further clarification is needed to better understand how age related differences (present or not) may affect ventilatory responses to an acute bout of poikilocapnic hypoxia.     11 1.3.2     Breathing patterns during hypoxia  Reductions in O2 saturation (SpO2) indexed by pulse oximetry and PETCO2 in response to hypoxia appear to be similar in children and adults (Scrase et al., 2009). Over the course of 5 days, 9 children aged 6-13 years slowly accented to 3500m, and proceeded to remain at this altitude for a further 9 days (i.e. slow acclimatization response). In comparison to baseline, ascending to 3500m was conjunctive with a ~7 mmHg fall in PETCO2 and a ~9% drop in SpO2 (Scrase et al., 2009; see Figure 1.3). In accordance, changes in nocturnal PETCO2 and SpO2 were also comparable between children and their fathers (Kohler et al., 2008). Despite these similarities, there does appear to be a difference in the way in which VE is mediated during hypoxia. In adults, increased fR and VT both contributed to the increased VE in response to hypoxia, yet the increase in VE in children was primarily attributed to increased fR, with minimal changes in VT (Kohler et al., 2008). This hyperventilation typically causes reductions in PaCO2 which has important implications for CBF and will be discussed in section 1.5 Cerebrovascular response to hypoxia in children.    Figure 1.3     Alterations in (A) end-tidal carbon dioxide (etCO2) and (B) peripheral oxygen saturation (SpO2) on ascent to altitude in healthy children and following acclimatization. Mean is indicated in bold. Child C and Child H represent the two extremes of response. Reproduced from Scrase et al., 2009 - ©permission received.  1.3.3     Are respiratory responses influenced by genetics?  Genetic inheritances have been prominent in underpinning the regulation of the hypoxic response  12 throughout the course of history. Several native populations have genetically evolved (via natural selection) over thousands of years to tolerate harsh conditions of very low O2. Of these populations, the Tibetans and Andeans have been living around 3500-4500m for ~25000 and ~11000 years, respectively, which makes them by far the longest high altitude inhabitants. Advantageous adaptations to ultimately increase O2 availability include changes in pulmonary ventilation, lung volume and pulmonary diffusion capacity and the transportation, diffusion and utilization capacity for O2 (reviewed in Frisancho, 2013). Whilst these responses have been adapted and passed down over thousands of years, there is considerable utility and growing evidence to suggest that the low altitude dwellers response to hypoxia is at least in part hereditary.   Kriemler and colleagues (2008) estimated systolic pulmonary artery pressure (the pressure of blood pumped though the arteries of the lung from the heart; PAP) from the pressure gradient of tricuspid regurgitation in 20 pre-pubertal children and their biological fathers following 2 days at 3450m. PAP has high clinical significance, since abnormal increases have been associated with a greater susceptibility of developing high-altitude pulmonary edema (HAPE, Maggiorini et al., 2001); the leading cause of death from high-altitude related illness (Hackett & Roach, 2001). Following day 1 at 3450m, PAP was greater in children (15.5 mmHg) than their fathers (6.4 mmHg), though this returned to adult levels by day 2 (Kriemler et al., 2008). The increase in PAP was correlated between children and their fathers (P = 0.02) suggesting a hereditary component to the hypoxic PAP response. Predicting how well individuals will respond to hypoxia from a respiratory stand point is extremely difficult, and although the magnitude remains to be investigated, these authors elude to being able to determine individual PAP responses in children by the response of their parents. Likewise, the same group (i.e. same expedition, participants and ascent profile) observed similar correlations among child-father pairs with respect to reductions in peak VO2 at 3450m (Kriemler et al., 2015).   The same expedition also examined the reduction in SpO2, PETCO2 and prevalence of nocturnal periodic breathing between children and their fathers, for which no correlation was found (Kohler et al., 2008). Periodic breathing refers to normal breathing interspaced with periods of apnea (temporary cessation of breathing, for usually no longer than 10 seconds) which is typically followed by several rapid, shallow breaths to reestablish adequate SaO2. First described by Mosso  13 (1898), nocturnal periodic breathing is common in adults exposed to hypoxia. Depending on the magnitude of the hypoxic response, increased VE can substantially lower PaCO2 leading to respiratory alkalosis which destabilizes breathing and induces apnoea (Berssenbrugge et al., 1983; Berssenbrugge et al., 1984). Reported side effects of periodic breathing include poor quality sleep with frequent arousal (Anholm et al., 1992) and the worsening of acute mountain sickness (AMS) symptoms (Erba et al., 2004). Children experienced fewer incidences of periodic breathing compared to their fathers on both the first (8 vs 34%, respectively) and second night (9 vs 22%, respectively) at 3450m altitude, which was attributed to a lower apnea threshold for CO2 (children, 27 mmHg; adults, 30 mmHg). Interestingly, although periodic breathing is described as maladaptive, the gasp-like breaths which occur during this cycle may actually defend SaO2 and protect against AMS (Nespoulet et al., 2012). In accordance, the lower prevalence of periodic breathing in children does correspond with a greater prevalence of AMS (children, 50%; fathers, 30%; Kohler et al., 2008). Hence, data on hereditary influences to the ventilatory response to hypoxia is mixed, and seem largely dependent on the specific variable in question. Given the documented correlation in PAP and peak VO2, it is prudent to recruit child-parent pairs for further investigations comparing the age dependent response to hypoxia to remove any potential confounding influence of genetics.    1.4     Cerebrovascular physiology in healthy children  To contextualize the cerebrovascular response to hypoxia, it is important to first acknowledge cerebrovascular physiology of a healthy child under normal environmental conditions (i.e. FIO2 = 0.21). Basic developmental trajectories of the brain will first be highlighted, followed by a description of cerebrovascular anatomy. Then a review of resting CBF, sex differences in cerebrovascular function and an overview of CBF regulation.  1.4.1     Basic development of the brain  Cerebral maturation is a highly complex and lengthy process. Whilst it is outside of the scope of this review to appropriately cover the changes which occur during pregnancy, it is worth noting that brain development begins as early as the third week of gestation and continues well into  14 adulthood. The most rapid period of growth takes place from birth to 5 years of age. The brain grows to approximately 80% of its adult mass in the first two years of life (Lenroot & Giedd, 2006). By the 5th year, brain growth reaches 90% of adult mass; four times the size at birth (Dekaban & Sadowsky, 1978). In the early 1980’s, Levene et al. (1982) performed one of the first magnetic resonance imaging (MRI) studies to investigate brain development. MRI scans were performed on four healthy children (ranging between 5 weeks and 5 years old) and they observed that progressive myelination (myelin sheath formation around a nerve which allows impulses to be transmitted faster) increased with age. Although this study was underpowered, it was a precursor for the expansion of research which followed. The first large scale study to longitudinally track brain development in children - the “Pediatric Brain Imaging project” - was conducted by the National Institute of Mental Health (NIMH; USA) in 1989. In 1996, they published the first developmental report containing over 100 healthy children/adolescents (14-18 years old), exploring the relationship between gender, age and brain morphology (Giedd et al., 1996). Cerebral and cerebellar (hindbrain area largely responsible for motor control) volumes scaled for height and weight were greater in boys than girls by 9% and 8%, respectively, but did not change significantly with age. In the subcortical structures, the putamen and globus pallidus (involved in regulating movement) remained larger in males after adjusting for cerebral size, whilst relative caudate size (associated with motor processes, learning and inhibitory control) was greater in females (Geidd et al., 1996). Moreover, age at peak total cerebral volume occurs earlier in girls (11.5 years) than boys (14.5 years; Geidd et al., 1999). This appropriately corresponds to age at peak height velocity; a marker of the trajectory of pubertal maturity, occurring at approximately 12 and 14 years in girls and boys, respectively (Tanner et al., 1966).  The central nervous system consists of grey and white matter. Grey matter has been labelled the ‘working tissue of the brains cortex’. It is found throughout the brain, brainstem, cerebellum and spinal cord and is made up of cell bodies, axon terminals and dendrites. White matter is made of axons which connect this grey matter together and is crucial for the coordination and relay of information between the various areas of the brain (Fields, 2008). Prior to the longitudinal NIMH study very little was known about the development of grey and white matter. Cross sectional observations had previously identified a linear increase in cortical white matter from early childhood up until ~20 years (e.g. approximate mean values at birth, 41mL; 5 years, 51mL; 10  15 years, 59mL and 20 years, 68mL; Pfefferbaum et al., 1994), which was confirmed by Giedd et al. (1999) in the NIMH study. This is likely due to ongoing myelination and axonal growth. Preliminary reports established that grey matter volume peaked in children around 4 years of age followed thereafter by steady decline (Pfefferbaum et al., 1994). This corresponds with the rapid cell growth, exuberant synaptogenesis (the mass formation of synapses between neurons that occurs during the first few years of life), arborization (branching of nerve fiber endings) and cell proliferations (an imbalance between cell division and cell loss which results in an overall increase in the number of cells), which characterizes early brain development. The following decline in grey matter volume is indicative of a pruning process which removes unused circuitry – subject to the experiences (or lack of) that a young child is exposed to (Stiles & Jernigan, 2010). This overproduction and pruning process in the early years of life was later confirmed by Giedd et al. (1999), but instead of a linear decline thereafter, they discovered a second surge of overproduction pre-puberty followed similarly by a stage of pruning.  1.4.2      Cerebrovascular anatomy   Blood flow to the brain is maintained via an intricate and interconnected network of arteries. Two bilateral pairs of extracranial internal carotid arteries (ICA) and vertebral arteries (VA) supply the initial feed to the brain. Branching cephalad from the common carotid artery (CCA) bifurcation, the ICA’s continue to run to the base of the brain where they ramify into a collating ring (named the Circle of Willis) alongside several other arteries. The ICA’s are responsible for delivering blood to the anterior portion of the brain, supplying approximately 70% of the total CBF volume (CBFV). The VA’s, which originate from the subclavian artery, run through the vertebral column forming the basilar artery (BA) at the base of the brain. They supply the posterior portion of the brain and account for the remaining ~30% of CBFV (Zarrinkoob et al., 2015; see Figure 1.4).    16  Figure 1.4     Anatomical course of the internal carotid and vertebral artery feed to the brain. This right-sided diagram is mirrored on the left side of the body.  The Circle of Willis is a remarkable inter-fed, inter-distributing cerebral structure that was originally identified in 1664 by Sir Thomas Willis (Willis, 1664). The ICA, BA and communicating arteries feed into this circle and blood flow is distributed via intracranial vessels to the surface of the cerebral cortex off the anterior cerebral arteries (ACA), middle cerebral arteries (MCA) and posterior cerebral arteries (PCA). Once these arteries leave the parenchyma they enter the pia matter and become known as pial vessels, before penetrating the brain tissue. Interestingly, the idealistic configuration portrayed in textbook is not always representative of actual human physiology. Aside from the distribution of CBF, the Circle of Willis plays an important role in protecting the brain against ischemia. If an area of the circle or supplying arteries becomes stenosed, redirecting blood flow from alternative blood vessels can help maintain cerebral perfusion. Magnetic resonance angiography of the Circle of Willis was performed on 150 volunteers (20-88 years old), with just 42% of subjects conforming to the typical, complete arrangement (Krabbe-Hartkamp et al., 1998). Variation of this anatomy in children remain largely unknown, though a genetic link for both the development and caliber of the circle has been postulated (Vasović et al., 2002).  1.4.3     Measurement of CBF  Angelo Mosso, an Italian physiologist from the 19th century, developed the first ever assessment  17 of CBF, marking the expansion of interest in the area and the techniques used to quantify this. Mosso’s neuroimaging invention, coined the ‘human circulation balance’ was capable of measuring brain pulsations, and thus indications of changes in CBF volume. After recording these pulsations in response to mental stimulation in patients admitted to hospital for neurological surgery, Mosso concluded that CBF volume was augmented by mental activity (Mosso, 1880). Succeeding advancements include the Kety-Schmidt technique (based upon the Fick principle) to measure CBF and oxygen metabolism using a nitrous oxide tracer. Though this method is highly invasive, requiring catheterization to sample the arterial blood entering the brain and venous blood leaving it (Kety & Schmidt, 1948).  Aaslid and colleagues (1982) later discovered that Doppler ultrasound; a method already employed to assess the peripheral vasculature, could also be used to assess cranial vessels non-invasively. Transcranial Doppler ultrasound (TCD) enables quantification of CBF velocity using a low frequency transducer probe to insonate the intracranial arteries though several thinner regions of the skull (see Figure 1.5, panel a). Thus, TCD can be used to assess baseline flow velocity or changes in flow velocity in the ACA, MCA, PCA and BA in response to experimental interventions (e.g. exercise or hypoxia) with excellent temporal resolution. For a more comprehensive description of TCD utility, readers are directed to Willie et al. (2011a). Although praised for its non-invasive qualities, this method is confounded by the inability to directly measure volumetric blood flow; instead using velocity as a surrogate, with the underlying assumption that vessel diameter remains constant. However, recent evidence has supported intracranial vessel diameter changes in response to acute and chronic hypoxia (Wilson et al., 2011), hypocapnia and hypercapnia (Ainslie & Hoiland, 2014), thus using TCD under these circumstances may not be accurate. An increasingly popular alternative is the volumetric assessment of CBF using duplex ultrasound (Thomas et al., 2015; see Figure 1.5, panel b) which will be discussed in more detail below.    18  Figure 1.5     TCD set up to analyze CBF velocity (a) and Duplex ultrasound analysis of extracranial blood flow (b). Images obtained in the Pediatric Exercise Research Laboratory, Kelowna. Parental permission received.     Duplex ultrasound  Duplex ultrasound is a method of simultaneously measuring both arterial diameter and CBF velocity in the extracranial vasculature (CCA, ICA and VA) using a multi-frequency array transducer probe (typically 10MHz) and high-resolution ultrasound machine (e.g. Terason 3000). Arterial diameter is obtained by B-mode imaging and CBF velocity by pulse wave velocity. A pulse is directed from the probe into the chosen vessel and the time between the transmitted signal and the returning signal is measured – referred to as the ‘Doppler shift’. In acquiring both arterial diameter and CBF velocity of a vessel, duplex ultrasound can quantify volumetric CBF where TCD cannot. Optimal location and insonation of the ICA and VA, the calculation of volumetric CBF and the software used to analyze these scans will be discussed, along with some potential methodological considerations for this technique.     Location and insonation of the ICA and VA   A schematic of the extracranial vasculature including both the ICA and VA can be revisited in Figure 1.4. A comprehensive set of technical recommendations for the acquisition of extracranial CBF using duplex ultrasound has been documented by Thomas et al. (2015), and will be summarized below. Readers are directed to this review for further detail.   To locate the ICA with a transducer probe, it is simplest to initially identify the chosen CCA (i.e. left or right side of the head) which is situated toward the base of the neck, in the transverse plane.  19 The CCA runs adjacent to the internal jugular vein (IJV), though these two vessels can be distinguished by applying light pressure; the IJV is compressible where the CCA is not. Once located, the bulb and bifurcation of the CCA (where it branches cephalad into the ICA and external carotid artery [ECA]), can be found by running the probe up along the neck toward the head. Rotating the probe 45° clockwise will bring the bulb, and branching ICA and ECA into a sagittal plane. From here, it is important to differentiate between the ICA and ECA to obtain the correct imaging scan (i.e. the ICA in this study) using the following steps proposed by Thomas et al. (2015); (i) In 95% of subjects, albeit in adults, the ICA is typically located posterior-laterally as opposed to the ECA which is positioned anterior-medially (Prendes et al., 1980). Pivoting the probe into a posterior-lateral position within the region of 10 to 40 ° should bring the ICA into longitudinal view. (ii) The ICA is usually larger in diameter (~6 mm) at the bifurcation compared to the ECA (~3-4 mm; Brandt, 2001). (iii) The ECA has several extracranial branches which can be visualized using color Doppler, while the ICA has none (Primozich, 2002) (iv) ICA and ECA spectral Doppler waveforms are different (see Figure 1.6) and (v) temporal tapping distorts an ECA waveform, whereas it does not always distort an ICA waveform. This is the least reliable method of distinction thus should be used with cation. Flow near the bulb is typically turbulent, thus it is prudent to ascertain measures of velocity and diameter at least 2 cm distal to this (Thomas et al., 2015).    Figure 1.6     Spectral Doppler trace differentiation between the ICA (A) and ECA (B). The ICA is a low-resistance waveform (gradual upstroke, broad systolic peak, continuous forward flow and high end-diastolic velocity) (A). The ECA is a high-resistance waveform (sharp upstroke, narrow systolic peak, low end-diastolic velocity) (B). Reproduced from Thomas et al, 2015 - ©permission not required.    20 The VA can also be located from a longitudinal view of the distal CCA. Pivoting the probe laterally (i.e. toward the ipsilateral ear) will bring sections of the VA into view, located deeper and more laterally than the CCA and IJV (Thomas et al., 2015). Since the VA runs between the vertebral processes, it can only be seen in sections (see Figure 1.7)   Figure 1.7     B-mode image of the vertebral artery. Viewed between the vertebral processes, deep to the common carotid (not in view) and internal jugular vein (IJV). Reproduced from Thomas et al, 2015 - ©permission not required.     Quantification of volumetric CBF    Doppler shift frequency is calculated using the following equation:   𝐷𝑜𝑝𝑝𝑙𝑒𝑟	𝑠ℎ𝑖𝑓𝑡	𝑓𝑟𝑒𝑞𝑢𝑒𝑛𝑐𝑦 = 𝑓𝑟 − 𝑓𝑡 = 2𝑣𝑓𝑡	𝑐𝑜𝑠𝜃	 ∙ 𝑐  Where ‘fr’ represents the received frequency, ‘ft’ represents the transmitted frequency, ‘v’ represents velocity of the blood and ‘c’ represents the speed of sound in the tissue. By rearranging the above equation, CBF velocity is calculated on the principle that the speed of the red blood cells traveling through the vessel lumen is directly proportional to the magnitude of Doppler shift:  𝑣 = 𝑓𝑑	𝑐	 ∙ 2𝑓𝑡	𝑐𝑜𝑠𝜃  Volumetric CBF can then be calculated by multiplying the velocity of the blood by the cross sectional area of the vessel (obtained from the analysis software as discussed in section  21 Analysis software). Cross sectional area is calculated as follows:  𝐶𝑟𝑜𝑠𝑠	𝑠𝑒𝑐𝑡𝑖𝑜𝑛𝑎𝑙	𝑎𝑟𝑒𝑎 = 	𝜋 0.5 ∙ 𝑑 2  Where ‘d’ represents the vessel diameter. Once velocity and cross sectional area have been obtained, volumetric CBF can be calculated as follows (Evans, 1985):   𝑣𝑜𝑙𝑢𝑚𝑒𝑡𝑟𝑖𝑐	𝐶𝐵𝐹 = (𝜋(0.5	 ∙ 𝑑)2) · ((1/2)(vmax))  Where ‘vmax’ represents the vessels maximum blood velocity.     Analysis software   Ultrasound video files are recorded, saved and stored for offline analysis using a custom-designed edge-detection and wall-tracking software (FMD/BloodFlow Software Version 4.0). Before this level of software materialized, sonographers would manually measure diameter and blood flow responses which was understandably confounded by observer bias and/or human error. On the other hand, the reliability and validity of the FMD/BloodFlow analysis software has been strongly advocated for the assessment of arterial diameter (Woodman et al., 2001) and blood flow responses (Black et al., 2008).   Individual files are uploaded to the FMD/BloodFlow program and the video is replayed on loop. Following an adapted set of procedures from Woodman et al. (2001), first diameter and velocity must be calibrated. A region of interest (ROI) is drawn around the B-mode depth scale and pulsewave velocity scale (i.e. known values) for which the software can standardizes against. An ROI can then be drawn over the entire waveform (for velocity determination) and most stable section of the arterial vessel (for arterial diameter determination). Subsequently, the software automatically detects peak envelope velocity and arterial diameter which is used in the calculation of volumetric CBF.     22     Methodological considerations  The analysis procedures for acquiring diameter, velocity and volumetric CBF are of modest difficulty, though the actual acquisition of adequate ICA and VA video images requires a highly proficient and trained sonographer. The duplex ultrasound machine has many complex settings which need to be adjusted to ascertain the most optimal image rendering this technique as highly operator-dependent. Previous literature has lacked conformity, with varying methodological protocols hindering the ability to draw comparisons between studies. Subsequently, Thomas et al. (2015) have recently released the first comprehensive set of technical guidelines for best practice in extracranial blood flow measurement and it is prudent that studies abide to these guidelines to ensure the best quality research. Nevertheless, these recommendations are intended specifically for adults. Some aspects may not be entirely accurate in children, such as anatomical locations and dimensions, thus exaggerated caution must be given to imaging the correct artery.   1.4.4     CBF in children  The child brain utilizes an astonishing 66% of resting metabolic rate (RMR) and 43% of the body’s daily energy requirement (DER) during periods of advanced growth (Kuzawa et al., 2014). Interestingly, this peak in total glucose uptake as a percentage of RMR or DER (glucoseRMR% and glucoseDER%, respectively) does not occur at birth, when the brain is at its largest size relative to body mass. Instead, peak glucoseRMR% and glucoseDER% is documented to occur between 4.2-4.4 years (Kuzawa et al., 2014; see Figure 1.8).    23  Figure 1.8     Glucose use of the human brain by age. (A) Grams per day in males. (B) Grams per day in females; dashed horizontal line is adult value (A and B). Peak daily glucose uptake by the brain (167 g/day and 146.1 g/day in males and females, respectively) occurs at 5.5 years of age; 1.88 and 1.82 times greater than the average daily glucose uptake in adults. GlucoseRMR% (solid line) and glucoseDER% (dashed line) in males (C) and females (D). Reproduced from Kuzawa et al., 2014 - ©permission received.  Tight regulation of CBF is therefore paramount for supplying the brain with the oxygen, glucose and nutrients needed to facilitate neural activity and ATP production via oxidative metabolism (Ainslie et al., 2016), particularly during key growth periods. In fact, the child brain is so demanding that glucoseRMR% and glucoseDER% are inversely related to body growth throughout maturation, implying that the high cost of brain development induces a compensatory reduction of body growth rate (Kuzawa et al., 2014; see Figure 1.9)    24  Figure 1.9     GlucoseRMR% and body-weight growth rate. GlucoseRMR% and weight velocities plotted as SD scores to allow unit-less comparison. GlucoseRMR% (red dots) and body-weight growth velocity by age (blue dots) in males (A) and females (B). Note: GlucoseDER% also follows the same pattern. Adapted from Kuzawa et al., 2014 - ©permission received.  The first documentation of CBF velocity in children was indirectly obtained by Harald Bode whilst reviewing the application of TCD use in children in 1988. MCA, ICA and ACA CBF velocity was commendably obtained in over 500 children (0-18 years old). They report that MCA velocity progressively increases, peaks around 6 years old (92 cm.s-1), and is then followed by a slow decline into adulthood. ICA velocity appears to peak slightly earlier at approximately 3-4 years old (98 cm.s-1), whilst in the ACA, peak velocity remains stable and plateaus around 60 cm.s-1 between 1-5 years which is followed by a gradual decline (Bode, 1988).   In 1996, Schöning & Hartig conducted one of the first large scale, non-invasive studies to assess developmental changes in global CBF (sum of flow volumes in both sides of the ICA and VA; gCBF) using duplex ultrasound. In 94 healthy individuals aged 3-18 years, gCBF was reported to increase from 687 ± 85 ml.min-1 to 896 ± 110 ml.min-1 between the age of 3-6.5 years, thereafter declining to adult levels (~700 ml.min-1) by 15 years of age. Furthermore, Takahashi and colleagues (1999) more invasively measured regional CBF, regional cerebral metabolic rate for oxygen (CMRO2) and regional oxygen extraction fraction (OEF) using the 15O steady-state inhalation method with C15O2, 15O2 and 15CO, respectively, and positron-emission tomography. Of the 24 children studied (aged 10 days to 16 years), regional CBF in all areas of the brain peaked around 7 years of age, similar to the previous findings from Schöning & Hartig (1996). Changes in regional CMRO2 did not occur simultaneously with changes in regional CBF, which continued  25 to gradually increase into adolescence whilst regional OEF was comparable throughout childhood (Takahashi et al,. 1999). Thus, peak CBF velocity during childhood is consistent with the increase in metabolic glucose and O2 demands of cerebral development (Takahashi et al., 1999; Kuzawa et al., 2014).     Sex differences in CBF   Sex differences in cerebrovascular function are apparent at a young age. Tontisirin and colleagues (2007) examined intracranial CBF velocity in 48 healthy children (24 boys, 24 girls) aged between 4 and 8 years. Although there were no sex differences in autoregulation, girls had greater MCA (99 vs 91 cm.s-1) and BA (70 vs 61 cm.s-1) velocities compared to boys. These differences persist throughout adolescence (Vavilala et al., 2005) in both vessels (MCA: girls, 87 cm.s-1 vs boys, 75 cm.s-1; BA: girls, 60 cm.s-1 vs boys, 50 cm.s-1) and continues into adulthood (Bakker et al., 2004). These observations have been speculated to derive from sex related differences in cerebral metabolic rate and/or cerebrovascular resistance (Tontisirin et al., 2007), supported by predominately higher glucoseRMR% and consistently higher glucoseDER% in girls compared to boys (Kuzawa et al., 2014).   Since CBF velocity is often used as a surrogate of CBF, the aforementioned data would elude towards girls having higher CBF than boys, as is the case in adults (Liu et al., 2012). However, recent advancements using arterial spin labeled MRI in 922 youths (8 to 22 years old) have provided more detailed insight into the maturation of CBF in girls and boys. Satterthwaite and colleagues (2014) have reported that cerebral perfusion is not uniformly higher in girls throughout the entire developmental trajectory. Instead, up until the age of ~13 years, boys had higher CBF than girls. This is followed by an age-related CBF decline in boys, and an age-related increase in CBF in girls, accentuating a mid-puberty sex divergence (Figure 2.0; Satterthwaite et al., 2014). The increase in CBF in girls has been speculated to coincide with increased estrogen levels, whilst the increase in testosterone (which may attenuate vascular dilation) could be linked to the progressive declines in CBF observed in boys (Gonzales et al., 2004; Satterthwaite et al., 2014). When the girls and boys were aligned by pubertal stage, CBF values were similar and declined in a similar manner early puberty. By mid-puberty there is marked sex-divergence with a higher CBF  26 velocity in girls, likely explained by a smaller arterial diameter.    Figure 2.0     Developmental pattern of CBF change in males (blue) and females (pink) by age, in multiple regions within the heteromodal association cortex. Whereas CBF values declines in males until late adolescence, CBF in females declined until midadolescence but increased thereafter. Reproduced from Satterthwaite et al., 2016 - ©permission received.  1.4.5     Regulation of CBF  The brain reserves a very limited intra-cellular storage capacity for energy, which is remarkable given that it is the most metabolically demanding organ in the body relative to size (Kety & Schmidt, 1946). Precise regulation of CBF is therefore crucial to continually match this demand and maintain adequate perfusion, for in the extreme case, cessation of CBF will induce irreversible damage to brain function within minutes (Hossmann, 1994). Three principle mechanisms responsible for CBF regulation, which have been measured in children, are (1) PaO2 and PaCO2 (i.e. arterial blood gasses; Levene et al., 1988; Gavlak et al., 2013; Leung et al., 2016), (2) cerebral metabolism (i.e. neurovascular coupling [NVC]; Rosengarten et al., 2003; Groen et al., 2012) and (3) cerebral perfusion pressure (i.e. cerebral autoregulation; Vavilala et al., 2003).    27     Regulation by arterial gasses  To protect against acid base balance fluctuations, the adult brain is exquisitely sensitive to the smallest changes in PaCO2. Thus, reductions in PaCO2 (hypocapnia) cause a decline in CBF to conserve what little PaCO2 remains, and elevations in PaCO2 (hypercapnia) cause an increase in CBF to expel excessively high levels of PaCO2 (Ainslie & Duffin, 2009; Willie et al., 2012). This capacity of the cerebral vessels to dynamically dilate or constrict in response to oscillations in arterial blood gases is termed cerebrovascular reactivity (CVR). Maintaining homeostasis in the pediatric brain is paramount for development, hence, a 44% increase in CBF velocity per 7.5 mmHg rise in PaCO2 is evident within just 24 hours of birth (Levene et al., 1988). Moving forward, the developmental trajectory of CVR to CO2 in children of broader age range has only recently been characterized. Using arterial spin labeling and blood-oxygen level-dependent MRI, Leung et al. (2016) measured CVR in 34 volunteers aged between 9 and 30 years old. Using an end-tidal system to alternate 60 second periods of normocapnia (PETCO2 = 40 mmHg) with 45 second periods of hypercapnia (PETCO2 = 45 mmHg), age-dependent changes in CVR to CO2 were observed, increasing throughout childhood and adolescence, and then decreasing into adulthood (see Figure 2.1). Although speculative, it is likely that the brains high metabolic and O2 demand during cerebral development (and thus increased CBF) attenuates the ability to further increase CBF on demand in response to hypercapnia.  Cerebrovascular responses to variations in PaO2 are also evident, though this response is less potent than that of PaCO2. In adults, an appreciable increase in CBF (to adequately oxygenate the brain) does not occur until PaO2 is reduced below ~50 mmHg (SpO2 <80%; reviewed in Willie et al., 2014; Hoiland et al., 2016). Limited data exists on the cerebrovascular response to hypoxia in the child. The first study to address this, which eludes towards some similarities in responses between children and adults, will be discussed in more detail in section 1.5 Cerebrovascular response to hypoxia in children. Alternatively, high levels of PaO2 (hyperoxia) induce mild reductions in CBF velocity from birth (Niijima et al., 1988) throughout adulthood (Willie et al., 2012).    28  Figure 2.1     Cerebral blood flow (CBF, black diamonds) and cerebrovascular reactivity to CO2 (CVR, white squares) values in grey matter in participants separated by age. Data were adapted from Biagi et al., 2007 and Leung et al., 2016. Reproduced from Ellis and Flück, 2016 - ©permission received.     Regulation by cerebral metabolism  Neurovascular coupling (NVC) is the mechanism by which increases in local neural activity of the brain (and therefore the demand for glucose and O2) are met with equivalent increases in CBF (Mosso, 1880; Aaslid, 1987; Willie et al., 2011b). This coupling of perfusion and metabolic need is essential for protecting the brain against dysfunction. The extent of CBF increase, and the location to which CBF is directed (lateralization) is indicative of specific neural stimuli, such as emotional stimulation (Mosso, 1880), visual task (Rosengarten et al., 2003) or exercise (Willie et al., 2011b). Although changes in neural activity and CBF are clearly correlated with one another, the actual mechanisms underlying this response have not been fully elucidated. Roy and Sherrington (1890) initially suggested that CBF was directly regulated by the energy demand of the brain, though this concept has since been deemed a little oversimplified. Instead, a complex feedforward mechanism involving the coordination of neurons, glia and vascular cells is likely responsible for regulating NVC, with a large controlling influence from astrocytes (reviewed in Attwell et al., 2010).   Cerebrovascular function in relation to metabolism has been investigated in children using several different invasive techniques, such as MRI and positron emission tomography. More recently, the  29 use of transcranial Doppler ultrasound (which was discussed in section 1.4.3 Measurement of CBF) has been advocated as a favorable, non-invasive alternative (Bakker et al., 2014). Briefly, the average increase in intracranial CBF velocity in response to a specific cognitive task can be quantified using this method. Groen et al. (2012) studied sixty healthy children (6-16 years old) and found that the coupling response to language tasks was not influenced by age or gender. Similarly, Rosengarten et al. (2003) found no differences in NVC using visual stimulation between participants aged 10 to 60 years old. In contrast, the strength of response to visuospatial memory function appears to increase with age and is stronger in boys than girls (Groen et al., 2012).     Regulation by cerebral perfusion pressure  Cerebral perfusion pressure (CPP) is the difference between mean arterial pressure (MAP) and intracranial pressure (ICP), which acts as a pressure gradient regulating CBF. The importance of maintaining constant CBF throughout a range of blood pressures was recognized decades ago in adults (Fog, 1937; Fog, 1939), though few studies exist on cerebral autoregulation in children (Udomphorn et al., 2008). Prior to the advancements of accurate CBF measurement, fluctuations in MAP anywhere between 60-150 mmHg, and CPP between 50-150 mmHg were believed to have minimal effect on CBF (Lassen, 1959; Paulson et al., 1990), in adults at least. In actuality, proceeding studies have revealed that CBF is much more dynamic than this (Tan, 2012; Tzeng & Ainslie, 2014; see Figure 2.2). When blood pressure is too low (i.e. hypotension), pial arteriolar diameter increases (Fog, 1937) to protect the brain from ischemic injury. By contrast, when blood pressure is to high (i.e. hypertension), pial arteriolar diameter decreases (Fog, 1939) to protect the brain from overperfusion and subsequent breakdown of the blood-brain barrier. Thus, brain function is greatly compromised if the ability to sufficiently autoregulate is impaired.   30  Figure 2.2     Stylized representation of the possible relationship between mean arterial pressure and cerebral blood flow. The left panel represents Lassen’s classic cerebral autoregulation curve. The curve on the right panel shows a more restricted autoregulatory plateau as indicated in recent studies (Tan, 2012). Reproduced from Tzeng & Ainslie, 2014 - ©permission not required.   Autoregulatory index (the speed of response change in MCA CBF velocity to change in MAP) appears to be similar between children (6 months-14 years old) and adults (18-41 years old) during steady state pharmacologically altered MAP (Vavilala et al., 2003). The average autoregulatory index in children was 0.85, and for adults 0.88 (0 being no autoregulation, 1 being perfect autoregulation). This contradicts their earlier finding which reports autoregulatory index to be physiologically lower in healthy adolescents (12-17 years old) compared to healthy adults (25-45 years old; Vavilala et al., 2002). Though this observation was tested during transient hypotension (i.e. dynamically measuring autoregulation via a thigh cuff) which is less robust, lacking control of both the degree and duration of hypotension following cuff release.   1.5     Cerebrovascular response to hypoxia in children  In adults, a reduction in PO2 below ~50 mmHg will trigger hypoxic cerebral vasodilation to increase CBF in the VA and ICA and maintain O2 delivery to the brain (Masamoto & Tanishita, 2009). When PO2 is further reduced to ~25 mmHg, these vessels have a remarkable ability to double resting CBF (Johnston et al., 2003). The cerebrovascular response to hypoxia in children however, is poorly understood.   Rapid fR dramatically reduces PaCO2 in the brain. Hyperventilation is not uncommon during  31 hypoxia and in turn causes hypocapnic cerebral vasoconstriction to protect the brain from further disturbances to the acid base balance (Dempsey & Forster, 1982). Since children rely predominantly on increased fR during hypoxia (Kohler et al., 2008) it could be postulated that they may experience exaggerated hypocapnic cerebral vasoconstriction. Furthermore, as previously mentioned, cerebral perfusion is 30-50% greater in children than adults (Leung et al., 2016). Although this helps to adequately supply the brain with a greater amount of oxygen and nutrients during the key developmental periods of growth, this greater perfusion is reflected in a reduced cerebrovascular reserve to increase CBF in response to hypercapnia. If the VA and ICA response to hypoxia follows a similar trend, it could be postulated that children may have a reduced cerebrovascular reserve to increase CBF during this stimulus also. This is problematic in maintaining O2 delivery to the brain and could explain why children typically report higher AMS scores than adults (Kohler et al., 2008).  The only study to date that has assessed the cerebrovascular response to hypoxia in children has partially contended this theory. Gavlak and colleagues (2013) measured CBF velocity in the anterior cerebral (MCA and ACA) and posterior cerebral circulation (BA and PCA) using TCD. Data was collected at sea level, 1300m and 3500m in nine children (8.8 ± 2.4 years) undertaking a 5-day ascent on a family trekking holiday in Nepal. Whilst they report no change in posterior circulation, a significant increase in left MCA (29%) and ACA (left, 65%; right, 109%) was observed from baseline to 3500m. Thus, during reductions in O2 availability, children seemingly display the capacity to increase anterior circulation, which is preferentially favored over posterior flow. Interestingly, the opposite response is observed in adults. Following isocapnic hypoxia, a drop in SpO2 to approximately 80% is associated with a 50% greater increase in the posterior circulation (indexed by the VA) compared to the anterior circulation (indexed by the ICA; Willie et al., 2012). Whilst this study marks the forefront of hypoxic cerebrovascular physiology in children, TCD as previously mentioned, is confounded by the inability to account for possible changes in arterial diameter.       32 1.6     Aims and hypotheses  In summary, many important aspects of hypoxic ventilatory and cerebrovascular physiology in children remain largely or entirely speculative. It is unclear from previous research whether the ventilatory response to hypoxia is developmentally mediated or not, and very little is known in the way of acute, short term exposure. Furthermore, extracranial CBF responses to hypoxia in children are unknown. Further research using Duplex ultrasound is needed to clarify whether the contrasting intracranial CBF distribution (obtained using TCD) between children and adults is accurate, or whether this observation was confounded by diameter change. Given the documented sex differences in CBF (Tontisirin et al., 2007) and ventilation (Aitken et al., 1986), we chose to limit our comparison to girls and their mothers. Since both the ventilatory and cerebrovascular systems are closely integrated, they should be measured in consort to tease out inclusive and important insight into the child’s physiological ability to contest low levels of O2.  Hence, the aim of the following study was to determine the ventilatory (VE, VT/TI), respiratory gas exchange and cerebrovascular (extracranial blood flow and vasodilation of the ICA and VA) responses to acute normobaric hypoxia in girls and women, and explore relationships between changes in CBF, SpO2 and PETCO2. We hypothesized that following one hour of normobaric hypoxia (i) the magnitude of change in VE and VT/TI would be similar between girls and women, (ii) elevations of CBF in girls would be mediated via greater elevations in flow in the ICA versus VA, compared to greater elevations in flow in the VA in their biological mothers and (iii) that increases in CBF would be correlated to declines in SpO2 and PETCO2.          33 Chapter 2 Cerebrovascular and ventilatory responses to acute normobaric hypoxia in girls and women  Laura E. Morris, Daniela Flück, Philip N. Ainslie and Ali M. McManus   Centre for Heart, Lung and Vascular Health, School of Health and Exercise Sciences,  University of British Columbia, Kelowna, Canada    2.1     Rationale          In adults, exposure to acute hypoxia results in rapid declines in the partial pressure of arterial oxygen (PaO2), a compensatory increase in ventilation (VE) and, as a consequence, declines in the partial pressure of arterial carbon dioxide (PaCO2) (Weil et al., 1970). These changes are closely coupled with the regulation of cerebral blood flow (CBF), such that when PaO2 falls below ~50 mmHg, at least in adults, cerebral vasodilation occurs and perfusion is augmented (Willie et al., 2012; Lewis et al., 2014; Hoiland et al., 2016). This vasodilatation occurs throughout the cerebrovascular tree, from the large extracranial and intracranial conduit arteries (e.g., the internal carotid [Willie et al., 2012] and middle cerebral arteries [Wilson et al., 2011; Imray et al., 2014]), to the arterioles in the pial mater (Wolff et al., 1930). Evidence of the ventilatory and cerebrovascular responses to hypoxia in children is sparse (Kohler et al., 2008; Gavlak et al., 2013), with much of our understanding extrapolated from adult data. This is problematic because of documented developmental differences in the ventilatory (Gratas-Delamarche et al., 1993) and cerebrovascular responses of the child (Schöning & Hartig, 1996).   Evidence that the ventilatory response to hypoxia is dependent on age is mixed. Using an isocapnic progressive hypoxic technique, no differences in the hypoxic ventilatory response was noted from 7 to 18 years of age (Honda et al., 1986).  In contrast, using the same isocapnic rebreathe technique, Marcus and colleagues (1994) reported a significantly increased hypoxic ventilatory response in children compared to adults; however the relationship with age was weak (r=0.34). Following one day at 3450m, an increased respiratory drive (VT/TI; VT, tidal volume; TI, inspiratory timing) and VE was found in both children and adults suggesting that ventilatory responses to hypoxia are  34 independent of age (Kohler et al., 2008). Further clarification is needed to better understand how age related differences (present or not) may affect ventilatory responses to an acute bout of hypoxia.  Cerebral perfusion shows distinct developmental patterns, peaking between the ages of 5 to 10 years, with values 30-50% greater than in adults, and a higher cerebral blood flow in girls compared to boys (Leung et al., 2016). This greater perfusion in children is reflected in a reduced cerebrovascular reserve, as indicated by an attenuated cerebrovascular vasodilation to hypercapnia (Leung et al., 2016). There is very limited data on the cerebrovascular response to hypoxia in the child, with one study demonstrating an increase in perfusion in the anterior cerebral circulation (indexed by increased middle and anterior cerebral artery blood velocity), but not in the posterior cerebral circulation (indexed by the basilar and posterior cerebral arteries) in children aged 6-13 years who completed a 5-day ascent to 3500m (Gavlak et al., 2013). A limitation of this approach is the assumption that vessel diameter is unchanged; however, since hypoxia may lead to dilation of the cerebral arteries (Wilson et al., 2011) this would result in an underestimation of CBF (Ainslie & Hoiland, 2014; Hoiland & Ainslie, 2016). In adults, increases in global cerebral perfusion in response to normobaric isocapnic hypoxia from about 80% oxygen saturation (SpO2), were a consequence of a greater increase in the posterior circulation (via the vertebral artery [VA]) compared to anterior flow (via the internal carotid artery [ICA]) (Willie et al., 2012; Hoiland et al., 2017). Regional CBF changes in response to acute hypoxia in the healthy child is unknown.  The purpose of this investigation therefore was to determine the ventilatory (VE, VT/TI), respiratory gas exchange and cerebrovascular (extracranial blood flow and vasodilation; ICA and VA) responses to acute normobaric hypoxia in girls and women, and explore relationships between changes in CBF, SpO2 and end-tidal carbon dioxide (PETCO2). Given the documented sex differences in CBF (Tontisirin et al., 2007) and potential hereditary influences on the cardiorespiratory response to hypoxia (Kriemler et al., 2015) we chose to limit our comparison to girls and their biological mothers. We hypothesized that following one hour of normobaric hypoxia (i) the magnitude of change in VE and VT/TI would be similar between girls and women, (ii) elevations of CBF in girls would be mediated via greater elevations in flow in the ICA versus VA, compared to greater elevations in flow in the VA in women and (iii) that increases in CBF would  35 be correlated to declines in SpO2 and PETCO2.  2.2     Methods  2.2.1     Participants  Ten healthy pre- and early-pubertal girls (9.9 ± 1.7 y) and their biological mothers (43.9 ± 3.5 y) were recruited. All participants were born, raised and resided at low altitude with no history of cardiorespiratory, circulatory or metabolic disease. All girls were classified as Tanner stage 1 or 2 by parental assessment of Tanner stage (Rasmussen et al., 2015; Appendix A). Informed consent was obtained from parents, and written assent obtained from children. Ethical approval was granted by the Clinical Research Ethics Board (H16-00855).  2.2.2     Procedures  Girl-mother pairs attended the laboratory once for 2.5 hours, located in Kelowna (344m). The girls and their mothers were tested together to help reduce any anxiety the child may have felt. Participants were asked to refrain from vigorous exercise and caffeine 12 hours prior to arrival, and visit at least 2 hours postprandial. After familiarization with the experimental procedures, initial anthropometric measurements were taken, followed by five minutes of rest, after which baseline normoxic SpO2, heart rate (HR), VE, VT/TI and PETCO2 were assessed. Diameter and flow of the ICA and VA were recorded using Duplex ultrasound. Participants then entered an air tight normobaric hypoxic chamber for 1 hour and all measures were repeated following 5 (5 HYP), 30 (30 HYP) and 60 (60 HYP) minutes of hypoxic exposure, with the exception of ICA and VA diameter and flow, which were assessed after 60 minutes only. Rating of acute mountain sickness (AMS) and cerebral symptoms were completed after 1 hour of hypoxic exposure before leaving the chamber. The fraction of inspired oxygen within the chamber was continuously sampled using a gas analyzer (ML206 Gas Analyzer, ADInstruments, Colorado) and the chamber was maintained at 12.58 ± 0.1% to simulate ~4000m altitude.    36 2.2.3     Primary measures  Respiratory function: Ventilation (breathing frequency [fR] and VT), VT/TI and PETCO2 were collected continuously for five minutes, by sampling breath-by-breath volumes and gas concentrations at the mouth using an online metabolic gas-analysis system (Oxycon Pro, Care Fusion, Hoechberg, Germany). The children and adults wore a mouthpiece and nose clip and flow volumes were measured with a low deadspace (40 mL) turbine. The practicality of using the equation ‘VT/TI’ to measure the drive to breathe has been advocated in children, and is directly related to mouth occlusion pressure – a more direct, but invasive measure of respiratory drive (Gaultier et al., 1981). Prior to measurement at normoxic baseline or hypoxia, the digital turbine volume sensor was calibrated with a 3 litre syringe and the gas analyzer was calibrated with a known concentration of gas. Data were converted from breath by breath to second-by-second over the 5-minute collection period and volumes were expressed as a ratio standard with body mass.  Extracranial blood flow: Volumetric blood flow of the right ICA and VA was measured using a 10MHz multi-frequency linear array probe and a high-resolution ultrasound machine (Terason 3000, Teratech, Burlington, MA, USA), while participants lay supine. B-mode imaging and pulse-wave velocity was optimized to obtain arterial diameter and blood flow velocity, respectively. We report an intra-tester reliability of 9.68%. All scans were recorded over at least 10 consecutive cardiac cycles, using custom-designed edge-detection and wall-tracking software to determine diameter and flow, as described in depth elsewhere (Thomas et al., 2015). All scans were analyzed blinded by two researchers. Results were compared, and where discrepancy occurred, scans were reanalyzed for agreement. Global cerebral blood flow (gCBF) was calculated using the following formula:  gCBF= 2 (ICA flow + VA flow)  2.2.4     Secondary measures  HR was assessed using telemetry (Polar T31, Polar Electro Oy, USA) and SpO2 by pulse oximetry (MD300K1 Pulse Oximeter, VacuMed, California). The Lake Louise Sickness Score (LLSS;  37 [Roach et al., 1993]; Appendix B) was used to assess altitude sickness symptoms. The category ‘difficulty sleeping’ was removed since participants did not sleep. A score ranging from 3-5 was considered mild AMS and any value exceeding this was considered severe. Participants also rated any cerebral headache they experienced on a scale from 0-100; 0 being no headache at all and 100 being the worst headache imaginable on the cerebral specific section of the environmental symptoms questionnaire (ESQ-CS; [Sampson et al., 1983]; Appendix C).  2.2.5     Data and statistical analysis  Descriptive data were expressed as means and SD.  Ventilatory, respiratory, HR and SpO2 responses to normobaric hypoxia were assessed using time (baseline, 5, 30 and 60 minute exposure) by age repeated measures analyses of variance (RM ANOVA). Where baseline values differed by age percentage change from baseline was calculated and used in the analyses. Extracranial blood flow and diameter were also examined using time (baseline and 60 minute) by age (girls, women) RM ANOVA; however, when percentage change from baseline was calculated a one-way ANOVA was used to compare the hypoxic response. Simple effects using t-tests were used to deconstruct main effects and interactions from the RM ANOVA where necessary. Statistical significance was set a priori at P £ 0.05. All statistical analysis was performed using SPSS (Statistical Package for Social Sciences).  2.3     Results  Participant characteristics are presented in Table 1. Eight of the ten girls were classified as Tanner stage 1 and two were Tanner stage 2. As expected, height and weight were greater in the women.  Of the ten girl-mother pairs, two children had incomplete ventilatory data; and, in one adult, ICA measures were not adequately obtained following 60 HYP.       38 Table 1. Descriptive characteristics. Data are mean (±SD).  Girls (n=10) Women (n=10) Child-Adult Difference Age (y) 9.9 (1.7) 43.9 (3.5) P < 0.05 Height (cm) 141.2 (11.3) 167.8 (6.3) P < 0.05 Weight (kg) 34.8 (6.2) 62.4 (11.7) P < 0.05  No significant change in HR was observed in either group (see Figure 2.3, panel a). SpO2 declined with increasing hypoxia (F(3,54)=74.049, P<0.01) similarly in girls and women (see Figure 2.3, panel b).   Figure 2.3     Heart rate (panel a) and arterial oxygen saturation (panel b) at baseline (BL) and following 5 (5 HYP), 30 (30 HYP) and 60 (60 HYP) minutes of hypoxia in girls (white circles) and women (black squares). * within subject change from baseline in girls, P < 0.05; ≠ within subject change from baseline in women, P < 0.05; # child-adult difference, P < 0.05.  There was no difference in LLSS reporting’s for girls (3 ± 2) or women (2 ± 2). Three girls presented with mild and one with severe AMS and four women presented with mild AMS. The ESQ-CS scores were also similar for girls (19 ± 27) and women (18 ± 24).     39 2.3.1     Ventilation  VE (D%) increased during hypoxia (F(1.897,30.345)=8.557, P<0.01), in a similar manner in girls and women (see Figure 2.4, panel a). Initial exposure to 5 HYP elicited increases of 25% in girls, although not significantly greater than baseline, whereas at 30 HYP and 60 HYP VE was significantly greater than baseline. In women VE was elevated 17% above baseline at 5 HYP (P < 0.05) which remained elevated at 30 HYP (P < 0.05) before returning toward baseline at 60 HYP. VE normalized to body mass is reported in Table 2.   VT (D%) increased during hypoxia (F(2.026, 32.421)=6.026, P<0.01), but in an age divergent manner (F(2.026, 32.421)=3.616, P<.05; see Figure 2.4, panel b). With initial hypoxia (5 HYP), no difference in VT (D%) was apparent between girls and women; however, with increasing hypoxic exposure VT (D%) remained elevated above baseline in girls, and higher than women, whereas VT declined back toward baseline in women (P’s<0.05). The pattern of change in fR (D%) with hypoxia is shown in Figure 2.4, panel c. The response was highly variable and as such neither the main effect for hypoxia (P=0.10), nor the interaction with age were significant (P=0.097).   2.3.2    Drive to breathe  VT/TI (D%) increased during hypoxic exposure (F(1.734,27.740)=10.134, P<0.01) similarly for girls and women. In the girls, VT/TI increased by 32% (P>0.05) from baseline to 5 HYP, becoming significant at 30 HYP (41%, P<0.05) and 60 HYP (40%, P<0.05). In women, VT/TI increased by 23% at 5 HYP (P<0.05), remained elevated at 30 HYP, but was not significantly greater than baseline at 60 HYP (Figure 2.4, panel d).    40  Figure 2.4     Relative change from baseline (BL) in ventilation (D%; panel a), tidal volume (D%; panel b), breathing frequency (D%; panel c) and respiratory drive (D%; panel d) following 5 (5 HYP), 30 (30 HYP) and 60 (60 HYP) minutes of hypoxia in girls (white circles) and women (black squares). * within subject change from baseline in girls, P < 0.05; ≠ within subject change from baseline in women, P < 0.05; # child-adult difference, P < 0.05.   2.3.3    Respiratory gas exchange  Declines in PETCO2 with hypoxic exposure (F(1.692,27.075)=86.946, P<0.001) were apparent in both girls and women at all time-points in comparison to baseline (P’s < 0.05; see Table 2).   A main effect for hypoxia was apparent for VO2 (F(3,48)=8.196, P<0.001), but there was no main effect for age (P>0.05) or an interaction (P=0.08; see Table 2). There was no difference between baseline VO2 and initial hypoxia (5 HYP) in girls, with VO2 rising at 30 HYP (P<0.05) and remaining elevated above baseline at 60 HYP (P<0.05).  In the women, VO2 was similar to baseline at all time points. Mean VCO2 was similar to baseline at all time points in both the girls and women.   41 The ventilatory equivalent for VO2 also altered with hypoxia (F(2.026,32.418)=5.849, P<0.01) and in an age specific manner (F (2.026,32.418)=3.310, P<0.05; see Table 2). In girls, VE/VO2 was similar to baseline at 5 HYP, 30 HYP and 60 HYP. In contrast, VVE/VO2 rose in women from baseline to 5 HYP (P<0.05), remaining elevated above baseline at 30 HYP (P<0.05). The ventilatory equivalent for VCO2 was also altered with hypoxia (F(2.127,34.037)=5.849, P<0.01; see Table 2), but in a similar manner in girls and women. VE/VCO2 was elevated above baseline at all time-points in both the girls and women (P’s <0.01).  Table 2. Ventilatory responses at baseline (BL), and following 5 (5 HYP), 30 (30 HYP) and 60 (60 HYP) minutes of hypoxic exposure in girls and women. Data are mean (±SD).    BL 5 HYP 30 HYP 60 HYP VE  (ml.kg.min-1) Girls  Women 241.8 (62.6) 119.6 (20.7)# 295.0 (80.3) 136.7 (19.9)*# 302.6 (78.0)* 139.3 (20.0)*# 295.3 (65.5)* 135.7 (14.3)*# PETCO2 Girls 34.7 (2.9) 30.1 (3.9)* 28.1 (3.1)* 26.6 (2.1)* (mmHg) Women 36.3 (2.7) 32.7 (3.0)* 30.5 (1.7)* 28.3 (1.9)* VO2  Girls 255.0 (52.1) 304.5 (69.6) 355.2 (72.6)* 333.9 (70.4)* (ml.min-1) Women 275.3 (69.2) 276.0 (65.6) 301.4 (58.0) 298.7 (55.6) VCO2  Girls 248.8 (65.2) 267.8 (64.9) 253.6 (50.9) 238.5 (53.7) (ml.min-1) Women 234.0 (67.6) 247.0 (57.2) 223.8 (41.0) 202.1 (26.4) VE/VO2  Girls 31.8 (4.7) 32.3 (3.7) 30.1 (2.8) 29.8 (2.8)  Women 24.7 (8.6) 28.3 (10.3)* 26.3 (9.3)* 26.2 (9.3) VE/VCO2 Girls 32.7 (2.8) 37.0 (5.0)* 39.8 (4.5)* 41.9 (4.4)*  Women 29.0 (10.4) 31.3 (11.4)* 35.2 (12.5)* 37.9 (13.6)* * within subject change from baseline, P < 0.05; # child-adult difference, P < 0.05. Girls, n=8; women n=10       42 2.3.4    Cerebral blood flow   ICA flow increased with hypoxia (F(1,17)= 9.645, P<0.01), but was consistently higher in the girls compared to women (F(1,17)=10.104, P<0.01). When expressed as a percentage change the increase in ICA flow after 60 HYP was similar in girls and women (36% vs. 38% respectively; see Figure 2.5). Elevations in VA mean flow were also noted after 60 HYP (F(1,18)=25.368, P<0.01), which did not differ between groups. In the girls, VA mean flow increased from 86 ± 21 ml min-1 at baseline, to 109 ± 37 ml min-1 at 60 HYP. In women, VA mean flow increased from 81 ± 41 ml min-1 at baseline, to 112 ± 50 ml min-1 at 60 HYP.    Figure 2.5     Relative change from baseline to 60 minutes of hypoxia in internal carotid artery blood flow (D%; panel a) and vertebral artery blood flow (D%; panel b) in girls (white circles) and women (black squares). * within subject change from baseline, P < 0.05.  Global CBF increased with hypoxia (F(1,17)=16.969, P<0.001), in a similar manner in girls and  43 women. When the contribution of extracranial artery flow to gCBF was considered, this was accounted for by ICA flow contributing to 75% of gCBF in the girls, but just 61% in the women at 60 HYP, while VA flow contributed 25% of gCBF in the girls, but 39% in the women (see Figure 2.6). The increase in gCBF was not correlated with the decline in SpO2 (r = -.101, P=.680) or PETCO2 (r = .458, P=.064).  Figure 2.6     Global cerebral blood flow distribution at baseline and after 60 minutes of hypoxia in girls (a) and women (b). The grey bars represent vertebral artery contribution and the white bars represent internal carotid artery contribution. * within subject change from baseline in, P < 0.05.  2.3.5    Cerebral blood velocity and arterial diameter    ICA blood velocity did not change in response to 60 HYP, whilst there was a significant increase in VA blood velocity (F(1,18)=6.252, P<0.05) which was similar between girls and women (see Table 3). In women, VA blood velocity significantly increased from baseline following 60 HYP (P<0.05), though in girls this failed to reach significance. There was a significant main effect for ICA diameter (F(1,17)=8.237, P<0.05) and interaction (F(1,17)=6.824, P<0.05), but not age. In women, ICA diameter significantly increased from baseline following 60 HYP (P<0.05), though in girls this failed to reach significance (see Table 3). VA diameter increased with hypoxia (F(1,18)=40.477, P<0.05) similarly between girls and women (see Table 3).    44 Table 3. Internal carotid and vertebral artery diameter at baseline (BL) and following 60 minutes (60 HYP) of hypoxic exposure in girls and women. Data are mean (±SD).  BL 60 HYP ICA diameter (cm) Girls (n=10) .471 (.066) .506 (.049)  Women (n=9) .419 (.045) .447 (.044) * VA diameter (cm) Girls (n=10) .374 (.040) .410 (.055) *  Women (n=10) .384 (.080) .422 (.080) * ICA velocity (cm.s-1) Girls (n=10) 48.8 (10.8) 56.8 (20.9)  Women (n=9) 37.4 (11.0) 41.4 (14.4) VA velocity (cm.s-1) Girls (n=10) 25.6 (3.7) 26.6 (4.6)  Women (n=10) 21.8 (6.1) 25.4 (7.2) * * within subject change from baseline, P < 0.05.     2.4     Discussion   We report, for the first time in pre- to early-pubertal girls, that cerebral perfusion of the extracranial arteries increases in response to acute normobaric hypoxia. Interestingly, the distribution of gCBF favours ICA flow in the girls, but VA flow in women. Additionally, increases in VE, VT/TI and decreases in SpO2 and PETCO2 are comparable between girls and women, although the pattern of breathing differed, with an increased VT in girls and an increased fR in women. These findings suggest that developmental differences exist in the way increased gCBF and ventilatory parameters are mediated in response to acute hypoxia.  2.4.1    Ventilatory responses to hypoxia in children    Similar to the findings of Kohler and colleagues (2008) in hypobaric hypoxia, we also observe comparable VE and VT/TI responses between girls and women. Although we did not directly assess hypoxic chemosensitivity, our findings provide support that hypoxic ventilatory chemoreflex response is not age dependent. VE following 5 minutes of hypoxia in girls was elevated by an  45 appreciable 25%, though this increase was non-significant. This is likely explained by the large inter-individual variation observed at this time point, since one girl had a much greater increase in VE. Interestingly, the decline in PETCO2 was no more than for other girls, who did not show such a marked increase in VE; most likely because the increase in VE was a result of increased VT as opposed to a more elevated fR. Children are notoriously ‘noisy’ breathers, so observing high inter-child variability is not unusual (Potter et al., 1999). Our baseline PETCO2 data is within normal resting range (Cooper et al., 1987) and although children regulate PaCO2 at a lower set point and breathe with a higher VE/VO2 at rest and during exercise (McMurray et al., 2003), we found no child-adult difference in PETCO2 following acute hypoxia. Additionally, VE/VO2 remained constant in the girls, but increased in the women following 5 and 30 minutes of hypoxia. Theoretically, if hypoxia-induced hyperventilation in the child induced further declines in PETCO2, this may result in hypocapnic-induced cerebral vasoconstriction and consequent attenuation of CBF (Kety & Schmidt, 1946). Yet, we do not observe this; therefore, since the variability of the chemoreflex changes in arterial blood gases can influence CBF (Willie et al., 2014; Hoiland et al., 2016), the comparable changes in blood gases might explain the similar changes in CBF and hence presumably adequate O2 delivery to the brain. Likewise, although directionally similar, the variability within the chemoreflex responses likely underpin the variability in the CBF responses.  An increase in ventilatory stress is primarily reported as an increase in VE, yet, the ability to achieve the same VE with varying contributions of VT and/or fR is often overlooked (Tipton et al., 2017). During exercise (an alternative ventilatory stressor), children typically rely on a greater increase in fR (Benchetrit, 2000) up until approximately 65-70% of maximal capacity, whereby further increases in VE are met with a rise in VT (Rowland & Green, 1990). This increase in VT increases the elastic work of breathing and reduces mechanical efficiency. It was therefore surprising that the girls in this study increased VE solely via an increase in VT in response to hypoxia. By contrast, increasing VE via increased VT can reduce the effect of physiological dead space for a given alveolar ventilation (Sheel & Romer, 2012; Tipton et al., 2017). It could be speculated that the increase in VT (opposed to fR) in girls helps to defend adequate gas exchange in conditions of very low O2. Additionally, a reduction in PaCO2 (which has a constricting effect on the cerebral arteries) is heightened by an increased fR. Instead, preferentially favoring an increase in VT could help to limit the reduction in PaCO2 and may explain why girls demonstrate  46 the ability to increase CBF.   2.4.2    Cerebral hemodynamic responses to hypoxia     Cerebral perfusion is known to be developmentally mediated, and peaks between the ages of 5 to 10 years (Leung et al., 2016). Consequently, these elevations in resting CBF have been linked to a reduced cerebrovascular reserve in response to hypercapnia (Leung et al., 2016), though little is known about the response to hypoxia. Under hypobaric hypoxic conditions, an increase in anterior CBF has been observed (indexed by middle and anterior cerebral blood velocity; [Gavlak et al., 2013]). Likewise, we report significantly elevated anterior flow of the extracranial vessels (indexed by the ICA); however, we also report significant elevations in extracranial posterior flow (indexed by the VA) which was not observed in the intracranial vessels (indexed by basilar and posterior cerebral blood velocity; [Gavlak et al., 2013]). It is possible that the use of transcranial Doppler cerebral velocity has clouded our understanding because of the inability to account for diameter changes. Similar to responses to hypoxia in adults (Wilson et al., 2011), we report significantly increased VA diameter, and although not significant, a trend toward an increase in mean ICA diameter in girls. Thus, despite a potentially lower cerebrovascular reserve, girls still demonstrate the ability to significantly increase both anterior and posterior flow under hypoxic conditions. This response is likely paramount for increasing cerebral O2 delivery, protecting the young brain against otherwise detrimental effects of hypoxia.  In adults, acute poikilocapnic hypoxia causes elevations in both ICA and VA blood flow, though the percentage increase in VA flow (~37%) is much higher than in the ICA (~18%) (Lewis et al., 2014). It is believed that this is likely because the cardiovascular and respiratory control centers located in the brainstem require greater oxygenation and hence an increase in posterior cerebral flow (Ogoh et al., 2013; Lewis et al., 2014). Instead, we report a preferential dependence of increased ICA flow in girls, supporting the observations described by Gavlak et al. (2013) using transcranial Doppler. It has been suggested that a smaller rise in posterior cerebral flow may compromise oxygen delivery to the parieto-occiptal cerebellar brain tissue, which may correspond to symptoms of nausea and dizziness (Gavlak et al., 2013). This may explain why children typically report a higher prevalence of AMS (Kohler et al., 2008), although no difference in AMS  47 was noted between the girls and women in this study, likely due to its short-term nature. Although speculative, it might be that the local metabolic demand (due to development) is less in the brainstem regions in children and hence could explain why the relative increase in VA flow is less when compared to adults. Although under normoxic resting conditions the distribution of anterior and posterior blood velocity in the intracranial vessels has been shown to be similar between children and adults (Schöning et al., 1993). Since both resting diameter and the relative dilation of the VA was similar between children and adults, it would seem these differences are not explained by anatomical influences on vessel structure.  During conditions of isocapnic hypoxia, an appreciable increase in CBF does not occur until PaO2 is reduced below 50 mmHg (SpO2 <80%; reviewed in Willie et al., 2014; Hoiland et al., 2016). What is interesting in the current study is that CBF was still markedly elevated when SpO2 was ~80%, even with evident hypocapnia (PETCO2 ~26 mmHg). Normally this level of hypocapnia alone would be expected to reduce CBF by ~30-40% (assuming a normal hypocapnic reactivity of 2-4% reduction in CBF per mmHg reduction in PETCO2). It is possible that it is reductions in oxygen content and/or hemoglobin deoxygenation (rather than arterial blood gases per se) during poikilocapnic hypoxia, that are mediating elevations in CBF as a way of maintaining oxygen delivery. This topic, and related mechanisms of action, has been recently reviewed in detail elsewhere (Hoiland et al., 2016).   2.4.3    Limitations   We chose to assess hypoxia under normobaric pressure in the interest of participant safety. As such, our findings cannot be directly related to environmental (hypobaric) hypoxia. The physiological effects of normobaric versus hypobaric hypoxia has been a topic of recent debate. SpO2 and VE appear to be lower following short-term exposure to hypobaric compared to normobaric hypoxia (Coppel et al., 2015), which may have led to an underestimation of VE and an overestimation of SpO2 in our study if these results were extrapolated to environmental hypoxia. Similarly, these findings are limited to an acute bout (1h) of hypoxia. Nothing is known of the extracranial blood flow response to longer-term exposure and further investigation is needed to extend our understanding of hypoxic exposure in the child. Menstrual cycle phase in women was  48 not controlled for. The rationale for this approach was to represent the average family, who are highly unlikely to plan a trip to high altitude around the mother’s hormonal schedule. Varying hormonal stages of the menstrual cycle are known to influence VE and VT/TI (Schoene et al., 1981), thus, if we had standardized for menstrual cycle phase, our results may have varied slightly.  This investigation has provided novel insight into the extracranial blood flow and diameter, and ventilatory responses to an acute 1h bout of normobaric hypoxia in children. Significant increases in ICA, VA and gCBF were noted in girls, and VA and gCBF in women, with regional distribution of CBF favouring the anterior circulation in girls and the posterior circulation in women. Furthermore, increases in VE and VT/TI, and declines in SpO2 and PETCO2 were comparable between girls and women, though the increase in VE was mediated via an increase in VT in girls, and an increased fR in women. These results imply that the fundamentals of increasing CBF and ventilatory parameters on exposure to hypoxia are similar between ages, but the way in which these responses are mediated are developmentally divergent.            49 Chapter 3 Conclusion  3.1     The importance of studying the effect of hypoxia in children   Endeavoring to better understand hypoxic physiology in children is paramount in accounting for the safety of those subjected to hypoxemic conditions. In Colorado, a staggering ~150,000 children <12 years of age sojourn to the mountains for ski holidays each year (Yaron & Niermeryer, 2008). Considering that ~25% of Colorado ski resort visitors are reported to develop AMS, thousands of children (as many as 30,000) would supposedly be included in this statistic for Colorado alone (Honigman et al., 1993). Hence, it is particularly worrying that we understand very little of this response. Since this study indicates that children preferentially favor increases in anterior flow, opposed to posterior flow (which would support O2 delivery to the cardiovascular and respiratory control centers of the brain), children may be more susceptible to nausea and dizziness (Gavlak et al., 2013). Thus, children are not exempt from hypoxia induced illness and the same level of research scrutiny should be available in this population, as seen in adults. Characterizing a ‘normal’ hypoxic response in children will help practitioners to prescribe medication or, alongside parents, make the correct judgement for when to descend from high altitude.  This study sought to characterize the physiological response to hypoxia in low altitude dwellers, but there is also considerable utility of this work for high altitude residents. The Tibetan population have successfully adapted to survive in conditions of very little oxygen, serving as an elegant and unique model to study long term adaptation to chronic environmental hypoxia (reviewed in: Gilbert-Kawai et al., 2014). CBF in Sherpa children is lower than in children residing at sea level (Flück et al., 2017). In accordance with unpublished literature on Sherpa adults (Hoiland et al., 2017), this may reflect a cerebral hemodynamic pattern of long term adaptation to chronic hypoxia (Flück et al., 2017). Improved comprehension of this adaptation could help advance the management of many childhood diseases characterized by a chronic lack of oxygen throughout the uptake, transportation and/or utilization pathway (reviewed in: Berger & Grocott, 2017).        50 3.2     Methodological considerations   Working with children is exceptionally difficult, presenting its own set of challenges that rarely need be considered when testing adults. To quantify the effect of acute hypoxia in this study, an in-laboratory chamber was used to simulate ~4000m, and the techniques employed to measure respiratory and cerebrovascular physiology were strictly non-invasive. Whilst we were still able to investigate novel aspects of physiology which have not previously been explored in children using these techniques, they are in-part restrictive. The inherent methodological limitations of such approaches will be discussed below and issues surrounding the scaling of data for differences in body size will be explored.  3.2.1     Laboratory versus field work studies   Two types of protocol – an in-laboratory or field based protocol – must be carefully considered during the conception and design phase of hypoxic investigations in children. Expeditions to regions of high altitude (hypobaric hypoxia; e.g. at the Swiss Jungfrau-Joch research station, Switzerland or Namche Bazaar, Nepal) provide the real-life setting needed to study the pinnacle of high altitude physiology. The mere handful of studies to have performed such feats with children must be commended for the complexity and exquisite planning required to pull something like this off. Nevertheless, a large proportion of these studies have materialized secondary to a pre-planned family trekking holiday (e.g. Scrase et al., 2009; Gavlak et al., 2013), and have been confounded by the unpredictability of human nature. It is not surprising that parents are overly cautious of their child’s wellbeing, with participants returning home due to AMS and homesickness (Kriemler et al., 2015) which greatly affects sample size. The capacity to transport equipment up a mountain and the overall cost of such trips are also fundamental limitations.    On the other hand, in-laboratory based research provides a safer, more controlled setting to study hypoxic responses in children, though this approach is also methodologically flawed. The hypoxic chamber used in this study was incapable of stimulating hypobaric hypoxia, the implications of which have been discussed in section 2.4.3 Limitations. Moreover, exiting the chamber (e.g. for a washroom break) would have mitigated the effect of hypoxia, but being confined to a single space  51 for a prolonged period can be challenging for children. The intention of this study was to address a gap in the literature and assess hypoxia acutely in a controlled setting, however, if we later wish to perform longer-duration studies, the feasibility of using this chamber would be questionable. Both in-laboratory and field work studies are therefore characterized by an individual set of advantages and detriments and while neither appear inherently better than the other, it is important to acknowledge the limitations of both.   3.2.2     Mechanistic limitations    Pediatric literature lacks the same level of experimental physiology seen in adults. For example, pediatric researchers are describing, for the first time, CBF responses to exercise (Ellis et al., 2017), even though this has been the focus of research in adults for decades. Stricter ethics, a lack of non-invasive measurement techniques and fewer pediatric researchers have confined the experimental bounds of pediatric physiology research. The development of Duplex ultrasound over the past 10 years has provided a leading non-invasive measurement of cerebral blood flow, with merit in its accuracy, portability and semi-affordability (compared to MRI). It does however require highly trained personnel. Further exploration of mechanistic physiology in the child is limited because much of this work requires invasive techniques which are unrealistic and arguably unethical in the healthy child. Arterial and venous sample acquisition would be extremely insightful to calculate cerebral oxygen delivery, cerebral metabolic rate for oxygen, oxygen extraction fraction and cerebral delivery of glucose and lactate as demonstrated by Smith and colleagues (2014) in adults. Nevertheless, this is obtained by arterial and jugular venous catheterization which is undisputedly risky and predictably unappealing for most children. This kind of measurement is constrained to pediatric patients and therefore the future of pediatric physiology will require elegant experimental manipulations that make the best use of the non-invasive techniques available.  3.2.3     Scaling for body size    The importance of accounting for body size is vital for the correct interpretation of physiological data, especially when comparing children and adults. Several methods of scaling exist in varying degrees of scrutiny, and despite recommendations for best practice, a lack of conformity is evident  52 within the presentation of data. This amplifies concerns around interpretation and limits the comparisons that can be drawn between studies.   To appropriately compare individuals of very different size (i.e. children and adults), the effect of body size must be removed since the relationship between variables such as VE and VT to anthropometric characteristics (namely mass and stature) has been well-established (Mercier et al., 1991). Ratio scaling (i.e. dividing by body weight) is the simplest and most commonly used method, however, this approach does not appropriately remove the effect of size (Tanner, 1949). Theoretically, dividing the physiological variable e.g., VE by the size variable e.g., body mass, assumes that both variables in question have complete proportionality. This is seldom the case with any physiological variable and has been described as a fallacious and misleading method of correction (Tanner, 1949). Alternatively, allometric scaling is a more thorough method of scaling (Welsman et al., 1996). Like ratio scaling, the physiological variable (y) and body size (x) elements are the same, with the additional incorporation of a body size exponent (b) in the equation ‘y = a · xb’. This can also be achieved using a log-linear approach from analysis of covariance, which also enables a time-varying size covariate to be statistically controlled for and the effect of size appropriately removed. A classic example highlighting the disparity between ratio and allometric scaling has been presented by Mercier et al. (1991). Body mass, height, arm span, lean body mass and body surface area were recorded in seventy-six untrained schoolboys (10.5-15.5 years old) who performed a maximal incremental exercise test to characterize the influence of anthropometrics on maximal VE and breathing pattern during growth. Amongst other variables, allometric scaling indicated that maximum VE, VT and VT/TI increase with age and anthropometric characteristics, whilst maximum fR decreases. This clearly indicates an interaction between exercise ventilation and breathing pattern, yet, when the same data was simply ratio scaled with lean body mass, age was not related to a change in maximal VE, VT or VT/TI. These conflicting conclusions from the same data highlight the impact the scaling approach used is on the interpretation of the findings. Ratio scaling was used for the ventilatory data, and the limitations of this approach are duly acknowledged.   Many researchers and practitioners look for simple approaches to account for differences in size, which likely explains why ratio scaling, although discouraged over 60 years ago, continues to be  53 used. There are also instances where the relationship between the physiological variable of interest and size is poorly understood (e.g. CBF), where reporting delta change is a viable means of comparing two groups that differ at baseline, a difference that may be accounted for by size, or equally by developmentally divergent physiology. This approach enables the relative effect of a stimulus (e.g., hypoxia) to be compared equally between children and adults.     3.3     Future studies  This study has demonstrated for the first time, that children are able to facilitate an increase in ICA, VA and gCBF to meet the augmented oxygen demands during acute hypoxia in a similar manor to adults. Yet, regional distribution of CBF favors an increase in anterior circulation (ICA) in girls, opposed to posterior circulation (VA) in women. The ventilatory response to normobaric hypoxia was similar between the children and women, although breathing patterns differed. Future studies should venture to expand our understanding of the mechanisms regulating regional CBF distributions, and the acclimatization profile of extracranial CBF and ventilatory responses to prolonged hypoxia in the child. Exploring the connection between breathing patterns and CBF responses to hypoxia would also be helpful.  3.3.1     Determining the mechanisms behind regional CBF differences with age    The differences in regional CBF distribution between children and adults during acute hypoxia are not explained by maturational variations in flow distribution (Schöning et al., 1993) or anatomical influences on the vessel structure. Even in adults, the mechanism(s) behind preferentially favoring an increase in posterior circulation are largely unknown, though it makes physiological sense to direct a larger proportion of flow to the important homeostatic regions of the brainstem. Thus, potential mechanisms to explain the anterior CBF increase in girls will be discussed below, alongside the techniques that could be used to elucidate this.   The most plausible explanation as to why girls favor a greater increase in ICA flow (i.e. anterior circulation) is that the smaller VA is simply maximally dilated at ~4000m of simulated hypoxia. To support the 30-50% greater cerebral perfusion (Leung et al., 2016) needed to facilitate  54 exponential brain growth and maturation, the child’s VA may already be functioning close to maximal dilation and/or velocity in normoxia. The physiological capacity of this vessel to accommodate further increases in response to low O2 may therefore be limited (as similarly noted by the CVR to hypercapnia; Leung et al., 2016). If so, a greater percentage increase in ICA flow would help to meet the augmented O2 demand and supply a greater reservoir of blood to the Circle of Willis - which can then be distributed to the intracranial arteries. Establishing true maximal dilation of a vessel is difficult, particularly using non-invasive techniques. Though, advancements could be made to test this speculation by pushing the system further using either a lower FIO2 or a high hypercapnic stimulus (e.g. 8% CO2). If the VA flow we report in this study is in fact representative of a true maximum, the percentage increase in VA CBF would remain the same despite receiving a greater hypoxic and/or hypercapnic stimulus.    An alternative speculation is that the need to supply a greater proportion of blood flow to the homeostatic posterior region is outweighed by the need to protect cognitive function in the young brain. Whilst most brain regions have fully matured by the early age of 5 years (Dekaban & Sadowsky, 1978), the prefrontal cortex undergoes a second wave of growth prior to puberty (Giedd et al., 1999). In an interview with Frontline, Jay Giedd (2002) described the frontal lobe as the CEO of the brain; involved with organization, attention and thinking. His primary hypothesis is the “use it or lose it” principle. It is therefore tenable to suggest that the frontal lobe requires a greater O2 supply to facilitate growth and protect the pruning of cells and connections which may be important for later life. This could explain why pre- to early-pubertal girls preferentially favor an increase in ICA flow during hypoxia. Rimoldi and colleagues (2016) have previously assessed executive function (inhibition, shifting, working memory), memory (verbal short term, verbal episodic, visuospatial) and verbal speed processing in 48 healthy non-acclimatized children and adolescents (13.6 ± 1.7 years old) 24 hours after arrival at 3450m. Significant cognitive impairment was noted in all but visuospatial memory and verbal processing speed function. To our knowledge, a comprehensive battery of neuropsychological assessment has yet to be performed on children and adults in response to hypoxia (i.e. the same ascent profile, timing and form of cognitive measurement). If this speculation is accurate, we would expect to see an even greater attenuation of cognitive function in adults compared to children.    55 It has also been speculated that adenosine A2 receptors may play a role in the regulation of CBF distribution (Lewis et al., 2014). Linked to the control of vascular tone, adenosine A2 receptor concentration is highest in the basal ganglia (albeit in rats; Jarvis & Williams, 1989) which is consequently located posteriorly. It is possible that peak adenosine A2 receptor contribution in the basal ganglia may not fully mature until adulthood which could lead to reduced vascular function in the VA – though this link between rats and humans is weak, with no noninvasive technique to measure this in the child.   3.3.2     Assessment of the acclimatization response to hypoxia in children  Both the CBF and ventilatory acclimatization profile to hypoxia are readily available in adults. Within seconds, an increase in VE is evident (see Figure 2.7), which is followed by a marked reduction in VE within minutes, known as the ‘hypoxic ventilatory decline’. The relative degree of hypoxia upon initial exposure (up to 1-3 days) outweighs any confounding influence of hypocapnia, resulting in an increase in CBF (Ainslie & Ogoh, 2010). Thereafter, a progressive rise in VE is observed (due to ventilatory acclimatization) allowing increases in PaO2 and decreases in PaCO2, which together attenuate CBF (see Figure 2.7).   Figure 2.7     Integrative changes in CBF, haematocrit (Hct), VE, cerebral capillary density and hypoxia-inducible factor-1 (HIF-1) during poililocapnic hypoxic exposure. During acute exposure to poililocapnic hypoxia, the hypoxia-induced hyperventilation and subsequent hypocapnia causes cerebral vasoconstriction, thereby reducing CBF. Ventilatory decline associated with the acute hypoxic exposure coincided with an increase in CBF. During chronic hypoxic exposure, the increase in CBF peaked after 1-2 days followed by a slow and progressive decline towards sea-level baseline. In addition, this decline coincided with a steady increase in basal ventilation and elevation  56 in cerebral capillary density from ~day 4. Furthermore, haematocrit concentration steadily increased with prolonged hypoxic exposure. Modified from Xu & LaManna (2006). Reproduced from Ainslie & Ogoh, 2010 - ©permission received.  At most, there is a report of fR, SpO2 and PETCO2 in children following 9 days spent at 3500m (Scrase et al., 2009). Coupled with our findings that overall ventilatory and CBF responses to hypoxia are comparable between girls and women, this suggests that the early acclimatization process in the child may be analogous with the adult. However, to adequately establish this profile in children, future studies should endeavor to remain at altitude for 2-4 weeks, studying a much more comprehensive set of variables over a continuous period.   Coupled with the inability to reside in the chamber long enough to assess acclimatization, and the financial and logistical strain of traveling to far-off, high altitude regions such as Nepal, other more feasible alternatives are research stations such as the Barcroft Research Station, at White Mountain, California. 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J Cerebr Blood F Met 35, 648-654.  70 Appendices Appendix A: Parental assessment of Tanner staging                                               Girls                                                          Boys                 71 Appendix B: Lake Louise Sickness Score Questionnaire       72 Appendix C: Cerebral Symptoms Questionnaire    Please indicate on the line below how severe your headache is at the PRESENT MOMENT IN TIME 		                   73 Appendix D: Certificate of Ethical Approval       The University of British ColumbiaOffice of Research EthicsClinical Research Ethics Board – Room 210, 828 West 10th Avenue, Vancouver, BC V5Z 1L8ETHICS CERTIFICATE OF EXPEDITED APPROVAL PRINCIPAL INVESTIGATOR: INSTITUTION / DEPARTMENT: UBC CREB NUMBER:Ali McManus UBC/UBCO Health & Social Development/UBCO Health and Exercise Sciences H16-00855INSTITUTION(S) WHERE RESEARCH WILL BE CARRIED OUT:Institution SiteUBC OkanaganOther locations where the research will be conducted:N/A CO-INVESTIGATOR(S):Thomas J. WarshawskiLaura MorrisPhilip Ainslie  SPONSORING AGENCIES:- Natural Sciences and Engineering Research Council of Canada (NSERC) - "Exercise Oxidative Metabolism in Children " PROJECT TITLE:The respiratory response to acute hypoxia in children and adults THE CURRENT UBC CREB APPROVAL FOR THIS STUDY EXPIRES:  May 31, 2017The UBC Clinical Research Ethics Board Chair or Associate Chair, has reviewed the above described research project, including associated documentation noted below, and finds the research project acceptable on ethical grounds for research involving human subjects and hereby grants approval.This approval applies to research ethics issues only. The approval does not obligate an institution or any of its departments to proceed with activation of the study. The Principal Investigator for the study is responsible for identifying and ensuring that resource impacts from this study on any institution are properly negotiated, and that other institutional policies are followed. The REB assumes that investigators and the coordinating office of all trials continuously review new information for findings that indicate a change should be made to the protocol, consent documents or conduct of the trial and that such changes will be brought to the attention of the REB in a timely manner. DOCUMENTS INCLUDED IN THIS APPROVAL: APPROVAL DATE:Document Name Version DateProtocol:Protocol 0.2 May 28, 2016Consent Forms:Consent 0.2 May 28, 2016Assent Forms:Assent 0.2 May 28, 2016Questionnaire, Questionnaire Cover Letter, Tests:May 31, 2016 74  


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