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Cerebral blood flow in heart transplant recipients at rest and during incremental exercise Smirl, Jonathan David 2011

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 i   CEREBRAL BLOOD FLOW IN HEART TRANSPLANT RECIPIENTS  AT REST AND DURING INCREMENTAL EXERCISE   by   JONATHAN DAVID SMIRL    B.Sc., The University of Victoria, 2004    A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF   MASTER OF SCIENCE   in   College of Graduate Studies   (Interdisciplinary Studies)   [Health and Exercise Sciences]    THE UNIVERSITY OF BRITISH COLUMBIA  (Okanagan)    September 2011   © Jonathan David Smirl, 2011   ii Abstract   Pathological impairments to cardiac output may impact cerebral blood flow (CBF). Prior studies on heart transplant recipients (HTR) have reported increases of 25-53% in CBF, 1-6 months following transplant. It is currently unknown if CBF is chronically altered in the years following transplant or during progressive exercise stress, when compared with aged- matched controls (AM).  Donor population controls (DC) were included to determine if the responses observed in HTR are related to the age of the donor rather than the individual.  The aim of this thesis was to examine the influence of long-term heart transplantation on the regulation of CBF velocity (CBFv) at rest and during incremental exercise. Two hypotheses were tested: 1) CBFv would be similar in HTR when compared to AM, but lower than DC; 2) that during incremental exercise, the HTR would have reduced elevations in CBFv compared with AM and DC. To address these hypotheses, HTR were tested who have a reported inability to acutely increase cardiac output during exercise.  Seven male clinically stable HTR (62 ± 9 yrs of age, 9 ± 7 yrs post-transplant), seven male AM (62 ± 7 yrs), and seven male DC (22 ± 3 yrs) were recruited for this study. Bilateral middle cerebral arteries were insonated using transcranial Doppler ultrasound to obtain an index of CBFv.  Data were obtained while seated and during an incremental cycling test to volitional exhaustion. A repeated measures ANOVA was applied to identify differences across exercise intensity. Comparisons between groups were performed with Fisher‟s LSD post hoc test. The main findings were: 1) Rest: CBFv was comparable between HTR and AM (40 vs. 41 cm/s), as expected, CBFv was 68% higher in the DC compared with the HTR and AM   iii groups (P<0.05).  2) Incremental exercise: mean CBFv was not significantly different between the HTR and AM groups across any of the exercise intensities.   In conclusion, the CBFv of long-term HTR are comparable to AM both at rest and during incremental exercise that despite a suppressed VO2 Peak (and likely Q) CBFv is well maintained during incremental exercise in long – term HTR.      iv Preface  This study was approved by the University of British Columbia Clinical Research Ethics Board (H11-02576 – CBF in HTR) and the University of Alberta Clinical Research Ethics Board. (Pro00011560). Chapters three, four and five are based on data collected in the Mazankowski Alberta Heart Institute at the University of Alberta by Dr. Philip Ainslie, Dr. Mark Haykowsky, Dr. Luis Altamirino-Diaz, Dr. Helen Jones, Kit Marsden, Mike Nelson and Jonathan Smirl.  The data were analyzed in the cardiovascular physiology laboratory at the University of British Columbia by Jonathan Smirl.  Dr. Ainslie and Dr. Haykowsky were responsible for the overall design and concept of the study, technical assistance, data collection, equipment acquisition, subject recruitment, and funding for the study.  Dr. Altamirino-Diaz was responsible for the collection and analysis of the echocardiograph images.  Dr. Jones, Kit Marsden and Mike Nelson assisted in the collection of the data.  Jonathan Smirl was responsible for assisting in the overall study design and study concept. Jonathan coordinated the data collection involved in this study, and analyzed all of the data, including statistical analyses and writing.  There has been a version of chapters three, four and five accepted for an oral  presentation at Physiological 2011 (annual scientific meeting of the Physiological Society).  Jonathan D. Smirl, Mark J. Haykowsky, Katelyn R. Marsden, Helen Jones, Michael D. Nelson, Luis A. Altamirano-Diaz, and Philip N. Ainslie. (2011) Cerebral blood flow in heart transplant recipients: rest and during exercise.  Jonathan Smirl was responsible for the data collection, data analysis, writing, and formatting of the abstract.      v  Table of Contents   Abstract…….. ........................................................................................................................... ii Preface…………… .................................................................................................................. iv Table of Contents ..................................................................................................................... v List of Tables .......................................................................................................................... vii List of Figures ........................................................................................................................ viii List of Abbreviations ............................................................................................................... ix Acknowledgements .................................................................................................................. xi Dedication ............................................................................................................................... xii  Chapter One: Introduction and Review of the Literature ...................................................... 1  1.1. Brief Background on Heart Transplantation .........................................................1 1.2. Regulation of CBF ...............................................................................................3 1.2.1.  Neurogenic Control of CBF............................................................................4 1.2.2.  Influences of Cardiac Function on CBF ..........................................................6 1.2.3.  Autoregulatory Control of CBF ......................................................................9 1.2.4.  Regulation of CBF by PaCO2 ....................................................................... 10 1.2.5.  Effects of Cerebral Metabolism on CBF at Rest and During Exercise ........... 11 1.3. Cognitive Impairment in CHF ............................................................................ 12 1.4. Influences of Cardiac Disease on CBF ............................................................... 13 1.4.1. Congestive Heart Failure .............................................................................. 13 1.4.2.  Ischemic Heart Disease ................................................................................ 14 1.5. Changes in CBF in HTR .................................................................................... 15  Chapter Two: Purpose, Aims and Hypotheses .......................................................................22  2.1. Purpose of Thesis ............................................................................................... 22 2.2. Aims .................................................................................................................. 22 2.3. Hypotheses ........................................................................................................ 23  Chapter Three: Methods .........................................................................................................24  3.1. Participants ........................................................................................................ 24 3.2. Instrumentation .................................................................................................. 27 3.3.   Transcranial Doppler Ultrasound........................................................................ 29 3.3.1. Validity of TCD ........................................................................................... 31 3.3.2.  Principle of TCD .......................................................................................... 31 3.3.3.  Technique of TCD........................................................................................ 32 3.4. Incremental Cycling Exercise Protocol ............................................................... 33 3.5. Statistical Data Analysis..................................................................................... 34     vi  Chapter Four: Results .............................................................................................................35  4.1. Participant Characteristics .................................................................................. 35 4.2. Incremental Exercise Test .................................................................................. 35 4.3. Rest.................................................................................................................... 38 4.4. VO2peak ............................................................................................................... 40 4.5. HRreserve Relationships ........................................................................................ 43 4.6. Mild, Moderate and Intense Exercise ................................................................. 45  Chapter Five: Discussion and Conclusion ..............................................................................49  5.1. Principle Findings .............................................................................................. 49 5.2. Influence of HT on CBF at Rest - Comparison with Previous Studies................. 49 5.3. Young Heart, Old Brain: Influence of Aging on CBF ......................................... 50 5.4. Influence of HTR on CBF Alterations During Exercise ...................................... 51 5.5. Influence of Aging on End-Tidal PCO2 .............................................................. 52 5.6. Differential Changes in Systolic MCA Velocity and Pulsatility Index ................ 53 5.7.  Limitations ........................................................................................................ 55 5.7.1. TCD Ultrasound ........................................................................................... 55 5.7.2. Activity Matching ........................................................................................ 56 5.8. Implications ....................................................................................................... 56 5.8.1. MCAv at Rest .............................................................................................. 56 5.8.2. MCAv During Incremental Exercise............................................................. 56 5.8.3. MCAv in HTR With and Without Subject #02 ............................................. 57 5.9. Future Studies .................................................................................................... 57 5.9.1. Effects of Long-Term Endurance Training on CBF in HTR .......................... 57 5.9.2. Longitudinal Study of CBF in HTR .............................................................. 58 5.9.3. Effects of Incremental Exercise on CBF in End-Stage Heart Failure Patients 58 5.10. Conclusion ......................................................................................................... 58  Bibliography ............................................................................................................................59  Appendices ...............................................................................................................................83  Appendix I: Participant Information Sheet ....................................................................... 83 Appendix II: Participant Consent Form ............................................................................ 86 Appendix III: Literature Review of Cardiac Output on Cerebral Blood Flow ................... 87 Appendix IV: Raw Data – Output from LabChart .......................................................... 102    vii List of Tables   Table 3.1. Participant Characteristics (all subjects) ....................................................................25  Table 3.2. Participant Characteristics (subject #02 excluded) .....................................................26  Table 4.1. Cardiovascular, Pulmonary and Cerebrovascular responses at rest and during the incremental exercise test at 50%, 70%, 90% and Peak VO2 Consumption                    (all subjects) ..............................................................................................................36  Table 4.2. Cardiovascular, Pulmonary and Cerebrovascular responses at rest and during the incremental exercise test at 50%, 70%, 90% and Peak VO2 Consumption                (subject #02 excluded) ...............................................................................................37     viii List of Figures   Figure 1.1. Factors and pathways that regulate CBF control........................................................ 5  Figure 1.2. The de-innervated donor heart is being sutured in the recipient during cardiac transplantation surgery. ............................................................................................ 7  Figure 1.3. Summary of the percent change in pre- and post-transplant CBF observed in    studies of HTR. .......................................................................................................17  Figure 1.4. Summary of pre- and post-transplant and control/normal CBF values in the       studies with HTR.. ..................................................................................................18  Figure 3.1. Instrumentation on subject during the incremental exercise protocol. .......................28  Figure 3.2. Image of (A) the TCD probe in place with the headband, (B) a frontal                     view of  the MCA insoniation, (C) MCA velocity waveform and envelope .............30  Figure 4.1. Resting values for (A) MCAv, (B) HR and (C) BP for HTR, AM and DC. ..............39  Figure 4.2. Relative VO2peak values for HTR, AM and DC. ........................................................41  Figure 4.3. (A) HRpeak and (B) HRreserve for HTR, AM and DC. .................................................42  Figure 4.4. (A) Overall relationship between HRreserve and age for all subjects, (B) the relationship between HRreserve and years post transplant. ..........................................44  Figure 4.5. (A) MCAv, (B) Mean BP, (C) PET CO2, and (D) CVR for HTR, AM and               DC throughout the incremental exercise test. ...........................................................47  Figure 4.6. (A) Systolic MCAv, (B) Diastolic MCAv, and (C) PI for HTR, AM and                  DC throughout the incremental exercise test. ...........................................................48       ix List of Abbreviations   ACA - Anterior Cerebral Artery  ACE - Angiotension-Converting Enzyme  AM - Age-Matched Control  BP -  Blood Pressure  BPM - Beats Per Minute  BMI  - Body Mass Index  CA - Cerebral Autoregulation  CBF - Cerebral Blood Flow  CHF  - Congestive Heart Failure  CO2 - Carbon Dioxide  CPP - Cerebral Perfusion Pressure  CVP  - Central Venous Pressure  CVR - Cerebrovascular Resistance  DC - Donor Population Control  ECG - Electrocardiogram  PET CO2 - Partial Pressure of End Tidal Carbon Dioxide  HF  - Heart Failure  HR - Heart Rate  HRpeak - Heart Rate Peak  HRreserve - Heart Rate Reserve  HT - Heart Transplant  HTR  - Heart Transplant Recipient   x  ICP - Intra-cranial Perfusion Pressure  IHD - Ischaemic Heart Disease  LVEF - Left Ventricular Ejection Fraction  MABP  - Mean Arterial Blood Pressure  MCA - Middle Cerebral Artery  MCAv - Middle Cerebral Artery Velocity  O2 - Oxygen  PCA - Posterior Cerebral Artery  PaCO2 - Partial Pressure of Arterial Carbon Dioxide  PI - Pulsitility Index  PNS - Parasympathetic Nervous System  Q - Cardiac Output  SNS - Sympathetic Nervous System  SV - Stroke Volume  TOR - Target of Rapamycin  VO2peak - Peak Oxygen Consumption xi  Acknowledgements    I would like to thank Dr. Philip Ainslie for his support, vast knowledge and guidance during my M.Sc.  Throughout the process he has taught me more than just many valuable research skills and techniques but, also social and interpersonal skills that extend far beyond the research lab.    A large thank you also goes to Dr. Mark Haykowsky from the University of Alberta and all of the subjects who participated in this study.  I would especially like to acknowledge the tremendous performances of the heart transplant recipients.  Without their efforts, this study would have never been able to occur.  Dr. Shieak Tzeng from the University of Otago, the time that you spent advising on the data analysis was much appreciated.  Kurt Smith and Chris Willie, thank you for the informal discussions throughout the writing process.  Thanks to my committee members Dr. Neil Eves, Dr. Gord Binsted and Dr. Gareth Jones.  I appreciated your contributions and feedback.  The efforts of Kit Marsden and Dr. Helen Jones were much appreciated during the data collection.  I would also like to thank my peers on the many informal physiological discussions that take place around the Human Kinetics lab at UBCO.  These talks have helped shape my ideas and philosophy on what it takes to be a researcher.  Last but not least, I would like to thank all of my friends and family for their support and encouragement.    xii Dedication  “The only place where success comes before work is in the dictionary.”   ~Vince Lombardi    1 Chapter One: Introduction and Review of the Literature  The heart transplant recipient (HTR) population represents a unique opportunity to observe the effects of cardiac output (Q) on cerebral blood flow (CBF) because the cerebrovasculature and cardiovasculature systems differ in chronological age. The cerebrovasculature is left intact and is therefore the same age as the HTR, where as the donor heart will typically be younger.  This unique population provides an opportunity to examine some of the conflicting results in the Q and CBF literature (refer to section 1.2.2.)  Namely, studying this population should enable the determination as to which is the key component of CBF:  is it the age of the brain and its cerebrovasculature or is it the age of the heart and its cardiovasculature? The following literature review describes a brief background on heart transplantation (HT) along with the factors associated with the regulation of CBF. The influences of cardiac function, aging and cardiac disease on CBF as it relates to the healthy human, congestive heart failure (CHF) and HTR populations are also considered.  Thereafter, there is also a brief summary of the sparse literature that has directly assessed CBF in HTR.  1.1.   Brief Background on Heart Transplantation    The first successful heart transplant was performed by Christiaan Barnard in 1967. The initial survival outcome was not very promising; the recipient, Louis Washkansky, survived for just 18 days1.  However, the 2nd heart transplant recipient (HTR) had a much better outcome and survived for 19 months1.  With improved surgical techniques and better medications, survival rates have continued to improve.  During the 1980s the median survival   2 was up to 8.3 years; by the 2000s it had increased to approximately 10.5 years2.  With improved survival rates, heart transplantation has moved beyond being an experimental medical procedure and is now an accepted therapy for extending end-stage heart failure patients‟ lives3.  Coinciding with the improved survival rates of HTR, there is an increased desire by the patients to return to a functional lifestyle and experience a good quality of life. However, pre-operatively many of the HTR have CHF and, because of this, undergo extended pre-transplant hospitalization.  During this time of bed-rest, their cardiovascular parameters - such as peak oxygen consumption (VO2 peak) - decrease approximately 26% 3, typically ranging from 10 to 14 mL/kg/min4, 5.  Yet, after HT, ventricular systolic function is improved to normal levels 6,7 as a result of the donor heart. Furthermore, through regular endurance training, peak heart rate (HRpeak) and VO2 peak can improve to 95% of age-matched norms3. As extreme examples; there has been a case of a HTR who completed Ironman™ Canada8; and a group of 14 HTR successfully competing in a 4-day 600km running relay from Paris to La Plagne9.  Despite this potential for improvement, many HTR experience VO2 peak values approximately 50-70% of their predicted age-matched counterparts 3, 10.  Possible reasons for the reduced VO2 peak include; depression 11-14, fatigue12, 14-16, abnormal vascular function17, 18, diastolic dysfunction19, 20, post-transplant immunosuppressive therapy3, 21, 22, cardiac allograft de-innervation5, 7, 10, 23, skeletal muscle dysfunction24 associated with the pre-transplant/post-transplant de-conditioning 3, 21, and physical pain12-14. The increased longevity after successful heart transplantation2 has led to a change in the long-term outcomes of HTR.  For example, neurological complications eventually develop in approximately 60-80% of all HTR14, 25, 26.  Tung et al.12, have reported that this may be due to side effects from the life-long post-operative use of anti-rejection   3 immunosuppressant therapies.  Over the course the post-transplant survival period, there is approximately a 14-18% occurrence rate of a cerebrovascular event, such as cerebral haemorrhage or ischaemic stroke occuring14, 26, 27. Zierer et al.28, have suggested another possible reason for the cerebrovascular events that are experienced in the HTR populations - that the cerebrovasculature in HTR may have a limited range of tolerance for changes in systemic blood pressure (BP).  This risk of a cerebrovascular events may be due to cerebral vessel remodeling in the face of chronic cerebral hypoperfusion that has been associated with heart failure (HF)25, 26, 28, 29.  However, with the possible increase in CBF associated with cardiac transplantation30-32 (refer to section 1.5.) there is a trade-off; an increased risk of hyper-perfusion related complications occurring over the short-term post-transplant28, 30.  In addition over the long-term, there is an increased likelihood that an ischaemic stroke will occur as a complication of cardiac transplantation25-27.  The end result for HTR is that they ultimately require extremely sensitive hemodynamic management and it is suggested they should be cautious of both hypo- and hyper-tensive episodes28. This avoidance is troublesome, since changes in BP occur in a myriad of everyday activities; postural change, coughing, laughing, defecation, exercise, and sexual activity, etc...  1.2.  Regulation of CBF   The human brain has a mass of approximately 1400g which equates to ~2% of the total mass of an average 70kg male33.  At rest, normal cerebral oxygen (O2) consumption is 3.5mL/100g brain tissue per minute34 equating to ~49mL O2 per min, or approximately 20% of the ~250mL O2 per min the body consumes at rest.  Thus, the brain has placed itself very prominently in the hierarchical positioning of organ demands35.  With particular emphasis on   4 the integrative perspective, the following section provides an overview of the factors that regulate of CBF in humans.  1.2.1.  Neurogenic Control of CBF  Neurogenic control refers to the influence on CBF of the sympathetic nervous system (SNS) [Figure 1.1. (1)] and parasympathetic nervous system (PNS) [Figure 1.1. (2)].  The cerebral vasculature is extensively innervated by sensory and autonomic neurons36-38.  Sensory neurons have been implicated in the transmission of afferent activity produced by the nociceptors to the central nervous system; however, their role in cerebral vasomotor control is unclear39.  Autonomic neurons from the SNS and PNS nervous systems innervate the cerebral vasculature. The SNS innervations arise from the superior cervical ganglia40; PNS innervations arise from the sphenopalatine and otic nerves41. The functional role for SNS and PNS control of the cerebral vasculature is controversial42-47.  The SNS contribution to CBF regulation is dependant on the rate of change in arterial blood pressure42, a response that has an important role in dynamic cerebral autoregulation (CA)48, 49.  As BP can surge during REM sleep, it has been suggested that elevations in SNS activity may act in a shielding/protective role and prevent cerebral hyperperfusion during the periods of increased BP50-52.  Although SNS control of CBF is not fully understood, it appears it may have a functional role during extreme hypo and hypertension.  The role of the PNS in CBF regulation is also still largely unknown, especially in humans.  However, a recent study; has implied that the PNS may have a role in   5 cerebrovascular system regulation during exercise53, a finding that was further discussed in a companion editorial41.               Figure 1.1.  Factors and pathways that regulate CBF control. Control of cerebrovascular tone through neurogenic means is controversial, however sympathetic (1) and parasympathetic (2) inputs have been implicated.  The quotient of cardiac output (Q; 3) and total peripheral resistance (TPR; 4) of the circulatory system is mean arterial blood pressure (MABP; 5).  The peripheral vasculature has an impact on CBF regulation by way of MABP. Cerebral perfusion pressure (CPP; 6) is the pressure gradient difference between MABP and intra-cranial pressure (ICP) under conditions where the central venous pressure (CVP), as measured at the jugular vein (7), is lower than ICP and is the driving force behind cerebral perfusion. Cerebral autoregulation is under the influence of modulating factors such as CO2 and brain metabolism (8).  (adapted from 47).       6 1.2.2.  Influences of Cardiac Function on CBF   The primary function of the heart is to adequately perfuse the organs of the body with blood.  The heart provides this function by contracting its muscular walls, increasing the pressure in the closed chamber (i.e. ventricle), when the internal pressure is greater than that of the attached artery (pulmonary or aorta), the blood is expelled through the valve and into the circulatory system.  The intrinsic rate of the sino-atrial node of the heart is approximately 100 beats per minute (bpm). In healthy humans the sino-atrial node is also extrinsically regulated by the PNS and SNS54.  Increased parasympathetic tone slows the heart rate (HR) below the basal rate whereas enhanced sympathetic tone increases HR towards peak levels with the secondary assistance of catecholamines54.  Heart transplant recipients represent a unique population to study the effects of cardiac function because, during the transplantation process, the nerve fibres to the heart are transected, resulting axonal degeneration and complete denervation of the heart (Figure 1.2)55, 56.  Surgical denervation results in the heart having increased sensitivity to the neurotransmitters that were lost, resulting in the transplanted heart being highly sensitive to norepinephrine and epinephrine55.  The end result is that the transplanted heart is, at least initially, completely denervated and dependent upon hormones (e.g. catecholamines) to control increases in HR.  This hormone dependence following transplant results in a delayed and blunted HR response and creates a greater reliance upon the pre-load to increase stroke volume (SV) and thus Q, especially during exercise7, 10, 57.  However, over time, re-growth of transected nerves can take place and result in some re-innervation of the heart56.  More rapid and complete re-innervation of the heart can occur if the allograft heart comes from a younger donor and if the transplantation procedure is fast and uncomplicated56.   7        Figure 1.2. The de-innervated donor heart (red) is being sutured in the recipient (blue) during cardiac transplantation surgery.  The major blood vessels of the pulmonary and systemic circuits are labeled for identification and orientation.    8 Cardiac output [Figure 1.1. (3)] is the total volume of blood expelled per minute and is determined by the quotient of HR and SV5, 58.  At rest in the healthy human population, SV is approximately 70-80 mL and is influenced by 3 main factors: diastolic stretch, contractility and BP58.  Diastolic stretch and contractility are influenced by the total peripheral resistance (TPR) [Figure 1.1. (4)] which is the sum of the vascular resistance across the entire circulatory system and as such has an impact on SV by way of the diastolic stretch58.  Alterations in BP influence venous return and thus will also have an affect on SV58, for example: an increase in TPR will lead to an increase in diastolic stretch. The relationship between Q and CBF is unclear in the current literature (Table 1.1. – Appendix IV).  From the 31 studies reviewed in Table 1.1., 9 studies showed no Q-CBF relationship, 9 studies showed a Q-CBF relationship and 5 studies showed conflicting results.  The Q-CBF relationship occurs under certain conditions [e.g., changes in body temperature, blood volume or alterations in BP and venous return due to increases in lower body negative pressure or medications] while other conditions do not show a relationship at all.   A possible reason for some of this confusion in the literature on the Q-CBF relationship is the counterintuitive nature of this concept. For example, neither of the mechanisms that mediate Q [SV or heart rate] are represented in Poiseuille‟s law.  Poiseuille‟s law is a major concept in the determination of CBF and is represented in the following equation:    F = (P1-P2)πr 4/8μL  Equation 1. Where: F=flow, P1=inflow pressure, P2=outflow pressure, r=radius, μ= viscosity of the fluid, L=length    9 The inflow pressure is BP; out flow pressure is the pressure in the internal jugular vein [Figure 1.1. (7)]. The length of the cerebral vasculature is not a physiological variable that changes or can be altered as it is consistent within each individual87.  So if those are not responsible for the observed changes in Q and CBF, what does and is there a population we could observe that may better assist us in understanding this relationship?    1.2.3.  Autoregulatory Control of CBF   A critical intrinsic response of the mammalian brain is known as cerebral autoregulation (CA) – the process by which adjustments are made to the cerebrovascular resistance to ensure that the CBF levels are matched to metabolic needs88.  There are two main components to CA: static and dynamic.  Static CA describes the tendency of CBF to be chronically maintained over a wide range of BP changes89 [Figure 1.1. (5)]; and dynamic CA describes the ability of the cerebral vasculature to resist acute changes in perfusion pressure, due to changes in BP over a short time-course of less than five seconds48.  If CA fails, the brain is put at risk of ischaemic damage (during low blood pressures) and of haemorrhage (during high blood pressure).  Impairment of CA has been associated to the risk of stroke90.   The classic description by Lassen91, and further re-emphasized in a review by Paulson et al.92, was that CBF has an autoregulatory range that is maintained over a wide range of BP (50-150 mmHg); however, this has been challenged by recent evidence that CBF is also dependant upon alterations to BP89.  Lucus et al.89 have reported that MCAv changes ~8% per 10 mmHg shift in BP across both hypo- and hypertension, suggesting that arterial baroreflex regulation of BP [Figure 1.1. (4)] may have a greater role in CBF control than traditionally thought.   Moreover, an inverse relationship between cardiac baroreflex   10 sensitivity and dynamic CA, suggests the presence of compensatory interactions between peripheral blood pressure and central CBF control mechanisms, that optimize CBF control has been reported93.  Such interactions may account for the divergent changes in CA and baroreflex sensitivity seen with normal aging, and in clinical conditions such as spontaneous hypertension94, autonomic failure95, and chronic hypotension96.  Because of related alterations in heart rate variability97, 98 and baroreceptor sensitivity99 following heart transplantation, unstable control of blood pressure may ensue.  Thus, dynamic CA may be a particularly important mechanism to protect the brain from alterations in BP.  However, to date no studies have assessed CBF or dynamic CA over the longer term (i.e. years) in HTR.   1.2.4.  Regulation of CBF by PaCO2  The downstream cerebral arterioles [Figure 1.1. (8)] are highly sensitive to changes in the partial pressure of arterial CO2 (PaCO2) 100.   Respiratory-induced changes in PaCO2 are tightly coupled with CBF and therefore play an important role in the regulation of CBF101, 102.   Cerebrovascular reactivity is the CBF response to changes in PaCO2 and is of vital homeostatic importance as it helps regulate, influence and maintain central pH 33, 101, 103, 104.   When there is an elevation in PaCO2 (hypercapnia) there is a corresponding increase in the CBF as a result of the vasodilation of the cerebral arteriole bed [Figure 1.1. (8)]33.  Conversely, when there is a reduction in PaCO2 (hypocapnia), vasoconstriction of the cerebral arteriole bed occurs and there is a subsequent reduction in the CBF33.  However, there are differences in the amplitude of CBF response to hyper- and hypocapnic events100, with hypercapnic events demonstrating a larger response over a wider array of CO2   11 challenges105.    This is possibly due to a large release of nitric oxide in the brain that coincides with hypercapnia, where as this response is not present in hypocapnia106.   Studies have shown that there is a link between cerebrovascular endothelial dysfunction and impairment in the reactivity of cerebral vascualture107-109 as well as cognitive decline109-111. These findings suggest that alterations in the peripheral vasculature, may also affect cerebrovascular function, including cognitive outcomes.  Congestive heart failure (CHF), which is one of the primary precursors to heart transplantation, is characterized by decreasing cardiac function112, 113.  This reduction in cardiac function along with cerebrovascular endothelium dysfunction and impaired reactivity may relate to the observed cognitive declines associated with this population114, 115.  1.2.5.  Effects of Cerebral Metabolism on CBF at Rest and During Exercise   Oxygen delivery to the brain is reliant upon the arterial O2 content and CBF.  Resting cerebral O2 consumption is maintained throughout a variety of PaCO2 alterations, as typically experienced during cerebrovascular reactivity challenges116-118, via increases in O2 extraction33.    It has been well- reported that CBF is elevated by 10-20% during sub-maximal exercise (reviewed in: 100, 119).  These increases in CBF are likely driven via the increased O2 requirements of the brain100, 120.  After the aerobic threshold (60-70% of VO2Peak), CBF returns toward baseline (and possibly lower) values due to hyperventilation-induced hypocapnia, regardless of increasing O2 demands within the brain 120, 121.  Exercise-induced hyperventilation (and related hypocapnia) therefore appears to be a stronger regulator of CBF than cerebral metabolism at exercise intensities above aerobic threshold (~70% VO2max) 122.    12 1.3.  Cognitive Impairment in CHF   Clinical populations with severe cardiovascular disease and end-stage heart failure have also been shown to have impaired cognitive function30, 31, 114, 123-128.  Approximately 25- 50% of CHF patients experience cognitive impairment, which is associated with a 5-fold increase in mortality among older adult hospitalized patients114, 129.  Structural and functional brain changes, including discrete losses in gray matter, brain volume, areas of silent stroke, and decreased CBF31, 130-133, have also been observed in CHF patients.   End-stage HF patients who have undergone heart transplantation have shown significant rapid improvements in cognitive functioning soon after transplants that were stable within 3 months127, 134, 135.  These studies indicate that a higher stroke volume, higher cardiac and lower right atrial pressure are all correlated with better cognitive function135.    Gruhn et al.31, argued that the neurological impairment in the CHF patients was due cerebral hypoperfusion associated with a CBF 30% below normal resting values31.  These conclusions are hindered, as there were no neuropsychological testing pre- or post- transplant31.  The 30% reductions in CBF  are above the aggregate experimental and human study data, which showed that non-human primates have a 22 mL/100g per min (40% below normal resting values) threshold before neuronal function is impaired136.  In humans, electroencephalography activity decreases if mean CBF falls below 23 mL/100g per min (54% below normal resting values) during carotid clamping137.  Ackerman noted clinical cognitive dysfunction is not necessarily related to a CBF threshold.  Rather, these studies support the notion that the brain may be more tolerant to hypoperfusion than early investigators had implied138.  However, a review paper by Roman139 concluded that circulatory conditions, such as cerebral hypoperfusion due to CHF, result in localized brain   13 injuries that lead to undiagnosed forms of cognitive decline in older adults.  Thus, further research with prospective studies and experimental models are still needed to determine the pathogenesis of cognitive dysfunction in end-stage HF patients and HTR.   1.4.   Influences of Cardiac Disease on CBF   According to the 2008 cardiac transplant update from the Canadian Cardiovascular Society Consensus Conference140: Cardiac transplantation is the treatment of choice for patients who have severe end-stage heart failure despite maximal medical therapy and/or complex congenital heart disease not amenable to surgical palliation at reasonable risk. In the 27th official adult heart transplant report by the Registry of the International Society for Heart and Lung Transplantation2, CHF represents 51.4% of all pre-transplant diagnosis, Ischaemic heart disease (IHD) 39.9%, re-transplant 2.4% and other causes making up the remaining diagnosis2.  1.4.1. Congestive Heart Failure   The major reductions in deaths from cardiovascular diseases (hypertension, coronary diseases and damage to the cardiac valves) have led to more and more people living with HF112, 113.  There are an estimated 5.7 million people living with HF and almost 300 000 annual deaths from HF complications in the United States of America alone113.  End-stage HF occurs after the myocardium has exhausted all of its reserve capacity and compensatory   14 mechanisms, leaving the possibility for salvage limited leading to terminal CHF112.  The greatest survival outcome for end-stage HF patients is a cardiac transplant. However, the supply of donor hearts is limited, with only 3000-5000 heart transplants take place worldwide each year 2, 113.   Reduced CBF has been reported in older adult males with CHF32, 141-144. However, there are conflicting opinions in the literature on the root cause of the reduced CBF.  Loncar et al.141 reported that older adult males with mild to moderate CHF have reduced CBF values independently associated with reduced left ventricular ejection fraction (LVEF).  Conversely, Choi et al.32 have shown that in cases of severe HF there is no correlation between LVEF and CBF.  This discrepancy could be due to experimental differences in the assessment of CBF and related complications associated with the functional classification of CHF, such as neurohormonal activation, having an influence on CBF141.  Regardless of the cause of the reduced CBF, both groups acknowledge a myriad of negative outcomes associated with reduction in CBF.  For instance, the authors speculated that the reduced CBF may result in structural changes occurring within the brain32, 141, 144; this effect could manifest in negative outcomes such as impaired cognitive function (as previously described), and autonomic nervous system dysfunction141.  Whilst speculative, these outcomes might be the reason for the increased risk of CHF patients also developing cognitive disorders such as Alzheimer disease or dementia141.    1.4.2.  Ischemic Heart Disease   Ischemic heart disease (IHD) is one of the major causes of death in the western world and will result in over 150 000 deaths in the United Kingdom in the next year145, 146.  IHD   15 involves a complex interaction between haematological, biochemical, immunological and physiological factors145, 146.  All of these combine to elevate certain clotting factors, such as fibrinogen, which result in paralyzing the heart due to the blocking blood flow to one or more of the major coronary arteries eventually resulting in coronary artery thrombosis and resultant myocardial ischemia146, 147.  Over time, the blockages build-up and lead to cellular death of the localized myocardial tissue resulting in a reduced ejection fraction and possible rupture of the myocardial wall147.  The two most effective and viable long-term treatments for end-stage IHD are cardiac surgery and heart transplantation146, 147.  Reduced CBF has been observed in approximately 75% of IHD patients148, 149, which may possibly explain why one of the major complications of cardiac surgery is ischemic stroke148, 149.  With the increased risk of ischemic stroke associated with cardiac surgery, Kawabori et al.148, have recommended that IHD patients should be preoperatively screened for MCA and internal carotid artery occlusion.  1.5.  Changes in CBF in HTR   Heart transplantation is a life saving surgical intervention for select individuals with end-stage HF.  Despite normal resting left ventricular systolic function after surgery, HTR have reduced Q during exercise6, 10.  The mechanism responsible for post-transplant impairment in Q reserve 6, 10 is attributed to surgical denervation of the transplanted heart10, diastolic dysfunction10, and peripheral vascular dysfunction10.  A consequence of a blunted Q reserve is that it may result in decreased CBF (refer to section 1.2.2.).  Currently, there have been only three studies that have examined CBF at rest in HTR30-32. Moreover, to date, no studies have reported how CBF might be altered during exercise.  The following section will   16 provide an overview of the salient methodological and experimental findings from these three studies.  It was reported150 that CBF might be reduced in patients with CHF, resulting in cognitive impairments, memory problems, confusion, lethargy and dizziness.  Many of these conditions were improved after heart transplantation127.  On this basis, Gruhn et al. 31 reasoned that CBF alterations may be occurring in patients with CHF who undergo heart transplantation. In their study, CHF patients had reported CBF baseline values of 35 ± 3 mL/min per 100g rising to 50 ± 3 mL/min per 100g using Xenon gas inhalation (Figure 1.3.) one month after undergoing successful heart transplantation (P<0.05), and the after transplantation the HTR were not significantly different from the control values 52 ± 5 mL/min per 100g (Figure 1.4.).  Thus, leading to the conclusion that CBF rapidly normalizes after heart transplantation.  A small subset of the HTR were re-examined at 6 months post transplant and it was reported that the CBF and MCAv values did not differ from those at one month31.      17       Figure 1.3. Summary of the percent change in pre- and post-transplant cerebral blood flow (CBF) observed in studies of heart transplant recipients (HTR). All three HTR studies30-32 reported statistically significant CBF increases post- transplant of between 25 and 53% (*denotes, P < 0.05).      Pre- and Post- Transplant CBF Pre Post C ha ng e ( % ) -40 -20 0 20 40 60 80 100 Massaro et al. Choi et al.  Gruhn et al. * * *  18       Figure 1.4. Summary of pre- and post-transplant and control/normal cerebral blood flow (CBF) values in the studies with heart transplant recipients (HTR).  CBF values were reported as mL/min/100g brain mass (Xenon inhalation) by Gruhn et al.31 and Choi et al.32, and as cm/s (transcranial Doppler) by Massaro et al.30. Gruhn et al.31, and Choi et al.32, reported age-matched (AM) control values, Massaro et al.30, did not provide this information, instead AM normal values were obtained from Ainslie et al. 151.  All studies reported statistically significant differences pre- and post-transplant as well as between HTR and AM comparison groups.  No statistically significant differences were reported between post-transplant and AM comparison groups. (*denotes, P < 0.05)  HTR Study Gruhn et al. Massaro et al. Choi et al. Ce re br al Bl oo d Fl ow  (m L/ m in /1 00 g or cm /s) 0 20 40 60 80 100 Pre-Transplant Post-Transplant Control-Normal * * * * * *  19 One difference noted was the end-stage HF patients had significantly (P<0.05) lower end tidal carbon dioxide (PET CO2) concentrations (34.5 ± 1.5 mmHg) than their control groups (39.0 ± 0.8 mmHg) 31.  Assuming normal cerebrovascular CO2 reactivity 33, the 4.5 mmHg reduction in PET CO2 would account for approximately 18% of the observed 30% reduction in CBF.  Hypocapnia is a well known condition associated with congestive heart failure (CHF) patients152-155, and chronic hypocapnic exposure can result in cerebral hemodynamic maladaptation 156.  Gruhn et al.31 reported that the increase in CBF following HT being strictly caused by the marked hypercapnia was questionable.  Therefore, they concluded that the decreases in CBF for patients with severe CHF may contribute to the neurological symptoms they experience31.  Another possibility to explain Gruhn‟s findings was noted in a companion Editorial138. This editorial suggested that the reported 30% increase in CBF following HT was perhaps due to a perisurgical fall in hematocrit, as well as putting forth the notion that the pre-surgery reduction in CBF may not be sufficient to cause neurological impairment.  Although Gruhn et al. 31 did not provide the hematocrit data for their subjects, it was estimated that net hematocrit levels fell by 8.3% 30 days post transplant138.  A reduction in hematocrit levels from 0.57 to 0.24 can result in an increase in red blood cell velocity of more than 50% due to the reduction in red blood cell viscosity157.  Such changes may explain the CBF changes found by Gruhn et al. 31 could have occurred due to blood viscosity changes as noted in Poiseuille‟s law (Equation 1.)138.    In the second study, Massaro et al.30 reported changes in CBF velocity (as index by transcranial Doppler - TCD) in CHF patients before and after heart transplantation in order to evaluate the intracranial hemodynamic features that are associated with the improvement of   20 cardiac output.  Twenty-six patients were preoperatively selected for their study.  Upon completion of neuroimaging examinations (cranial CT or MRI), 4 patients were excluded because of silent ischaemic brain lesions and 20 for signs of cerebral atrophy30.  The twenty- two patients included in the study had a mean age of 45.3 years, MCAv of 45.1 ± 10.6 cm/s (Figure 1.3.), and a mean hematocrit of 38.8 ± 5.5%30.  Fourteen patients underwent successful heart transplantation. Findings showed a 53.3% increase in MCAv (P<0.0001) in MCAv of 53.3% (Figure 1.2.) and a lower mean hematocrit levels (31.2 ± 2.0%) was observed30.  Pre-operative and post-operative neuropsychological evaluations were not performed during the study30.  When evaluating their data for the 53.3% improvement in MCAv the authors were unable to observe any MCAv-hematocrit correlation138.  The lack of correlational data in the pre- and post-transplant measures of MCAv and hematocrit, led Massaro et al. 30 to conclude that the CBF increases observed in their studies were probably not due to the changes in hematocrit levels.  Overall, these findings allowed the authors to report that the main mechanism for the increase in MCAv after successful heart transplantation was most likely due to the improved Q.  In support, Baufreton et al.158 have recently confirmed in healthy humans there is no MCAv - hematocrit relationship.   The focus of the final study by Choi et al. 32 was to investigate factors that represent the chronicity and level of severity of HF, but not associated with exercise capacity or the LVEF, in respect to global CBF changes observed in CHF patients, rather than the effects of heart transplantation on CBF.  Overall, fifty-two CHF patients took part in their study, four of which underwent heart transplantation.  In these four, it was noted that global CBF at rest increased (35.5 ± 1.6 to 44 ± 3.2 mL/100g per min: Figure 1.3.) and left ventricular ejection   21 fraction (LVEF) normalized (19.8 ± 6.8% to 66.8 ± 3.3%) for these subjects32.  However, the authors did not expand upon or make any reference to these findings in their discussion.  These results, in conjunction with the findings by Gruhn et al. 31, support the conclusion by Massaro et al. 30, in that the main mechanism for the increase in CBF post-transplant is likely due to the increased Q that the „new‟ heart provides.   22 Chapter Two: Purpose, Aims and Hypotheses  2.1.  Purpose of Thesis  A limitation of the prior studies of CBF in HTR30-32, is that CBF was only examined at rest during the acute post-transplant time period (up to 6 months), and was also not examined during exercise stress, when Q reserve is challenged6, 21.   The HTR population has a reported inability to increase Q during exercise due to cardiac denervation159-161.    However, it has also been reported that the exercise HR response will vary with the length of time after transplant and this may be due to the possible re- innervation of the heart6.  Furthermore, the role that this possible cardiac re-innervation and concomitant improvement in exercise Q associated with heart transplantation has on CBF is unknown100.   How these possible alterations in the long-term HTR (cardiac re-innervation and improvements in exercise Q) will affect CBF at rest and during exercise stress is currently unknown.  2.2.  Aims   The aim of this thesis was enhance the current literature of CBF in HTR by being the first study to examine the influence of long-term heart transplantation on the regulation of CBF at rest as well as being the first study to assess the effects of exercise stress on CBF in HTR.    23 2.3.  Hypotheses   1. That CBF (indexed using TCD) would be similar in HTR when compared to age and activity-matched controls, but CBF would be attenuated relative to donor population controls. 2. During incremental cycling exercise, to exhaustion, HTR would have reduced elevations in CBF compared with age and activity-matched controls (AM) and donor population controls (DC).        24 Chapter Three: Methods  3.1.  Participants  Seven male clinically stable HTR (62 ± 9 yrs), years post transplant (9 ± 7 yrs), from the University of Alberta Heart Transplant Clinic, seven male AM (62 ± 7 yrs), and seven male DC (22 ± 3 yrs) were recruited for this study (Table 3.1). All of the HTR subjects were clinically stable and had no clinical or biopsy evidence of rejection.   The AM were recruited to match with the HTR on an individual basis for both age and activity level, as there is a decrease in MCAv of approximately 1% per year across the aging spectrum, and a 17% increase in MCAv that is associated with long-term endurance training, irrespective of aging151.  The DC were included in the study to offset a study limitation of Kao et al.10, which indicated that the exercise response observed in HTR may be more closely related to the age of the donor rather than the age of the individual.  The age of the DC group was chosen to represent the most common heart transplant donor group. For example, in North America over 50% of all donors are between the ages of 18-342. All subjects were asked to abstain from caffeine, smoking and alcoholic beverages for a period of 12 hours prior to the study and all medications were maintained for the study. Each subject underwent a familiarization of the laboratory and testing protocols before the initiation of the incremental peak exercise protocol. This study and was approved by the University of Alberta Health Research Ethics Board (Pro00011560), and all participants provided written informed consent before the experiment.    25 Included in the study was the only HTR to have successfully completed the Ironman™ endurance race (subject #02)8.  This subject was also the only HTR participant to have a non-ischemic pre-surgery etiology. Statistical analysis were performed with and without HTR subject #02 in order to establish if the increased fitness and training levels associated with completion of an Ironman Triathlon and/or the effects of the non-ischemic pre-surgery etiology influenced the main findings of the study (Table 3.2).  Table 3.1. Participant Characteristics (all subjects)   HTR (n=7) AM (n=7) DC (n=7) Age (years) 62 ± 9 62 ± 7      22 ± 3 †‡ Body Mass Index (kg/m2) 27 ± 5 26 ± 4 25 ± 3   Resting BP (mmHg) 99 ± 4  99 ± 15 91 ± 6 Resting Mean MCAv (cm/s)  40 ± 12 41 ± 7     69 ± 9 †‡ Resting Cerebrovascular Resistance (mmHg/cm/s)  2.6 ± 0.7  2.5 ± 0.5       1.3 ± 0.2 †‡ Resting Pulsitility Index (AU)  0.9 ± 0.2  1.1 ± 0.3  1.1 ± 0.2 Peak O2 Consumption (ml/kg/min)  25 ± 10 35 ± 9     51 ± 7 †‡ Years after transplantation  9 ± 7   Medications    Corticosteroid  2   Antiproliferative agent  4   Calcinerurin inhibitor  4   mTOR inhibitor  4   Ca2+ channel blocker (diltiazem)  5  1  ACE inhibitor  4  1  Diuretic  3  1  Aspirin  6  1  Lipid –lowering agent  4    Values are means ± SD. All subjects were ischemic pre-surgery etiology except HTR subject #02 who was non-ischemic etiology. Heart transplant recipient (HTR); age-matched (AM); donor population control (DC); blood pressure (BP); target of rapamycin (TOR); angiotensin-converting enzyme (ACE).  Statistical significance was set at P < 0.05, †denotes significance between HTR vs. DC, ‡denotes significance between AM vs. DC.     26         Table 3.2. Participant Characteristics (without HTR subject #02 and matched controls)   HTR (n=6) AM (n=6) DC (n=6) Age (years) 64 ± 8 63 ± 7      21 ± 2 †‡ Body Mass Index (kg/m2) 28 ± 4 26 ± 4 26 ± 2   Resting BP (mmHg) 98 ± 3  98 ± 16 90 ± 7 Resting Mean MCAv (cm/s)  41 ± 13 42 ± 8     67 ± 7 †‡ Resting Cerebrovascular Resistance (mmHg/cm/s)  2.6 ± 0.7  2.5 ± 0.4       1.4 ± 0.3 †‡ Resting Pulsitility Index (AU)  0.9 ± 0.3  1.1 ± 0.3  1.0 ± 0.2 Peak O2 Consumption (ml/kg/min)  22 ± 4 33 ± 8*     50 ± 7 †‡ Years after transplantation  7 ± 4   Medications    Corticosteroid  2   Antiproliferative agent  4   Calcinerurin inhibitor  4   mTOR inhibitor  4   Ca2+ channel blocker (diltiazem)  4  1  ACE inhibitor  4  1  Diuretic  3  1  Aspirin  6  1  Lipid –lowering agent  4    Values are means ± SD. All subjects were ischemic pre-surgery etiology. Heart transplant recipient (HTR); age-matched (AM); donor population control (DC); blood pressure (BP); target of rapamycin (TOR); angiotensin-converting enzyme (ACE).  Statistical significance was set at P < 0.05, *denotes significance between HTR vs. AM, †denotes significance between HTR vs. DC, ‡denotes significance between AM vs. DC.      27 3.2. Instrumentation   Whenever possible, both the right and left middle cerebral arteries were insonated by placing a 2-MHz Doppler probe (Spencer Technologies, Seattle, WA, USA) to obtain bilateral CBF velocity and are securely locked in place with an adjustable head-band (Spencer Technologies, Seattle, WA, USA), enabling continuous measures of CBFv throughout the incremental cycling test.  Heart rate was recorded with a three-lead electrocardiogram (ECG).  Blood pressure was monitored in the arm by electrosphygmomanometry (SunTech Medical, Morrisville, NC, USA), with a microphone placed over the brachial artery and the Korotkoff sounds gated to the ECG.  Expired O2 and CO2 gases were measured by an online gas analyzer (Sensormedics Metabolic Cart, 2900BZB, Sensormedics Corporation, Homestead, FL, USA and Vmax Encore VIASYS Healthcare Inc. Yorba Linda, CA, USA) and were calibrated with standard gas of a known concentration before each exercise test.  All data were recorded and stored for subsequent analysis using commercially available software (LabChart version 7.0, ADInstruments, Colorado Springs, CO, USA). Echocardiograph images (Vivid-i, GE Healthcare, USA) were recorded and assesed by a cardiologist in order to directly compare the Q with the CBFv.  Unfortunately upon assessment of the recorded images, the data were deemed unacceptable for valid measurement due to subject movement during the incremental exercise test.   An example of the instrumentation setup for the incremental exercise protocol is shown in Figure 3.1.   28    Figure 3.1. Instrumentation setup on subject during the incremental cycling exercise protocol.  Transcranial Doppler ultrasound (TCD), Electrocardiograph (ECG).   TCD     Gas Exchange  ECG         Sphygmomanometer         Echocardiograph                        Imaging   29 3.3.   Transcranial Doppler Ultrasound  Transcranial Doppler ultrasound (TCD) was first introduced into the clinical and research areas by Aaslid162 in 1982.  This technique has several advantages for the assessment of measuring CBF velocities in humans; it is non-invasive, has high resolution, allows for continuous monitoring, and repeated measures101, 163.  Transcranial Doppler ultrasound functions are based on the principle of Doppler shift; as such, TCD is able to detect the red blood cell flow velocities in a variety of cerebral vessels (MCA, anterior cerebral artery, posterior cerebral artery).  This study utilized the MCA as it is readily available through the temporal acoustic window, has a trajectory that is parallel with the insonation probe101, 162 (Figure 3.2.) and has been shown to carry approximately 70-80% of the blood volume into the respective hemisphere164.  It is key to be aware that TCD gives a measure of blood flow velocity (cm/s) and not blood flow (mL/min/100g) per se.  However, provided that the insonated vessel remains constant in diameter, these two principles are closely related as shown by the following equation163:  Cerebral blood flow velocity = blood flow volume / blood vessel diameter Equation 2.      30        Figure 3.2. Image of the transcranial Doppler ultrasound (TCD) probe being held in place with the headband (A), a frontal view of the insonation of the middle cerebral artery (MCA) (B), image of the MCA velocity waveform and envelope (C).  A B C  31 3.3.1. Validity of TCD   In the literature it has been shown that the diameter of the MCA remains relatively unchanged over a range of 23-60 mmHg for PaCO2 163, 165-167.  Additionally, there have been several studies on humans that have compared TCD estimates of MCAv with several other measures of CBF: Kety-Schmidt method168, magnetic resonance imaging 167 and the Fick principle169.  All of these studies have shown a good correlation between the measures of CBF with the assessment of MCAv via TCD; as such the findings of these studies collectively support the use and validity of MCAv as an indirect measure of CBF.  3.3.2.  Principle of TCD   The basic principle of TCD is that a transmitter inside a Doppler probe emits a 2 MHz pulsed-Doppler ultrasound beam through the thin bone acoustic window to a given target vessel.  Some of the ultrasound beam is then reflected off of the red blood cells and recorded by the receiver in the Doppler probe.  The Doppler shift is the difference between the transmitted and received signals and determines the velocity of the red blood cells in the target vessel and is calculated in the following equation170:  Doppler frequency shift = 2 x V x Ft x cosθ/C Equation 3.  Where:  V=velocity of the reflector (red blood cells), Ft=transmitted frequency (2 MHz), cosθ=correction factor based on the angle of insoniation, and C=speed of sound in the blood (1540m/s)    32  In TCD, since both the transmitted frequency and speed of sound in the blood are constants, the frequency of the Doppler shift is ultimately dependant upon the velocity of the red blood cells and the angle of insonation of the Doppler probe170.  The angle of insonation is vital to the accuracy of the measure.  It is recommended that the angle of insonation is kept less than 30°; as the cosine will vary between 1 and 0.86 in this range, thus giving a maximum error of less than 15%162.  An advantage to measuring the MCA, as opposed to other cerebral vessels is that the angle of insonation is that the flow of the vessel is parallel to the probe (Figure 3.2.-B), thus resulting in a lower insonation angle and therefore less error170.  The final Doppler signal is the summation of all of the signals reflected from the velocity of each of the red blood cells within the sample volume of the target vessel170.  The processing unit in the TCD uses spectral analysis to extract the 3-dimensional Doppler data into a 2-dimensional Doppler waveform (Figure 3.2.-C)170.  This waveform and its subsequent spectral envelope are used to determine the MCAv, which is recorded for future analysis.  3.3.3.  Technique of TCD   The approach to insonating a cerebral vessel has been described in detail elsewhere101, 162, 170 and is summarized as follows. First, acoustic gel is placed on the probe and temporal window, this aids in signal conduction. Second, the Doppler probe is positioned over the acoustic window and held in place with an adjustable headband.  Lastly, the MCAv signal is obtained and secured by an experienced research technician using search methods that have been previously described101, 162.   33  The flow-volume MCAv waveform was obtained from the spectral envelope and displayed in LabChart in real time.  From the flow-volume waveform, systolic and diastolic MCAv were obtained.  Mean MCAv was calculated as follows:  Mean MCAv = 1/3 Systolic MCAv + 2/3 Diastolic MCAv Equation 4. Where:  Mean MCAv = mean middle cerebral artery velocity (cm/s), Systolic MCAv = systolic middle cerebral artery velocity (cm/s), Diastolic MCAv = diastolic middle cerebral artery velocity (cm/s).   In addition, Cerebrovascular Resistance (CVR) was calculated as: MAP/MCAv. MCAv Pulsitility Index (PI) was calculated as: (Systolic MCAv-Diastolic MCAv)/Mean MCAv.  3.4. Incremental Cycling Exercise Protocol  An electrically braked cycle ergometer (Monark 894E, Varberg, Sweden) was utilized for the incremental cycling exercise test.    Baseline measures were recorded in a seated position on the cycle ergometer, after the subjects began the exercise protocol.  The first stage was begun at 50 watts (W) and the workload for all subsequent stages was increased 25W every 2 min until the respiratory exchange ratio exceeded 1.00, subsequently the workload increased 25W every minute until volitional exhaustion. Upon completion of the protocol VO2peak was determined from the highest 20-second average expired O2 value. This protocol was modified for the HTR to begin at 25W due to the lower expected VO2peak values as described by Scott et al.6.   34 The criteria for completing the exercise protocol were: leveling off of VO2, respiratory exchange ratio greater than 1.15, a rate of perceived exertion of 20 (on the 6-20 Borg scale), or heart rate at age-predicted maximal values.   3.5. Statistical Data Analysis  Statistical analyses were performed using PASW version 18.0 for Windows (PASW, Inc. Chicago, Illinois). A one-way repeated measures ANOVA with group by intensity comparisons was applied to identify differences across intensity.  Upon establishing the normality of the data using a skewness-kurtosis normality test, comparisons between groups were performed with Fisher‟s LSD post hoc test.  Linear Regression was used to determine the relationship between heart rate reserve (HRreserve) and age, and years post transplant. Data are presented as means ± standard deviation (SD), and significance was set at P < 0.05. All statistical tests were run with and without HTR subject #02.     35 Chapter Four: Results  4.1. Participant Characteristics  Participant characteristics are shown in Table 3.1.  There were no statistical significances in body mass index (BMI), resting BP or resting pulsitility index (PI).  By design the DC were younger than the HTR and AM groups.  Differences were observed between the HTR and DC as well as the AM and DC groups for resting MCAv and cerebrovascular resistance (CVR).  The HTR (inclusive of subject #02) had a lower VO2peak than the AM and DC comparison groups by 27% and 50% respectively (Table 3.1.). When subject #02 and matched controls were excluded, the HTR had a lower VO2peak than the AM and DC comparison groups by 34% and 56% respectively (Table 3.2.). Removal of this subject, however, did not influence any other variables.   4.2. Incremental Exercise Test  Cerebrovascular, cardiovascular and pulmonary responses at rest and during the incremental exercise test are summarized in Table 3.3. (all subjects) and Table 3.4. (without HTR subject #02 and matched controls).   36 Table 4.1.  Cardiovascular, Pulmonary and Cerebrovascular responses at rest and during the incremental exercise test at 50%, 70%, 90% and peak VO2 (all subjects)   MCAvmean (cm/s) MCAvsys (cm/s) MCAvdia (cm/s) HR (bpm) HRreserve (bpm) BPmean (mmHg) BPsys (mmHg) BPdia (mmHg) PET CO2 (mmHg) CVR (mmHg/cm/s) PI (AU)  Rest HTR 40 ± 12 63 ± 20 28 ± 10 93 ± 9 0 ± 0 99 ± 4 123 ± 5 87 ± 7 28 ± 5 2.7 ± 0.7 0.9 ± 0.2 AM 41 ± 7 70 ± 12 27 ± 7 74 ± 10* 0 ± 0 104 ± 19 134 ± 26 82 ± 12 29 ± 3 2.6 ± 0.6 1.0 ± 0.3 DC 69 ± 9†‡ 119 ± 18†‡ 45 ± 7†‡ 72 ± 13† 0 ± 0 91 ± 6 117 ± 11 78 ± 8 37 ± 4†‡ 1.3 ± 0.2†‡ 1.0 ± 0.2 50% VO2peak HTR 46 ± 10 80 ± 19 28 ± 7 107 ± 8 14 ± 5 107 ± 6 149 ± 12 86 ± 7 33 ± 3 2.5 ± 0.6 1.1 ± 0.2 AM 52 ± 10 97 ± 16 29 ± 9 100 ± 10 26 ± 19 110 ± 12 169 ± 21* 81 ± 10 37 ± 3 2.2 ± 0.3 1.3 ± 0.3 DC 84 ± 11†‡ 159 ± 23†‡ 47 ± 9†‡ 122 ± 19†‡ 51 ± 7†‡ 99 ± 6‡ 143 ± 11‡ 77 ± 8 43 ± 4†‡ 1.2 ± 0.2†‡ 1.3 ± 0.2 70% VO2peak HTR 47 ± 11 87 ± 22 28 ± 7 125 ± 3 32 ± 11 118 ± 12 176 ± 24 88 ± 10 34 ± 2 2.6 ± 0.8 1.2 ± 0.2 AM 54 ± 8 107 ± 12* 27 ± 8 125 ± 19 51 ± 28 120 ± 13 197 ± 28 81 ± 10 37 ± 2* 2.2 ± 0.4 1.5 ± 0.3* DC 84 ± 11†‡ 162 ± 18†‡ 45 ± 10†‡ 164 ± 13†‡ 92 ± 7†‡ 105 ± 6†‡ 163 ± 13‡ 76 ± 8† 44 ± 4†‡ 1.3 ± 0.3†‡ 1.4 ± 0.2 90% VO2peak HTR 46 ± 11 88 ± 24 25 ± 7 142 ± 7 49 ± 11 126 ± 11 196 ± 17 91 ± 12 32 ± 2 3.0 ± 1.0 1.4 ± 0.3 AM 54 ± 6 112 ± 8* 25 ± 8 143 ± 19 69 ± 28 127 ± 11 214 ± 20 83 ± 10 35 ± 5 2.4 ± 0.4 1.6 ± 0.3 DC 75 ± 8†‡ 148 ± 20†‡ 38 ± 6†‡ 185 ± 8†‡ 113 ± 15†‡ 109 ± 7†‡ 178 ± 9†‡ 74 ± 8† 36 ± 4† 1.5 ± 0.2†‡ 1.5 ± 0.2 100% VO2peak HTR 45 ± 11 88 ± 25 23 ± 7 152 ± 10 60 ± 15 128 ± 9 202 ± 16 91 ± 13 28 ± 3 3.1 ± 1.0 1.4 ± 0.3 AM 53 ± 8  109 ± 13  24 ± 7  154 ± 18 80 ± 28 129 ± 13  221 ± 17* 84 ± 17  32 ± 4 2.5 ± 0.5 1.6 ± 0.2 DC 63 ± 9† 132 ± 24† 28 ± 8 196 ± 5†‡ 124 ± 12†‡ 111 ± 7†‡ 187 ± 12‡ 73 ± 8† 28 ± 5 1.8 ± 0.2† 1.7 ± 0.3  Values are means ± SD. Heart transplant recipient (HTR); age-matched (AM); donor population control (DC); middle cerebral artery velocity (MCAv); heart rate (HR); blood pressure (BP); end tidal CO2 (PET CO2); cerebrovascular resistance (CVR) and pulsitility index (PI). Statistical significance was set at P < 0.05, *denotes significance between HTR vs. AM, †denotes significance between HTR vs. DC, ‡denotes significance between AM vs. DC.       37 Table 4.2.  Cardiovascular, Pulmonary and Cerebrovascular responses at rest and during the incremental exercise test at 50%, 70%, 90% and peak VO2 (without HTR subject #02 and matched controls)   MCAvmean (cm/s) MCAvsys (cm/s) MCAvdia (cm/s) HR (bpm) HRreserve (bpm) BPmean (mmHg) BPsys (mmHg) BPdia (mmHg) PET CO2 (mmHg) CVR (mmHg/cm/s) PI (AU)  Rest HTR 41 ± 13 64 ± 22 29 ± 10 93 ± 10 0 ± 0 98 ± 3 124 ± 5 85 ± 6 27 ± 5 2.6 ± 0.7 0.9 ± 0.3 AM 42 ± 8 71 ± 12 27 ± 8 76 ± 10* 0 ± 0 98 ± 16 133 ± 28 80 ± 12 29 ± 3 2.5 ± 0.4 1.1 ± 0.3 DC 67 ± 7†‡ 113 ± 13†‡ 44 ± 7†‡ 71 ± 14† 0 ± 0 90 ± 7 117 ± 12 77 ± 9 37 ± 4†‡ 1.4 ± 0.3†‡ 1.0 ± 0.2 50% VO2peak HTR 46 ± 11 79 ± 21 28 ± 8 106 ± 9 13 ± 5 106 ± 7 150 ± 13 84 ± 7 33 ± 3 2.5 ± 0.7 1.1 ± 0.2 AM 51 ± 11 97 ± 18 28 ± 9 99 ± 10 24 ± 19 106 ± 7 165 ± 20 78 ± 5 36 ± 3 2.1 ± 0.4 1.4 ± 0.3 DC 82 ± 10†‡ 154 ± 20†‡ 46 ± 9†‡ 120 ± 20‡ 50 ± 7†‡ 98 ± 6 143 ± 12‡ 76 ± 8† 43 ± 4†‡ 1.2 ± 0.2†‡ 1.3 ± 0.2 70% VO2peak HTR 47 ± 12 85 ± 24 28 ± 7 125 ± 3 32 ± 12 117 ± 13 174 ± 25 90 ± 10 33 ± 2 2.7 ± 0.8 1.2 ± 0.2 AM 54 ± 9 107 ± 13* 27 ± 8 123 ± 20 48 ± 30 118 ± 13 195 ± 30 79 ± 7* 37 ± 2 2.2 ± 0.4 1.5 ± 0.3* DC 82 ± 11†‡ 159 ± 17†‡ 44 ± 11†‡ 163 ± 14†‡ 92 ± 8†‡ 104 ± 6 163 ± 14‡ 75 ± 8† 43± 4†‡ 1.3 ± 0.3†‡ 1.4 ± 0.2 90% VO2peak HTR 46 ± 13 86 ± 26 25 ± 7 142 ± 8 49 ± 12 125 ± 12 203 ± 18 92 ± 12 32 ± 2 3.0 ± 1.1 1.3 ± 0.2 AM 55 ± 7 113 ± 8* 25 ± 8 143 ± 21 66 ± 30 127 ± 12 218 ± 17 83 ± 11 34 ± 5 2.3 ± 0.4 1.6 ± 0.3* DC 75 ± 9†‡ 149 ± 22†‡ 38 ± 7†‡ 186 ± 9†‡ 115 ± 15†‡ 109 ± 7†‡ 179 ± 10‡ 74 ± 9† 36 ± 4 1.5 ± 0.3†‡ 1.5 ± 0.2 100% VO2peak HTR 45 ± 12 85 ± 26 24 ± 7 151 ± 10 59 ± 16 130 ± 8 203 ± 17 94 ± 12 28 ± 2 3.2 ± 1.1 1.4 ± 0.2 AM 53 ± 8  111 ± 14  24 ± 7  152 ± 20 77 ± 29 124 ± 6  218 ± 17 78 ± 8*  32 ± 5 2.4 ± 0.3 1.6 ± 0.2 DC 64 ± 9† 136 ± 25† 28 ± 8 195 ± 6†‡ 125 ± 13†‡ 111 ± 8†‡ 187 ± 14‡ 73 ± 8† 29 ± 5 1.8 ± 0.2† 1.7 ± 0.3†  Values are means ± SD. Heart transplant recipient (HTR); age-matched (AM); donor population control (DC); middle cerebral artery velocity (MCAv); heart rate (HR); blood pressure (BP); end tidal CO2 (PET CO2); cerebrovascular resistance (CVR) and pulsitility index (PI). Statistical significance was set at P < 0.05, *denotes significance between HTR vs. AM, †denotes significance between HTR vs. DC, ‡denotes significance between AM vs. DC.     38 4.3. Rest  Resting mean (Figure 4.1.-A), systolic and diastolic MCAv were not significantly different between the HTR and AM groups; however, these variables were lower in the HTR and AM groups when compared to DC (Table 4.1.).  At rest, HR (Figure 4.1.-B) was significantly different for the HTR as compared to both the AM and DC.  No significant differences were reported in any of the resting mean, systolic or diastolic BP (Figure 4.1.-C).   The DC had higher (P<0.05) PET CO2 levels and lower CVR than both the HTR and AM.  No other significant differences were found at rest between the groups (Table 4.1.).  When subject #02 was excluded from the analysis, all of the previous findings were unchanged (Table 4.2.).     39   Figure 4.1. Resting values for (A) mean middle cerebral artery velocity (MCAv), (B) heart rate (HR) and (C) mean arterial blood pressure (MABP) for the heart transplant recipient (HTR), age-matched (AM) and donor population control (DC) groups.  Statistical significant differences between HTR vs. DC and AM vs. DC were observed in MCAv and HR, no statistical differences were noted between HTR vs. AM for any condition, nor between any of the HTR, AM or DC groups MABP. (Values are means ± SD. *denotes, P < 0.05)   HTR AM DC MA BP ( mm Hg ) 80 90 100 110 120 130 Heart Transplant Recipient Age-Matched Control Donor Population Control C HTR AM DC HR  (bp m) 60 80 100 120 B HTR AM DC Me an M CAv ( cm /s) 20 40 60 80 100 A * * * *  40 4.4. VO2peak  Relative VO2peak for all subjects (Figure 4.2.-A; Table 3.1) values for the HTR (25.4 ± 10.4 mL/kg/min) and AM (35.0 ± 8.7 mL/kg/min) were significantly lower than the DC (50.6  ± 6.7 mL/kg/min) (Table 3.1.). The HRpeak (Figure 4.3.-A) and HRreserve (Figure 4.3.-B) for the DC were significantly higher than both the HTR and AM groups, and there was a trend toward lower relative VO2peak and HRreserve between HTR and AM (P = 0.055, and P = 0.071 respectively).    When subject #02 was excluded from the analysis, there was a significant difference observed in the VO2peak between the HTR and AM groups (Figure 4.2.-B; Table 3.2.). At VO2peak, mean and systolic MCAv were significantly lower in the HTR as compared to the DC, but not significantly different from the AM.  The DC had lower MABP than both the HTR and AM (P<0.05).  Systolic BP was higher in the AM group, compared with both the HTR and DC.  The only other significant difference in BP at VO2peak was the lower diastolic BP for the DC when compared with the HTR.  The DC also had lower (p<0.05) CVR when compared to the HTR.  All other cerebrovascular, cardiovascular and cardiorespiratory VO2peak responses were not different between the HTR, AM and DC groups with all subjects included (Table 4.1.).  The exclusion of subject #02 resulted in the same findings except the significant differences observed between the HTR and AM groups in BP did not occur in the mean BP; rather, they occurred in both diastolic BP at both 70%and 100% of VO2peak (Table 4.2.).      41     Figure 4.2.  Relative VO2peak for the heart transplant recipient (HTR), age-matched (AM) and donor population control (DC) groups. (A) Data shown with the inclusion of all subjects, (B) Data shown with the exclusion of HTR subject #02. Statistical significant differences were observed between HTR vs. DC and AM vs. DC.  Values are means ± SD. *denotes, P < 0.05.   HTR AM DC R el a tive Pe a k V O 2  ( m L/kg/ m in ) 0 20 40 60 80 * * HTR AM DC R el a tive Pe a k V O 2  ( m L/kg/ m in ) 0 20 40 60 80 Heart Transplant Recipient Age-Matched Control Donor Population Control * ** B A A B   42    Figure 4.3. (A) Peak heart rate (HRpeak) and (B) heart rate reserve (HRreserve) for the heart transplant recipient (HTR), age- matched (AM) and donor population control (DC) groups.  Statistically significant differences observed between the HTR vs. DC and AM vs. DC in both HRpeak and HRreserve.  Although there were no statistically significant differences for either condition between HTR vs. AM, a lower trend was observed in HRreserve (P = 0.071). Significance between the HTR vs. AM groups was not reached upon the exclusion of subject #02 (not shown). Values are means ± SD. *denotes, P < 0.05. HTR AM DC He art  R ate  R ese rve  (b pm ) 0 50 100 150 200 Heart Transplant Recipient Age-Matched Control Donor Population Control B HTR AM DC Pe ak  H ear t R ate  (b pm ) 120 140 160 180 200 220 A * * * *  43 4.5. HRreserve Relationships  There was a significant negative relationship associated with HRreserve and age for the life-span of the entire subject pool (Figure 4.4.-A).   There was also a significant individual group relationship between HRreserve and age for the AM group (R 2 = 0.79, P = 0.008; data not shown).  There was no HRreserve relationship for either the HTR (R 2 = 0.13, P = 0.429) or DC (R2 = 0.02, P = 0.778).  The HRreserve for the DC were significantly higher than both the HTR and DC throughout the incremental exercise test (Table 4.1. and Table 4.2.).  The HTR also showed a lower trend in HRreserve as compared to the AM during the test; however significance was never reached (mild, P = 0.080; moderate, P = 0.070; intense, P = 0.075).  There was no evident relationship between HRreserve with years after transplant (Figure 4.4.- B) with or without HTR subject #02.    44    Figure 4.4. (A) Overall relationship between heart rate reserve (HRreserve) and age of all subjects - heart transplant recipients (HTR), age-matched (AM) and donor population controls (DC); and (B) the relationship between HRreserve and years post transplant.  Although not plotted, the relationship between age and HRreserve for each group (A) are as follows: HTR (R2 = 0.13, P = 0.429), AM (R2 = 0.79, P = 0.008), DC (R2 = 0.02, P = 0.778). Red circle indicates subject #02. Heart Rate Reserve (bpm) 30 40 50 60 70 80 90 Ye ars Pos t Tr ans pla nt 0 5 10 15 20 25 Age (years) 20 40 60 80 He art  R ate  R ese rve  (b pm ) 20 40 60 80 100 120 140 160 Heart Transplant Recipient Age-Matched Control Donor Population Control R 2  = 0.18, P = 0.341BA R 2  = 0.73, P < 0.001  45 4.6. Mild, Moderate and Intense Exercise  During mild (50% of VO2peak), moderate (70% of VO2peak) and intense (90% of VO2peak) exercise, MCAvmean (Figure 4.5.-A) was higher (P<0.05) in the DC compared to both the HTR and AM. However, MCAvmean was not significantly different between the HTR and AM groups.  From rest to mild exercise the MCAvmean for the DC demonstrated a large increase, plateaued as intensity increased to the moderate level, followed by a sharp decline as peak intensity was reached.  The HTR and AM groups demonstrated a different trend; both groups had a steady increase in MCAvmean up to the moderate intensity level, after which a plateau was obtained that continued to the end of the test to VO2Peak (Figure 4.5.-A). The HTR, AM and DC groups all demonstrated a trend of BP increasing with exercise intensity (Figure 4.5.-B).  During mild exercise the AM was significantly higher than the DC; however, HTR was not significantly different from either the AM or the DC.  In moderate exercise, intense and peak exercise the HTR and AM group were not different in BP; however, they were higher than the DC. The PET CO2 response (Figure 4.5.-C) for all three groups showed a similar trend to one another from rest to mild exercise, at which point the DC were significantly higher than both the HTR and AM.  All three groups experienced a small increase during moderate exercise and were significantly different from each other with DC and HTR having the highest and lowest values respectively.  The DC group experienced a sharper decline in PET CO2 during intense exercise than the HTR and AM groups; the only significant difference during intense exercise was between the HTR and DC.  There were no differences in PET CO2 during peak exercise (Table 4.1. and Table 4.2.).   46 Across majority of the incremental exercise test spectrum, the CVR of the HTR and AM were higher than the DC (Figure 4.5.-D; P<0.05).  Only at peak exercise intensity did this change, at which point only the CVR of the HTR was significantly higher than the DC; there were no differences between the HTR and AM groups.  There where no significant changes to the results when subject #02 was excluded from the analysis (Table 4.2.). During mild exercise, systolic MCAv (Figure 4.6.-A) for the DC was significantly higher than the HTR and AM.   All three groups were different during moderate and intense exercise; DC was higher than AM, and the HTR were lower than AM.  There was a slightly different pattern demonstrated in the diastolic MCAv (Figure 4.6.-B). For example, throughout the entire incremental exercise test, there were no differences between HTR and AM; however, the diastolic MCAv for DC was higher (P<0.05).  Only at peak exercise did the diastolic MCAv for the DC decrease enough that there was no longer any significant differences amongst the groups.  The only difference in PI throughout the incremental exercise test occurred during moderate exercise.  At which point the HTR were lower (P<0.05) than the AM (Figure 4.6-C; Table 4.1).    When subject #02 was removed from the analysis, there were no significant changes in the systolic or diastolic MCAv data (Table 4.2.).  However, during the analysis of the PI there were two changes to the significance findings: a difference between the HTR and AM at 90% VO2peak and between the HTR and DC at 100% of VO2peak.   47    Figure 4.5. (A) Mean middle cerebral artery velocity (MCAv), (B) mean blood pressure (BP), (C) end tidal CO2 (PET CO2), and (D) cerebrovascular resistance (CVR) values for the heart transplant recipient (HTR), age-matched (AM) and donor population control (DC) groups across the incremental exercise test. (Values are means ± SD. *denotes between HTR vs. AM, †denotes significance between HTR vs. DC, ‡denotes significance between AM vs. DC. Statistical significance was set at P < 0.05).  Data shown is for all subjects. Base 50% 70% 90% Peak En d  T id al  C O 2  ( m m H g ) 20 25 30 35 40 45 50 C Base 50% 70% 90% PeakCe rebro v ascu la r R es is tanc e (m m H g /c m /s ) 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 D Base 50% 70% 90% Peak M ean BP  ( m m H g ) 80 90 100 110 120 130 140 150 Heart Transplant Recipient Age-Matched Control Donor Population Control B Base 50% 70% 90% Peak M ean MC A v  ( cm /s ) 20 30 40 50 60 70 80 90 100 A †‡ †‡ †‡ †‡ †‡ ?†‡ †‡ † † †‡ †‡ †‡ †‡ † †‡ †‡ †‡ ‡ A B C D   48  Figure 4.6.  (A) Systolic middle cerebral artery velocity (MCAv), (B) Diastolic MCAv, and (C) Pulsatility Index (PI) values for the heart transplant recipient (HTR), age- matched (AM) and donor population control (DC) groups across the incremental exercise test.  (Values are means ± SD. *denotes between HTR vs. AM, †denotes significance between HTR vs. DC, ‡denotes significance between AM vs. DC. Statistical significance was set at P < 0.05). Data shown is for all subjects.  Base 50% 70% 90% Peak Pu ls iti lity  I n d e x 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 Heart Transplant Recipient Age-Matched Control Donor Population Control C * Base 50% 70% 90% Peak Diast o lic M CAv (c m /s ) 10 20 30 40 50 60 B †‡ †‡ †‡ †‡ Base 50% 70% 90% Peak S y st o lic M CAv (c m /s ) 0 40 80 120 160 200 A †‡ ?†‡ † ?†‡ †‡  49  Chapter Five: Discussion and Conclusion  5.1. Principle Findings  The two novel principle findings of this study were: 1) Although the general pattern of change in mean MCAv was comparable during exercise in HTR compared to AM, systolic MCAv was selectively reduced in the HTR during moderate and intense exercise; and 2) When compared to DC, a comparable „relative‟ elevation in mean MCAv was evident from rest to light intensity exercise; however, HTR and AM controls did not display comparable declines in MCAv at exercise intensities 70% of VO2peak and greater.  At these higher intensities the HTR and AM MCAv declines were similar to each other but blunted when compared to the DC.  These findings were unaltered with and without the inclusion of the highly trained HTR; subject #02 (Table 4.1. and 4.2.). Collectively, the similarities in the CBF response between HTR and AM during exercise are remarkable and highlights that a „younger‟ denervated donor heart does not adversely impact on CBF at rest or during exercise, even with the presence of immunosuppressive therapy and likely stiffer vasculature in HTR. Nevertheless, the influence of medication and/or arterial stiffness in the HTR may underpin the apparent differences in systolic MCA velocity and pulsation.   5.2. Influence of HT on CBF at Rest - Comparison with Previous Studies   To date, as mentioned, only three studies have quantified CBF following HT30-32. Gruhn et al.31 reported that CBF was reduced in patients with severe chronic heart failure by approximately 30% and that these levels were normalized with heart transplantation one month after transplant.  When the patients were re-examined at six months post transplant,   50 the CBF values did not differ from those observed one-month post. Ackerman138 wrote an editorial in response to the 2001 Gruhn et al.31 study and criticized the lack of data on hematocrit levels, as they have observed that hematocrit levels are lower in HTR – it was suggested that the lower hematocrit levels would reduce the viscosity in the blood and be able to account for virtually all of the increase in CBF.  Massaro et al.30 did report increases in CBF in all 14 of their subjects one month after receiving the HT; however, they were unable to correlate the 53% increase in CBF to the lower mean hematocrit levels. In the final study, Choi et al. 32 presented data on four patients with chronic heart failure that underwent heart transplantation; global CBF was raised by 25% two to four months post surgery.  Our study is the first report of how CBF is altered over the longer term (i.e. years – 1 to 22 years) following HT. Our findings show that CBF (as indexed by TCD) in HTR is similar to age- matched controls at rest (Table 4.1. and Table 4.2.). Based on the lack of correlational data between hematocrit and MCAv in the findings by Massaro et al.30 (HTR) and the confirmation by Baufreton et al.158 (healthy humans), we feel that MCAv values that were observed in the current study were not influenced by the hematocrit levels of either the HTR or their controls.  5.3. Young Heart, Old Brain: Influence of Aging on CBF  Over the course of normal human aging, physiological and psychological changes occur that result in the structural and functional alterations of the cardiovascular and cerebrovascular systems171, 172.  These alterations result in observed decreases in CBF over that possibly reflect a global decrease in cerebral perfusion151.  Ainslie et al.151, quantified this decline in CBF as a decrease in MCAv of approximately 1% per year through a   51 comprehensive study of 307 healthy male subjects.  Another key finding from this study was that regular endurance exercise training results in an increase in MCAv of approximately 17% across the aging spectrum151.  It was speculated that the underlying mechanisms for these improvements in CBF were due to the numerous cardiovascular benefits that are associated with regular physical training151.  These results also demonstrate that „normal‟ is really only a point of reference for comparison as living a healthy and active lifestyle can potentially delay pathological disorders that result in age-associated cerebrovascular related brain diseases151.  With healthy aging, CBF declines 25-30% between 20 and 80 years of age151, 173. Similarly, a longitudinal study by Fotenos et al.174 has shown there to be a reduction of 0.45% per year in total brain volume after 30 years of age. As expected, resting MCAv (Figure 4.1.-A; Table 4.1. and Table 4.2.) was higher in the DC compared with the AM and HTR participants173. Interestingly, despite a „younger‟ heart, the HTR had comparable declines in CBF as the AM controlled (Figure 4.5.-A). Thus, this apparent reduction in resting MCAv with age is likely due to the reduction in brain volume174 and therefore metabolism and blood flow175 rather than age- and HTR-related differences in cardiac function. In other words, an „old brain‟ is the fundamental cause of cerebral hypoperfusion rather than a „young heart‟ as the CBF in the HTR are responding in a similar fashion to the AM, even though their heart is more similar in age to the DC.  5.4. Influence of HTR on CBF Alterations During Exercise   MCAvmean was elevated ~20% in response to sub-maximal exercise in the young and older individuals with and without HT (Figure 4.5.-A). This magnitude of increase is similar to that observed by other authors in young participants100 and recently in old176. It is well   52 established, at least in healthy young humans, that MCAv is intensity dependent up until ~70% of VO2 max, after which it declines to near resting levels due to hyperventilation- induced hypocapnia120, 121. Below the ventilatory threshold the increase in CBF with exercise is likely driven by increases in PaCO2 and cerebral metabolism, as a result of increased functional activation with motor activity100; however, above this intensity, the large disproportional increase in ventilation results in a reduction in PaCO2 and concomitant reductions in CBF despite progressive elevations in the cerebral metabolism.  In contrast, in both HTR and age matched controls, we did not observe comparable declines in MCAv at maximal exercise intensity as evident in the AM (Figure 4.5.-A, Table 4.1. and Table 4.2.); thus, this influence seems to be due to age per se rather than related autonomic and vascular changes associated with HT.  The mechanisms by which older adults are less able to exhibit comparable levels of hypocapnia (mediated via exercise-induced hyperventilation) are not clear, but may be as a result of factors such as reduced lung and chest wall compliance177, 178, respiratory muscle fatigue179, and differences in age-related chemosensitivity180, 181.  5.5. Influence of Aging on End-Tidal PCO2  A number of factors may explain the apparently age-related reductions in PET CO2 (Table 4.1. and Table 4.2.).  Fundamentally, arterial PCO2 can be altered via respiratory mechanisms (alveolar ventilation) and renal (or acid-base) mechanisms (bicarbonate filtration/reabsorbtion).  In the respiratory system, arterial PCO2 diffuses from the blood stream into the alveoli where it can be expelled directly from the body.  In the renal system, bicarbonate is filtered by the glomeruli into the renal tubules, when bicarbonate levels are   53 below 24 mM/L, virtually all of the bicarbonate is reabsorbed, when bicarbonate levels exceed 28 mM/L, the excess above this value is expelled in the urine182.   With aging, there is decrease in the steady-state bicarbonate ion concentration and an increase in steady-state blood proton concentration183.  Decreases in PaCO2 with aging can be attributed to the progressive metabolic acidosis occurring due to the normal decline in renal function: reduction in size and number of glomeruli, impairments in electrolyte transfer in the renal tubules, and a decrease in renal blood flow184 which result in less bicarbonate filtration occurring. As well as the subsequent respiratory adaptations: less compliant chest wall, decrease in strength of respiratory muscles, alveoli dilatation, enlargement of airspaces, decrease in exchange surface area which combine to result in a decrease in air exchange within the lungs185.  These myriad of factors, alone or in combination, would seems to explain the higher baseline PET CO2 in DC as compared with the HTR and AM populations (Figure 4.5-C).    5.6. Differential Changes in Systolic MCA Velocity and Pulsatility Index  The superior location of the brain with respect to the heart means that a higher pressure, pulsatile flow may be more effective at overcoming the effect of gravity and maintaining CBF186.  Systolic MCAv was generally lower in the HTR across all exercise intensities but only reached statistical significance with the AM at 70% and 90% of the VO2peak (Table 4.1. and Table 4.2.; Figure 4.6.-A) i.e., a intensity close to or above the aerobic threshold. However, when subject #02 was removed from the analysis, the PI of the AM controls was also significantly higher than the HTR at 70% and 90% (Table 4.2.).  The higher trends shown systolic BP for the AM (Table 4.1. and Table 4.2.) in conjunction with   54 the PI and systolic MCAv indicate that there may be a stiffening of the vasculature that is responsible for these changes. Alternatively, the related reductions in BP at the higher exercise intensities in HTR were similar to the findings of Braith et al.3.  These reductions may be explained by the structural alternations experienced in the HTR during their low-flow state of CHF3; such changes may have led to an impairment in peripheral vasoconstrictor responsiveness post transplant3. Recently, Laurent et al.187, when observing the effects of systolic blood pressures in the circulatory system it is best to utilize propagative models (i.e. the Moens-Korteweg equation) as they assume the velocity at which the pulse wave travels along the vessel will have a finite value. Laurent et al.187 suggested that applying propagative models will represent a more realistic approach to how the arterial tree functions as a propagative model that is composed of a simple distensible vessel that terminates at the peripheral resistance and has elastic properties, which allow for the generation of pressure waves.  Using this notion, it is possible to understand how the increase in arterial stiffness would increase the cerebral perfusion pressure, and result in an increase in the systolic BP which would most likely influence the systolic MCAv and PI as noted in Table 4.1. and Table 4.2.   Furthermore, the PI (Figure 4.6.-C) was increased in response to exercise in the all of the groups (HTR, AM and DC). These increases in PI may be a result of changes in vascular tone that are due to the increased systolic BP and/or alterations in PET CO2 which indirectly influences this variable (Table 4.1. and Table 4.2.).  A decreased resistance/higher compliance system is beneficial, reducing the capacity of the vascular bed to respond to transient alterations in blood flow. It appears as though the HTR are well adapted to maintain   55 cerebral perfusion during exercise as well as both the AM and DC (Table 3.3.). This is a novel finding and warrants further investigation.   5.7.  Limitations  5.7.1. TCD Ultrasound  A limitation of TCD ultrasound is that it only measures CBF velocity (cm/s) and is not a measure of true CBF volume (mL/min/100g).  However, numerous studies have shown that the cross-sectional area of the conduit vessels in the brain remain constant163, 165-167 and as such the changes that are observed in CBFv are directly proportional to the global CBF both at rest and during exercise100,101.  It is recognized that changes in vessel diameter will significantly alter blood flow (radius to the fourth power, Equation 1.). However, several studies have measured vessel diameter directly measured in humans, over a wide range of mean arterial pressures and PET CO2, with diameter remaining relatively stable 163, 165-167. The quality of the TCD ultrasound signal is also dependant upon sufficient penetration of the temporal acoustic window. As we age, there is a general reduction in the quality of the acoustic window, Marinoni et al.188 have suggested that over time there may be changes to the reflection, scattering and absorption qualities, due to the effects of osteoporosis resulting in an increase in the amount of inadequate windows from 3.0% of males younger than 30 to 7.6% of males over 60 years old188.     56 5.7.2. Activity Matching  The HTR and AM controls were matched for BMI and activity levels; however, the young controls were recruited from a university based population and were not matched based on activity; they were only matched with BMI.  This may have resulted in a slight portion of the elevated MCAv values reported for the DC, as previous studies have shown that there can be up to a 17% increase in MCAv associated with long-term endurance training, irrespective of age151. However, subject #02 did not show an increased MCAv as compared with the rest of the HTR and AM which is probably due to a sample size issue as a larger N (>45) is typically needed to show a fitness influence on CBF189.  5.8. Implications   5.8.1. MCAv at Rest   The MCAv at rest in the HTR is comparable with their age and activity matched controls. When compared with the DC, the MCAv in both the HTR and AM groups were significantly lower, suggesting that the „older‟ brain and cerebrovasculature of the HTR plays a greater role than that of the „younger‟ heart.  5.8.2. MCAv During Incremental Exercise   During the incremental exercise test, the MCAv in HTR responded in a similar fashion as that of the AM, with and without the inclusion of subject #02.      57 The MCAv responses in both the HTR and AM did not decrease at the same rate as the DC during the higher exercise intensities (90% and 100% of VO2peak).  During the incremental exercise test there was very little variation in the PET CO2 and MCAv for both the HTR and AM.  However, the DC responded in a similar fashion up to aerobic threshold; at higher exercise intensities, the DC experienced a dramatic decrease in both the PET CO2 and MCAv (Figure 4.5.; Table 4.1. and Table 4.2.).  This finding highlights the powerful role of PaCO2 on MCAv, irrespective of the age of the brain or heart.  5.8.3. MCAv in HTR With and Without Subject #02   The removal of the „fit‟ HTR did not alter any of the main MCAv findings of this study.  Unfortunately, with only one highly fit HTR, it is not possible to examine the long- term training effects on MCAv in HTR as there was simply not a large enough sample size (n>45; 189) to show the training effects on MCAv.  A further study should be conducted with a much larger n to determine the effects of long-term training in HTR on MCAv.  5.9. Future Studies  To further understand the relationship between CBF and HTR, the following studies are proposed:  5.9.1. Effects of Long-Term Endurance Training on CBF in HTR  This study is proposed because the removal of the long-term trained HTR did not alter any of the main MCAv findings of this study.  This was most likely due to the   58 extremely low n (1) and a larger sample (n>45) would be most likely to show the training effects of HTR on MCAv.  5.9.2. Longitudinal Study of CBF in HTR  This study only provides a cross-sectional insight in the effects of long-term HT on CBF.  A study that follows a group of HTR throughout their post-transplant experience would enable a broader picture of the true long-term effects of HT on CBF regulation.  5.9.3. Effects of Incremental Exercise on CBF in End-Stage Heart Failure Patients  This study is proposed because the literature has shown that there is a known decrease in CBF in patients with end-stage heart failure32, 141-144.  Also, a VO2peak of less than 15.0 mL/kg/min is one of the criteria for receiving a HT.  It would be interesting to compare the CBF results of a population with a greatly reduced VO2peak to the HTR individuals in this study.  This could provide more insight into the benefits of HT on CBF.  5.10. Conclusion   During both rest and throughout the incremental exercise test, the HTR and AM had similar responses in their CBF, and these responses were different from DC. These findings held true with and without the inclusion of subject #02. Thus leading to the conclusion, that despite a suppressed VO2 Peak (and likely Q) cerebral blood flow is well maintained during incremental exercise in long – term heart transplant recipients.    59 Bibliography   1. Toledo-Pereyra LH. Heart transplantation. J Invest Surg. 2010; 23: 1-5.  2. Stehlik J, Edwards LB, Kucheryavaya AY, Aurora P, Christie JD, Kirk R, Dobbels F, Rahmel AO, Hertz MI. 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Tymchak, MD, FRCPC   U of A, Division of Cardiology    (780) 407-1574 I. Paterson, MD, FRCPC   U of A, Division of Cardiology                (780) 407-7729 R. Thompson, Ph.D.    U of A, Dept. of Biomedical Engineering               (780) 492-8665 P. Ainslie, Ph.D.                        UBCO, Human kinetics     (250) 807-8089  BACKGROUND AND PURPOSE Heart transplant recipients have reduced cardiac output (pumping capacity of the heart) during exercise or cardiac (un)loading (as occurs when going from lying to standing position quickly). The effect that reduced cardiac output reserve has on blood flow to the brain (called cerebral blood flow) has not been studied in heart transplant recipients. The aim of this study is to assess the acute effect of a ventilator challenge, cardiac unloading, and cycle exercise on cardiac function, cerebral blood flow and arterial  blood pressure in heart transplant recipients and age-matched healthy individuals.    DESCRIPTION OF THE STUDY If you decide to participate in this study, the total time commitment will be one extra visit (4 hours) at the Alberta Cardiovascular and Stroke Research Centre in the Mazankowski Alberta Heart Institute. Refusal to participate in this study will not affect your treatment at the University of Alberta Hospital.   RESEARCH PROCEDURES Assessment of cardiac function, cerebral blood flow and arterial blood pressure during ventilatroy challenge. Prior to this test, we will measure your height and weight. A number of electrodes (electrical contacts) will be placed on your chest and connected to a computer to measure your heart rate. A small cuff will be placed on your finger to measure your blood pressure.  A special headpiece with an ultrasound probed attached to it that will be positioned on your head to measure the blood flow to your brain.  Some gel and an ultrasound probe will be placed on your chest to measure your cardiac function. You will also be fitted with a nose clip and will breathe through a special mask that will be attached to a computer that will analyze your oxygen uptake and carbon dioxide (CO2) production. After resting measures are obtained, you will breathe a gas mixture (5% CO2 in 21% O2) for 3 min, followed by a brief recovery. You will then be asked to increase your breathing rate and depth for 3 minutes.    84  Assessment of cardiac function, cerebral blood flow and arterial blood pressure during cardiac unloading tests. After a 10-15 minute rest period of lying flat on your back, you will be instructed to sit up and stand as quickly as possible (within 3 seconds) during which time your heart rate, blood pressure and cerebral blood flow will be measured.   After a brief rest period, you to lie on your back in a comfortable, relaxed position with your legs inside  a custom built lower body pressure chamber. The chamber will be sealed snuggly around your waist. During the test, the pressure inside the chamber will be reduced with a household vacuum to alter the amount of blood returning to the heart. After a 5-miunte rest period, you will undergo three (-10 mmHg,   -20mmHg and -40 mmHg for 10 minutes at each stage) levels of decreased pressure during which time your heart rate, blood pressure, heart function and cerebral blood flow will be measured. If you become lightheaded, dizzy, or your blood pressure begins to drop the test will be stopped.  Assessment of cardiac function, cerebral blood flow and arterial blood pressure, during cycle exercise.  After resting measures of your heart rate, blood pressure, cardiac function, cerebral blood flow and oxygen uptake (using a special mouthpiece and collection tube attached to a computer that measures your O2 consumption) are obtained, you will begin cycling at a comfortable speed and the resistance that you will pedal against will become more difficult every two minutes until you feel you are no longer able to continue cycling. This test will be test will be completed in 10-15 minutes.   A specially trained health care worker will supervise the above tests.   POSSIBLE BENEFITS This study will determine your heart function, cerebral blood flow and blood pressure during a ventilatory challenge, cardiac unloading and during cycle exercise.   POSSIBLE RISKS During the lying to stand test you may feel lightheaded. During the lower body negative pressure test you may feel lightheaded or become unconscious, however, your heart rate, blood pressure, cardiac function and cerebral blood flow will be carefully monitored to prevent this from occurring. In addition, the pressure will be returned to normal immediately to reduce the feeling of light-headedness. The inflated finger cuff used to measure your blood pressure may result in a numb feeling that will disappear when the cuff is removed. The exercise that you will perform is considered safe. All testing and exercise sessions will be performed under appropriate supervision.  Data from individuals with/without heart disease suggests that the likelihood of having a heart attack or dying during an exercise test is 1 in 10,000 tests. The mouthpiece that is used during the exercise test may make your mouth feel dry. You may also experience temporary muscle soreness after exercising. There are no adverse effects associated with cardiac or cerebral ultrasound.  COSTS You will not have to pay for the tests that you will perform in this study. However, you may be coming to the University of Alberta more often than if you were not participating in this study. You will be reimbursed for your parking and food costs associated with participating in the study.    CONTACTS Please contact the investigators listed below if you have any questions or concerns:  M.  Haykowsky, PhD at (780) 492-5970  W. Tymchak, MD FRCPC (780) 407-1574     85 CONFIDENTIALITY Personal records will be kept confidential. Only the persons listed above will have access to your data. Any report published as a result of this study will not identify you by name.  For this study, the researchers will need to access your personal health records for health information such as medical history and test results.  The health information collected as part of this study will be kept confidential unless release is required by law, and will be used only for the purpose of the research study.  By signing the consent form you give permission to the study staff to access any personally identifiable health information which is under the custody of other health care professionals as deemed necessary for the conduct of the research.   In addition to the investigators listed, the Health Research Ethics Board may have access to your personal health records to monitor the research and verify the accuracy of study data.      By signing the consent form you give permission for the collection, use and disclosure of your medical records.  Study information is required to be kept for 7 years.  Even if you withdraw from the study, the medical information which is obtained from you for study purposes will not be destroyed.  You have a right to check your health records and request changes if your personal information is incorrect.  VOLUNTARY PARTICIPATION  You are free to withdraw from this study at any time without giving a reason. If knowledge gained from this study or any other study becomes available which could influence your decision to continue, you will be promptly informed.  If you have any questions or concerns about any aspect this study, you may contact the Health Research Ethics Board at 492-9724.  This office has no affiliation with the study investigators.    86 Appendix II: Participant Consent Form  CONSENT FORM CARDIAC FUNCTION, CEREBRAL BLOOD FLOW AND ARTERIAL BLOOD PRESSURE DURING VENTILATORY CHALLENGE, CARDIAC UNLOADING AND CYCLE EXERCISE, IN HEART TRANSPLANT RECIPIENTS    INVESTIGATORS  M. Haykowsky, Ph.D.   U of A, Dept. of Physical Therapy   (780) 492-5970 W. Tymchak, MD FRCPC   U of A, Division of Cardiology    (780) 407-1574 I. Paterson, MD, FRCPC   U of A, Division of Cardiology                (780) 407-7729 R. Thompson, Ph.D.    U of A, Dept. of Biomedical Engineering               (780) 492-8665 P. Ainslie, Ph.D.                        UBCO, Human kinetics     (250) 807-8089  Please answer the following questions:  Yes No Do you understand that you are being asked to be in a research study? ___ ___  Have you read and received a copy of an attached information sheet? ___ ___  Do you understand the benefits and risks in taking part in this research study? ___ ___  Have you had an opportunity to ask questions and discuss this study? ___ ___  Do you understand that you are free to withdraw from the study at any time without having to give a reason and without affecting your future medical care? ___ ___  Has the issue of confidentiality been explained to you, and do you understand who will have access to your medical records? ___ ___  Do you want the investigators to inform you family doctor that you are participating in this study? ___ ___  Who explained this study to you?      _______________________________________________  Yes No I agree to take part in this study: ___ ___  ________________________________________________  _________________ Signature of Participant Date  ________________________________________________ Printed Name of Participant ________________________________________________  __________________ Signature of Witness Date  I believe that the person signing this form understands what is involved in the study and voluntarily agrees to participate. ________________________________________________  __________________ Signature of Investigator or Designee Date   87 Appendix III: Literature Review of Cardiac Output on Cerebral Blood Flow Table 1.1. Summary of the current literature observing effects of cardiac output on cerebral blood flow Studya Population (n) CBF Technique Intervention (n) Findings Q-CBF Relationship Schienber g 195059 CHF (14) (patients with anaemia were excluded) Kety and Schmidt60 N2O technique General observations CHF patients when compared with normal population showed:   CBF ↓ 39% (40 vs 65 mL/min/100g), a-v O2 difference ↑ 41% (8.6 vs 6.1 volumes per cent).  The Author noted: the means may not be directly comparable as the CHF mean age was 40 Male and female and normal were all under 30 and healthy males. NF Schieve  et al. 195161 Human subjects of varying health (14) Kety and Schmidt 60 N2O technique Adrenocorto -tropic Hormone intake With Adrenocortotropic Hormone intake:   CBF ↓ 18% (61 to 50 mL/min/100g), MABP ↑ 9% (90 to 98 mmHg), CVR ↑ 32% (1.6 to 2.1 units). NF Sensen- bach et al. 196062 CHF (37): mild to moderate (24), severe (13)  (patients with anaemia, pulmonary disease, renal disease or cerebral arterioscleros is were excluded) Kety and Schmidt60 N2O technique Observation of CBF as compared to CHF classification state in lucid patients CHF patients as compared to normal data:   Mild to moderate CHF: ↔ CBF (51 vs 48 mL/min/100g), MABP ↑ 30% (121 vs 94 mmHg), CVR ↔ (2.49 vs 2.35 units).  Severe CHF: ↓ 21% CBF (39 vs 48 mL/min/100g), MABP ↔ (100 vs 94 mmHg), CVR ↑ 13% (2.66 vs 2.35 units). NF   88 Studya Population (n) CBF Technique Intervention (n) Findings Q-CBF Relationship Einsberg et al. 196063 Severe CHF (24) (patients with mental disease, pulmonary disease, renal disease or cerebral arterio- sclerosis were excluded) Kety and Schmidt60 N2O technique Observation of CBF as compared to CHF classification state in confused patients Confused severe CHF patients as compared to; normal controls and lucid severe CHF patients:   CHF vs normal: ↓ 46% CBF (26 vs 48 mL/min/100g), MABP ↔ (98 vs 94 mmHg), CVR ↑ 54% (3.61 vs 2.35 units).  Confused vs Lucid: ↓ 40% CBF (26 vs 43 mL/min/100g), MABP ↑ 15% (106 vs 92 mmHg), CVR ↑ 62% (3.54 vs 2.18 units). NF Andrews  et al. 196964 Unanesthet- ized Rats (14); Anesthetized Rats (14)  Fractional uptake of Iodo- antipryrine -133I  Temperature change Unanesthetized Rats showed a 50% ↓ in CBF with temperature ↓:  0.77 (37°C) to 0.38 mL/min (25°C), Hematocrit values were reported to ↑ from 0.45 (37°C) to 0.59 (25°C), Q ↓ 48% from 286 (37°C) to 149 mL/kg/min (25°C);  Anesthetized Rats showed a small ↓ in CBF with temperature ↓:  CBF, Hematocrit and Q values were not reported. + Shapiro and Chawla 196965 Human patients with complete heart block (5) Kety and Schmidt60 N2O technique Cardiac pacemaker controlled HR set at 30- 40, 60, 70, 90 and 100 beats per minute HR 30-40: Q (2.8 L/min), CBF control (100%), CVR control (100%), PaCO2 (38 mmHg) HR 60: Q (3.6 L/min), CBF control (118%), CVR control (88%), PaCO2 (41 mmHg) HR 70: Q (3.4 L/min), CBF control (118%), CVR control (94%), PaCO2 (40 mmHg) HR 90: Q (3.4 L/min), CBF control (118%), CVR control (93%), PaCO2 (41 mmHg) HR 100: Q (3.6 L/min), CBF control (124%), CVR control (98%), PaCO2 (39 mmHg) +   89 Studya Population (n) CBF Technique Intervention (n) Findings Q-CBF Relationship Davis and Sundt 198066 Cats (70) 133Xenon washout Hypovolumi c (10); Propanol (10); Isoproterenol (10); Hyper- volumic (10);  Angiotensio n (10); Propanolol- Angiotensio n (10); Phenoxy- benzamine- angiotension (10) Hypovolumic: Q ↓ 32%, CBF ↓ 24%, MABP ↓ slightly    Propanol: HR ↓ 28%, Q ↓ 23%, CBF ↓ 30%, MABP not reported   Isoproterenol: HR ↑ 20%, Q ↑ 38%, CBF ↔, MABP not reported  Hypervolumic: HR ↓ slightly, Q ↓ 7%, CBF ↓ 22%, MABP ↓ slightly    Angiotension: induced changes in MABP had varied results in CBF, overall a slight ↑ in CBF and ↓ in Q with ↑ in MABP (changes less than 10%)  Propanolol-Angiotension: MABP ↓ 6%, Q ↓ 26%, CBF ↓ 12%  Phenoxybenzamine-angiotension: MABP ↓ 24%, Q ↔, CBF ↓ 15% + / - Moustafa and Hopewell 198167 Female rats (28) 125Iodo- antipyrine extraction technique Age Changes: 6 months (6) 9 months (4) 12 months (6) 15 months (6) 18 months (6) 6 months (6): Q 257 mL/min/kg body weight, CBF 6.7 mL/min/kg body weight;  9 months (4): Q 183 mL/min/kg body weight, CBF 6.0 mL/min/kg body weight;   12 months (6): Q 208 mL/min/kg body weight, CBF 4.8 mL/min/kg body weight;   15 months (6): Q 163 mL/min/kg body weight, CBF 4.1 mL/min/kg body weight;   18 months (6): Q 157 mL/min/kg body weight, CBF 3.5 mL/min/kg body weight +   90 Studya Population (n) CBF Technique Intervention (n) Findings Q-CBF Relationship Cook et al. 198368 Healthy males (7) 133Xenon inhalation Intravenous admin- istration of Epoprostenol Pre infusion: ABPsys 123 mmHg, ABPdia 76 mmHg, HR 73 bpm, Q 4.2 L/min, CBF 46.0 mL/min/100g 15 minutes post: ABPsys 122 mmHg, ABPdia 73 mmHg, HR 79 bpm, Q 4.4 L/min, CBF 40.7 mL/min/100g 30 minutes post: ABPsys 123 mmHg, ABPdia 73 mmHg, HR 79 bpm, Q 4.1 L/min, CBF 38.9 mL/min/100g - Hermanse n et al. 198469 Newborn dogs (13) Radioactiv e microspher e reference organ technique Metabolic Acidosis With increasing acidosis, there was a 27% ↓ in Q, ↔ in HR, ↔ in CBF. Concluded that decrease in Q due to ↓ in SV and had no effect on CBF. - Barringto n et al. 198770 Adult monkeys (6) 133Xenon clearance Negative pressure ventilation via external high frequency oscillation Control (mechanical ventilation) vs. external high frequency oscillation:  ↔ Q (2.83 vs 3.03 L/min) ↔ CBF (43.9 vs 39.0 mL/min/100g) ↔ MABP (119 vs 113 mmHg) NF   91 Studya Population (n) CBF Technique Intervention (n) Findings Q-CBF Relationship Mutch et al. 199071 New Zealand white Rabbits (16) Summing weighted flows in all brain regions and comparing to brain weight of the decapitated rabbit Isoflurane injection (8); Halothane injection (8) Isoflurane vs. Halothane at each injection stage:  Baseline: MABP (64.3 vs 67.2 mmHg), Q (472 vs 506 mL/min), CBF (0.7 vs 0.9 mL/g/min), PE injection (N/A), ICP (2.9 vs 2.1), CPP (61.4 vs 65.5) Flow 2: MABP (79.2 vs 81.5 mmHg), Q (385 vs 363 mL/min), CBF (0.9 vs 1.1 mL/g/min), PE injection (8.5 vs 15.3 μg/kg/min), ICP (3.1 vs 2.4 mmHg), CPP (76.1 vs 79.5 mmHg) Flow 3: MABP (89.6 vs 94.7 mmHg), Q (388 vs 263 mL/min), CBF (1.0 vs 1.8 mL/g/min), PE injection (12.8 vs 28.6 μg/kg/min), ICP (3.4 vs 3.6 mmHg), CPP (86.2 vs 91.5 mmHg) Flow 4: MABP (105.4 vs 106.6 mmHg), Q (341 vs 226 mL/min), CBF (1.2 vs 2.5 mL/g/min), PE injection (16.9 vs 63.5 μg/kg/min), ICP (3.7 vs 5.1 mmHg), CPP (101.7 vs 102.0 mmHg) Flow 5: MABP (116.6 vs 115.8 mmHg), Q (325 vs 150 mL/min), CBF (1.4 vs 3.8 mL/g/min), PE injection (21.1 vs 137.3 μg/kg/min), ICP (5.0 vs 6.7 mmHg), CPP (111.7 vs 109.8 mmHg) - van der Giessen et al. 199072 Conscious cross-breed pigs (14) Inspection of the brain during dissection Nimodipine injection Highest nimodipine dosage as compared to baseline data:  HR ↑ 42%, Q ↑ 54%, CBF ↔, CVR ↔, MABP ↓ 9%  -   92 Studya Population (n) CBF Technique Intervention (n) Findings Q-CBF Relationship Bouma and Muizelaar 199073 Human patients with intact or impaired cerebral autoregulatio n (35) 133Xenon inhalation or 133Xenon injection Phenyl- ephrine, Arfonad and Mannitol admin- istration  Pre-drug vs pos-drug comparison for intact and impaired cerebral autoregulation:                      (Absolute values not reported for Q)  Intact cerebral autoregulation:                      Phenylephrine: CBF (36 vs 35 mL/100g/min, ↓ 1%), MABP (96 vs 127 mmHg, ↑ 32%), ICP (18 vs 20 mmHg, ↑ 11%), Q (↑ 7%)  Arfonad: CBF (41 vs 40 mL/100g/min, ↓ 2%), MABP (111 vs 86 mmHg, ↓ 23%), ICP (18 vs 21 mmHg, ↑ 17%), Q (↓ 10%) Mannitol: CBF (37 vs 37 mL/100g/min, ↔), MABP (101 vs 100 mmHg, ↓ 1%), ICP (18 vs 13 mmHg, ↓ 28%), Q (↑ 17%)  Impaired cerebral autoregulation:   Phenylephrine: CBF (21 vs 32 mL/100g/min, ↑ 53%), MABP (92 vs 123 mmHg, ↑ 34%), ICP (18 vs 16 mmHg, ↓ 11%), Q (↑ 15%)  Arfonad: CBF (66 vs 46 mL/100g/min, ↓ 31%), MABP (108 vs 77 mmHg, ↓ 29%), ICP (16 vs 18 mmHg, ↑ 13%), Q (↑ 22%) Mannitol: CBF (20 vs 27 mL/100g/min, ↑ 40%), MABP (94 vs 90 mmHg, ↓ 4%), ICP (17 vs 16 mmHg, ↓ 6%), Q (↑ 1%) -   93 Studya Population (n) CBF Technique Intervention (n) Findings Q-CBF Relationship Levine et al. 199474 Healthy males (13) MCAv via TCD Orthostatic challenge (lower body negative pressure) Arrows indicate changes from rest at –15, -30, -40 and –55 mmHg  Rest: Q (5.7 L/min), non-fainter MCAv (58 cm/s), fainter MCAv (52 cm/s), HR (58 bpm), SV (98 mL), MABP (82 mmHg) -15 mmHg: Q (5.7 L/min, ↔), non-fainter MCAv (61 cm/s, ↔), fainter MCAv (52 cm/s, ↔), HR (63 bpm, ↑ 9%), SV (90 mL, ↓ 9%), MABP (83 mmHg, ↔) -30 mmHg: Q (5.0 L/min, ↓ 12%), non-fainter MCAv (56 cm/s, ↔), fainter MCAv (50 cm/s, ↔), HR (68 bpm, ↑ 17%), SV (73 mL, ↓ 11%), MABP (83 mmHg, ↔) -40 mmHg: Q (4.2 L/min, ↓ 24%), non-fainter MCAv (55 cm/s, ↔), fainter MCAv (45 cm/s, ↓ 14%), HR (76 bpm, ↑ 31%), SV (55 mL, ↓ 33%), MABP (84 mmHg, ↔) -55 mmHg: Q (3.5 L/min, ↓ 39%), non-fainter MCAv (50 cm/s, ↓ 14%), fainter MCAv (N/A), HR (90 bpm, ↑ 55%), SV (90 mL, ↓ 57%), MABP (88 mmHg, ↑ 6%) -   94 Studya Population (n) CBF Technique Intervention (n) Findings Q-CBF Relationship Ide et al. 199875 Healthy human volunteers (9) MCAv via TCD Effect of reducing the ability to change Q at rest, during handgrip and cycling exercise with metroprolol (β1 adrenergic blockade) Control vs Metroprolol at rest and during Handgrip and cycling at (83, 113, 147, and 186 watts)  Rest: MCAv (59 vs 56 cm/s), percent change in Q (100 vs 92 %), HR (67 vs 65 bpm), MABP (94 vs 86 mmHg), PaCO2 (5.1 vs 4.9 kPa)  Handgrip: MCAv (67 vs 63 cm/s), percent change in Q (116 vs 100 %), HR (77 vs 71 bpm), MABP (106 vs 100 mmHg), PaCO2 (5.1 vs 5.0 kPa)  83 watts: MCAv (72 vs 64 cm/s), percent change in Q (218 vs 185 %), HR (113 vs 99 bpm), MABP (105 vs 96 mmHg), PaCO2 (5.2 vs 5.3 kPa) 113 watts: MCAv (72 vs 66 cm/s), percent change in Q (260 vs 222 %), HR (135 vs 114 bpm), MABP (112 vs 103 mmHg), PaCO2 (5.1 vs 5.2 kPa) 147 watts: MCAv (69 vs 64 cm/s), percent change in Q (315 vs 258 %), HR (158 vs 128 bpm), MABP (119 vs 106 mmHg), PaCO2 (4.9 vs 4.9 kPa) 186 watts: MCAv (66 vs 62 cm/s), percent change in Q (349 vs 293 %), HR (169 vs 130 bpm), MABP (122 vs 112 mmHg), PaCO2 (4.4 vs 4.6 kPa) + Larsen et al. 200076 Fulminant hepatic failure patients (9) MCAv via TCD Norepin- ephrine infusion Measurements before and during Norepinephrine infusion:  MCAv (49 vs 63 cm/s, ↑ 30%), Q (5.7 vs 7.1 L/min, ↑ 25%), MABP (75 vs 97 mmHg, ↑ 32%), SV (59 vs 71 mL, ↑ 20%) +   95 Studya Population (n) CBF Technique Intervention (n) Findings Q-CBF Relationship Gruhn et al. 200131 Congestive heart failure (12); Heart transplant recipients (5) 133Xenon inhalation Pre- and post- heart transplant- ation Measurements taken pre- and post-transplant:  CBF (36 vs 50 mL/min/100g, ↑ 39%), CI (2.5 vs 2.4 L/min/m2, ↔), MABP (76 vs 93 mmHg, ↑ 22%) NF Van Lieshout et al. 200145 Healthy young adult humans (10) MCAv via TCD 5 minutes of Standing, followed by 2 minutes of leg tensing and 2 final minutes of standing  Stand 1: Q (4.2 L/min), MCAv (58 cm/s), MABP (75 mmHg), PaCO2 (4.7 kPa)  Tensing: Q (4.5 L/min), MCAv (64 cm/s), MABP (79 mmHg), PaCO2 (5.0 kPa)  Stand 2: Q (4.2 L/min), MCAv (58 cm/s), MABP (77 mmHg), PaCO2 (4.7 kPa) + Wilson et al. 200277 Healthy humans (9) MCAv via TCD Normo- thermic tilt and whole body heating tilt (with and without pre- cooling) Measures are pre and during experimental condition:  Normothermic – no cooling: MCAv (62 vs 54 cm/s), Q (6.5 vs 5.2 L/min), MABP (87 vs 82 mmHg), HR (57 vs 72 bpm) Normothermic – pre-cooling: MCAv (61 vs 60 cm/s), Q (6.3 vs 5.8 L/min), MABP (86 vs 93 mmHg), HR (57 vs 63 bpm) Heating – no cooling: MCAv (55 vs 43 cm/s), Q (8.0 vs 5.7 L/min), MABP (83 vs 71 mmHg), HR (88 vs 126 bpm) Heating – pre-cooling: MCAv (54 vs 59 cm/s), Q (8.2 vs 5.2 L/min), MABP (88 vs 93 mmHg), HR (87 vs 72 bpm) + / -   96 Studya Population (n) CBF Technique Intervention (n) Findings Q-CBF Relationship Joseph et al. 200378 Human patients with vasoplasm after subarachnoid hemorrhage (16) Xenon CT system Hyper- volemia: Phenyl- ephrine to ↑ MABP (5); Dobutamine to ↑ Q (5) Measures are pre and post treatment:  Phenylephrine: CBF (19.2 vs 33.3 mL/100g/min, ↑ 73%), MABP (103.4 vs 132.0 mmHg, ↑ 28%)  Dobutamine: CBF (24.8 vs 35.7 mL/100g/min, ↑ 44%), CI (4.0 vs 6.0 L/min/m2, ↑ 50%)  + Brown et al. 200379 Human Adults (13) MCAv via TCD Orthostatic challenge: Lower body negative pressure Lower body negative pressure settings of 0, -10, -20, -30, -40, -50 mmHg (% change to baseline measure)  0 mmHg: MCAvmean (71 cm/s), Q (6.86 L/min), SV (116 mL), HR (60 bpm), MABP (86 mmHg) -10 mmHg:  MCAvmean (67 cm/s, ↓ 6%), Q (6.37 L/min, ↓ 7%), SV (109 mL, ↓ 6%), HR (59 bpm, ↔), MABP (90 mmHg, ↑ 5%) -20 mmHg:  MCAvmean (70 cm/s, ↔), Q (5.60 L/min, ↓ 18%), SV (93 mL, ↓ 20%), HR (61 bpm, ↔), MABP (89 mmHg, ↔) -30 mmHg:  MCAvmean (70 cm/s, ↔), Q (4.65 L/min, ↓ 32%), SV (74 mL, ↓ 36%), HR (65 bpm, ↑ 8%), MABP (86 mmHg, ↔) -40 mmHg:  MCAvmean (61 cm/s, ↓ 14%), Q (4.19 L/min, ↓ 39%), SV (63 mL, ↓ 44%), HR (71 bpm, ↑ 18%), MABP (89 mmHg, ↑ 3%) -50 mmHg:  MCAvmean (57 cm/s, ↓ 20%), Q (3.80 L/min, ↓ 45%), SV (48 mL, ↓ 59%), HR (83 bpm, ↑ 38%), MABP (91 mmHg, ↑ 6%) + / - Kusaka et al. 200580 Newborn infant humans (17) Mulitchan nel Near infrared spectrosco py Observa- tional Significant positive relation between Q and CBF:  Linear regression line equation: CBF = 0.03Q + 8.71, R2 = 0.70, P = 0.002 +   97 Studya Population (n) CBF Technique Intervention (n) Findings Q-CBF Relationship Ogoh et al. 200581 Healthy human males (7) MCAv via TCD Rest and Exercise with: infusions of albumin to ↑ Q; lower body negative pressure to ↓ Q Rest and exercise values for: lower body negative pressures at 8 Torr and 16 Torr, albumin infusion 1 and 2.  (% changes are to control values).  Rest: 8 Torr: MCAv (63 cm/s, ↓ 5%), Q (5.8 L/min, ↓ 11%), HR (70 bpm, ↑ 6%), MABP (96 mmHg, ↔), PaCO2 (39 mmHg, ↔) 16 Torr: MCAv (62 cm/s, ↓ 6%), Q (5.3 L/min, ↓ 18%), HR (74 bpm, ↑ 12%), MABP (99 mmHg, ↔), PaCO2 (40 mmHg, ↔) Albumin 1: MCAv (71 cm/s, ↑ 8%), Q (8.2 L/min, ↑ 26%), HR (82 bpm, ↑ 24%), MABP (92 mmHg, ↓ 4%), PaCO2 (41 mmHg, ↔) Albumin 2: MCAv (73 cm/s, ↑ 11%), Q (8.5 L/min, ↑ 31%), HR (84 bpm, ↑ 27%), MABP (91 mmHg, ↓ 5%), PaCO2 (41 mmHg, ↔)  Exercise: 8 Torr: MCAv (70 cm/s, ↔), Q (14.1 L/min, ↔), HR (123 bpm, ↑ 8%), MABP (104 mmHg, ↓ 5%), PaCO2 (42 mmHg, ↔) 16 Torr: MCAv (68 cm/s, ↔), Q (13.7 L/min, ↔), HR (130 bpm, ↑ 14%), MABP (106 mmHg, ↔), PaCO2 (41 mmHg, ↔) Albumin 1: MCAv (74 cm/s, ↔), Q (16.5 L/min, ↑ 12%), HR (129 bpm, ↑ 13%), MABP (105 mmHg, ↓ 4%), PaCO2 (41 mmHg, ↔) Albumin 2: MCAv (74 cm/s, ↔), Q (18.5 L/min, ↑ 26%), HR (133 bpm, ↑ 17%), MABP (106 mmHg, ↔), PaCO2 (41 mmHg, ↔) + / - Massaro et al. 200630 Congestive heart failure humans (22), heart transplantatio n subset (14) MCAv via TCD A subset (14) under heart transplant- ation Pre and 2-4 month post transplant measures:  CBF (45.1 vs 69.1 cm/s, ↑ 53%), MABP (75.2 vs 94.6 mmHg, ↑ 26%), hematocrit (38.8 vs 31.2 %, ↓ 20%) NF   98 Studya Population (n) CBF Technique Intervention (n) Findings Q-CBF Relationship Choi et al. 200632 Advanced heart failure humans (52), underwent heart transplant- ation (4) Radionucli de angiograph y small subset (4) under heart transplant- ation Pre and 2-4 month post transplant measures:  CBF (35.5 vs 44.3 mL/100g/min, ↑ 25%) LVEF (19.8 vs 66.8 %, ↑ 237%) NF Drombro wski et al. 200682 Adult male dogs (31) Stable isotope labeled microspher es with post mortem tissue evaluation Induced chronic obstructive hydro- cephalus Average CBF and Q for baseline, 2 weeks, 4-6 weeks, 8-12 weeks and 16+ weeks  (% change is in relation to current measure vs baseline)  Chronic Hydrocephalus:  CBF (mL/min/g): base (0.587), 2 weeks (0.317, ↓ 46%), 4-6 weeks (0.298, ↓ 49%), 8-12 weeks (0.384, ↓ 34%), 16+ weeks (0.242, ↓ 59%) Q (mL/min): base (4.97), 2 weeks (3.52, ↓ 29%), 4-6 weeks (3.46, ↓ 30%), 8-12 weeks (3.91, ↓ 21%), 16+ weeks (3.06, ↓ 38%)  Surgical Control:  CBF (mL/min/g): base (0.661), 2 weeks (0.312, ↓ 53%), 4-6 weeks (0.403, ↓ 39%), 8-12 weeks (0.408, ↓ 38%), 16+ weeks (0.582, ↓ 12%) Q (mL/min): base (4.10), 2 weeks (2.86, ↓ 30%), 4-6 weeks (2.62, ↓ 36%), 8-12 weeks (3.35, ↓ 18%), 16+ weeks (3.77, ↓ 8%) +   99 Studya Population (n) CBF Technique Intervention (n) Findings Q-CBF Relationship Ogoh et al. 200783 Healthy males (8) MCAv via TCD Moderate and heavy exercise before and after cardio- selective β1- adrenergic blockade (metro- prolol) Effects of rest, moderate and heavy exercise under control and metropolol conditions.  (Q and SV data are presented as % change from control rest, other values are absolute measures)  Control:  Rest: MCAv (60 cm/s), Q (0 %), SV (0 %), HR (66 bpm), MABP (92 mmHg), PaCO2 (5.2 kPa) Moderate: MCAv (75 cm/s), Q (182 %), SV (40 %), HR (130 bpm), MABP (104 mmHg), PaCO2 (5.7 kPa) Heavy: MCAv (67 cm/s), Q (276 %), SV (46 %), HR (171 bpm), MABP (115 mmHg), PaCO2 (5.4 kPa)  Metropolol:  Rest: MCAv (52 cm/s), Q (-5 %), SV (-6 %), HR (67 bpm), MABP (85 mmHg), PaCO2 (5.1 kPa) Moderate: MCAv (59 cm/s), Q (146 %), SV (35 %), HR (117 bpm), MABP (93 mmHg), PaCO2 (5.4 kPa) Heavy: MCAv (56 cm/s), Q (235 %), SV (46 %), HR (163 bpm), MABP (109 mmHg), PaCO2 (5.1 kPa) -   100 Studya Population (n) CBF Technique Intervention (n) Findings Q-CBF Relationship Ogawa et al. 200784 Healthy human males (12) MCAv via TCD Orthostatic challenge: lower body negative pressure; ↑ central blood volume with saline injections Data for baseline, lower body negative pressure (-15 and –30 mmHg), saline injection (15 and 30 mL/kg), (% changes are to baseline):  Baseline: MCAv (67 cm/s), Q (4.16 L/min), SV (72.9 mL), HR (58 bpm), MABP (80 mmHg), PET CO2 (41 mmHg) -15 mmHg: MCAv (67 cm/s, ↔), Q (3.64 L/min, ↓ 12%), SV (64.3 mL, ↓ 12%), HR (61 bpm, ↔), MABP (80 mmHg, ↔), PET CO2 (40 mmHg, ↔) -30 mmHg: MCAv (68 cm/s, ↔), Q (2.80 L/min, ↓ 33%), SV (44.2 mL, ↓ 39%), HR (68 bpm, ↑ 17%), MABP (77 mmHg, ↔), PET CO2 (40 mmHg, ↔) 15 mL/kg: MCAv (72 cm/s, ↑ 7%), Q (4.73 L/min, ↑ 14%), SV (78.0 mL, ↔), HR (61 bpm, ↔), MABP (86 mmHg, ↔), PET CO2 (40 mmHg, ↔) 30 mL/kg: MCAv (73 cm/s, ↑ 9%), Q (5.08 L/min, ↑ 22%), SV (75.5 mL, ↔), HR (68 bpm, ↑ 17%), MABP (82 mmHg, ↔), PET CO2 (40 mmHg, ↔) + / - Ogoh et al. 201085 Healthy human males (9) MCAv via TCD Acute hypotension by releasing thigh cuffs before and after; metropolol and glycol- pyruvate plus metropolol Data for control, metropolol, and glycopyruvate plus metropolol,  (% changes are to control):  Control: MCAv (73 cm/s), Q (6.8 L/min), SV (103 mL), HR (67 bpm), MABP (85 mmHg), PET CO2 (39.7 mmHg) Metropolol: MCAv (70 cm/s, ↔), Q (6.0 L/min, ↓ 12%), SV (105 mL, ↔), HR (58 bpm, ↓ 13%), MABP (85 mmHg, ↔), PET CO2 (39.8 mmHg, ↔) Glycopyruvate+: MCAv (72 cm/s, ↔), Q (8.6 L/min, ↑ 27%), SV (92, ↓ 11%), HR (95 bpm, ↑ 42%), MABP (86 mmHg, ↔), PET CO2 (39.1 mmHg, ↔) -   101 Studya Population (n) CBF Technique Intervention (n) Findings Q-CBF Relationship Deegan et al. 201086 Healthy human volunteers (19) MCAv and ACAv via TCD Transient systemic hypoper- fusion induced by thigh cuff deflation (supine and seated) Supine and Seated data (% changes are to condition baseline):  Supine: Baseline: MCAv (81.7 cm/s), ACAv (62.2 cm/s), Cardiac Index (2.2 L/min/m2), HR (64 bpm), MABP (90 mmHg) Pre-release: MCAv (83.7 cm/s, ↑ 3%), ACAv (62.8 cm/s, ↑ 1%), Cardiac Index (2.7 L/min/m2, ↑ 17%), HR (73 bpm, ↑ 14%), MABP (101 mmHg, ↑ 12%) Post-release: MCAv (79.2 cm/s, ↓ 3%), ACAv (58.5 cm/s, ↓ 6%), Cardiac Index (3.0 L/min/m2, ↑ 36%), HR (78 bpm, ↑ 22%), MABP (84 mmHg, ↓ 7%)  Seated: Baseline: MCAv (75.2 cm/s), ACAv (59.7 cm/s), Cardiac Index (2.5 L/min/m2), HR (73 bpm), MABP (91 mmHg) Pre-release: MCAv (77.6 cm/s, ↑ 3%), ACAv (60.3 cm/s, ↑ 1%), Cardiac Index (3.0 L/min/m2, ↑ 20%), HR (78 bpm, ↑ 7%), MABP (98 mmHg, ↑ 8%) Post-release: MCAv (71.1 cm/s, ↓ 5%), ACAv (52.9 cm/s, ↓ 11%), Cardiac Index (3.2 L/min/m2, ↑ 28%), HR (88 bpm, ↑ 21%), MABP (79 mmHg, ↓ 13%) - a Studies are in chronological order. n = number; CHF = congestive heart failure; CBF = cerebral blood flow; a-v O2 = atrial-venous Oxygen difference; MABP = mean arterial blood pressure; ABPsys = systolic arterial blood pressure; ABPdia = diastolic arterial blood pressure; N2O = Nitrogen Oxide; CVR = cerebrovascular resistance; Q = cardiac output; HR = heart rate; SV = stroke volume; CI = cardiac index; PaCO2 = partial pressure of arterial Carbon Dioxide; ICP = intracranial pressure; LVEF = left ventricular ejection fraction; MCA = middle cerebral artery; MCAv = middle cerebral artery velocity; ACA = anterior cerebral artery; ACAv = anterior cerebral artery velocity; TCD = transcranial Doppler.  ↑ indicates increase; ↓ indicates decrease; ↔ indicates no change.  + indicates positive Q-CBF relationship; - indicates no Q-CBF relationship; NF = no findings for Q-CBF relationship.  102 Appendix IV: Raw Data – Output from LabChart      MCAv       Systolic Relative Diastolic Relative Mean Relative Change to      (cm/s) (%) (cm/s) (%) (cm/s) (%) Baseline AM 1 Base 69.450 0.000 35.490 0.000 46.810 0.000 0.000 AM 2 Base 71.120 0.000 30.630 0.000 44.127 0.000 0.000 AM 3 Base 84.700 0.000 18.830 0.000 40.787 0.000 0.000 AM 4 Base 69.090 0.000 21.580 0.000 37.417 0.000 0.000 AM 5 Base 51.770 0.000 19.200 0.000 30.057 0.000 0.000 AM 6 Base 58.600 0.000 26.590 0.000 37.260 0.000 0.000 AM 7 Base 82.080 0.000 36.220 0.000 51.507 0.000 0.000 HTR 1 Base 50.980 0.000 26.050 0.000 34.360 0.000 0.000 HTR 2 Base 57.400 0.000 23.770 0.000 34.980 0.000 0.000 HTR 3 Base 63.680 0.000 33.860 0.000 43.800 0.000 0.000 HTR 4 Base 78.720 0.000 22.110 0.000 40.980 0.000 0.000 HTR 6 Base 101.270 0.000 47.500 0.000 65.423 0.000 0.000 HTR 7 Base 43.550 0.000 21.100 0.000 28.583 0.000 0.000 HTR 8 Base 48.480 0.000 22.440 0.000 31.120 0.000 0.000 DC 1 Base 123.000 0.000 43.000 0.000 69.667 0.000 0.000 DC 2 Base 91.000 0.000 38.000 0.000 55.667 0.000 0.000 DC 3 Base 105.000 0.000 54.000 0.000 71.000 0.000 0.000 DC 4 Base 149.000 0.000 49.000 0.000 82.333 0.000 0.000 DC 5 Base 120.000 0.000 40.000 0.000 66.667 0.000 0.000 DC 6 Base 119.000 0.000 37.000 0.000 64.333 0.000 0.000 DC 7 Base 125.000 0.000 52.000 0.000 76.333 0.000 0.000 AM 1 50% 87.490 0.260 32.500 -0.084 50.830 0.086 4.020 AM 2 50% 88.600 0.246 27.160 -0.113 47.640 0.080 3.513 AM 3 50% 103.290 0.219 17.800 -0.055 46.297 0.135 5.510 AM 4 50% 82.340 0.192 21.160 -0.019 41.553 0.111 4.137 AM 5 50% 91.790 0.773 26.260 0.368 48.103 0.600 18.047 AM 6 50% 97.380 0.662 32.590 0.226 54.187 0.454 16.927 AM 7 50% 130.550 0.591 45.450 0.255 73.817 0.433 22.310 HTR 1 50% 60.560 0.188 27.970 0.074 38.833 0.130 4.473 HTR 2 50% 86.990 0.516 25.470 0.072 45.977 0.314 10.997 HTR 3 50% 75.830 0.191 28.790 -0.150 44.470 0.015 0.670 HTR 4 50% 98.290 0.249 25.670 0.161 49.877 0.217 8.897 HTR 6 50% 105.690 0.044 41.330 -0.130 62.783 -0.040 -2.640 HTR 7 50% 51.760 0.189 18.130 -0.141 29.340 0.026 0.757 HTR 8 50% 80.640 0.663 31.510 0.404 47.887 0.539 16.767 DC 1 50% 172.000 0.398 48.000 0.116 89.333 0.282 19.667 DC 2 50% 132.000 0.451 36.000 -0.053 68.000 0.222 12.333 DC 3 50% 145.000 0.381 57.000 0.056 86.333 0.216 15.333 DC 4 50% 189.000 0.268 52.000 0.061 97.667 0.186 15.333   103 DC 5 50% 131.000 0.092 46.000 0.150 74.333 0.115 7.667 DC 6 50% 165.000 0.387 35.000 -0.054 78.333 0.218 14.000 DC 7 50% 177.000 0.416 54.000 0.038 95.000 0.245 18.667     MCAv       Systolic Relative Diastolic Relative Mean Relative Change to      (cm/s) (%) (cm/s) (%) (cm/s) (%) Baseline AM 1 70% 98.140 0.413 32.670 -0.079 54.493 0.164 7.683 AM 2 70% 104.360 0.467 24.550 -0.198 51.153 0.159 7.027 AM 3 70% 111.220 0.313 15.620 -0.170 47.487 0.164 6.700 AM 4 70% 95.700 0.385 21.820 0.011 46.447 0.241 9.030 AM 5 70% 103.330 0.996 24.950 0.299 51.077 0.699 21.020 AM 6 70% 105.830 0.806 30.490 0.147 55.603 0.492 18.343 AM 7 70% 132.450 0.614 39.790 0.099 70.677 0.372 19.170 HTR 1 70% 63.870 0.253 26.980 0.036 39.277 0.143 4.917 HTR 2 70% 100.080 0.744 25.590 0.077 50.420 0.441 15.440 HTR 3 70% 81.930 0.287 27.590 -0.185 45.703 0.043 1.903 HTR 4 70% 113.120 0.437 27.500 0.244 56.040 0.367 15.060 HTR 6 70% 109.550 0.082 37.280 -0.215 61.370 -0.062 -4.053 HTR 7 70% 54.210 0.245 15.660 -0.258 28.510 -0.003 -0.073 HTR 8 70% 85.960 0.773 33.300 0.484 50.853 0.634 19.733 DC 1 70% 176.000 0.431 53.000 0.233 94.000 0.349 24.333 DC 2 70% 133.000 0.462 30.000 -0.211 64.333 0.156 8.667 DC 3 70% 156.000 0.486 54.000 0.000 88.000 0.239 17.000 DC 4 70% 183.000 0.228 48.000 -0.020 93.000 0.130 10.667 DC 5 70% 146.000 0.217 42.000 0.050 76.667 0.150 10.000 DC 6 70% 168.000 0.412 34.000 -0.081 78.667 0.223 14.333 DC 7 70% 172.000 0.376 53.000 0.019 92.667 0.214 16.333 AM 1 90% 115.990 0.670 37.290 0.051 63.523 0.357 16.713 AM 2 90% 108.430 0.525 19.450 -0.365 49.110 0.113 4.983 AM 3 90% 117.720 0.390 14.720 -0.218 49.053 0.203 8.267 AM 4 90% 107.490 0.556 22.450 0.040 50.797 0.358 13.380 AM 5 90% 104.580 1.020 25.100 0.307 51.593 0.717 21.537 AM 6 90% 102.430 0.748 26.990 0.015 52.137 0.399 14.877 AM 7 90% 126.000 0.535 31.830 -0.121 63.220 0.227 11.713 HTR 1 90% 61.990 0.216 22.440 -0.139 35.623 0.037 1.263 HTR 2 90% 97.650 0.701 18.520 -0.221 44.897 0.283 9.917 HTR 3 90% 87.420 0.373 27.020 -0.202 47.153 0.077 3.353 HTR 4 90% 117.530 0.493 25.960 0.174 56.483 0.378 15.503 HTR 6 90% 112.250 0.108 33.800 -0.288 59.950 -0.084 -5.473 HTR 7 90% 53.460 0.228 13.500 -0.360 26.820 -0.062 -1.763 HTR 8 90% 84.440 0.742 32.540 0.450 49.840 0.602 18.720 DC 1 90% 182.000 0.480 41.000 -0.047 88.000 0.263 18.333 DC 2 90% 127.000 0.396 28.000 -0.263 61.000 0.096 5.333 DC 3 90% 129.000 0.229 47.000 -0.130 74.333 0.047 3.333 DC 4 90% 142.000 -0.047 38.000 -0.224 72.667 -0.117 -9.667   104 DC 5 90% 147.000 0.225 38.000 -0.050 74.333 0.115 7.667 DC 6 90% 168.000 0.412 32.000 -0.135 77.333 0.202 13.000 DC 7 90% 143.000 0.144 41.000 -0.212 75.000 -0.017 -1.333     MCAv       Systolic Relative Diastolic Relative Mean Relative Change to      (cm/s) (%) (cm/s) (%) (cm/s) (%) Baseline AM 1 Peak 108.090 0.556 28.910 -0.185 55.303 0.181 8.493 AM 2 Peak 107.340 0.509 21.720 -0.291 50.260 0.139 6.133 AM 3 Peak 120.620 0.424 15.550 -0.174 50.573 0.240 9.787 AM 4 Peak 101.220 0.465 22.460 0.041 48.713 0.302 11.297 AM 5 Peak 95.520 0.845 20.520 0.069 45.520 0.514 15.463 AM 6 Peak 99.160 0.692 23.860 -0.103 48.960 0.314 11.700 AM 7 Peak 133.070 0.621 36.330 0.003 68.577 0.331 17.070 HTR 1 Peak 60.610 0.189 20.730 -0.204 34.023 -0.010 -0.337 HTR 2 Peak 105.170 0.832 15.390 -0.353 45.317 0.296 10.337 HTR 3 Peak 91.080 0.430 24.520 -0.276 46.707 0.066 2.907 HTR 4 Peak 115.070 0.462 25.900 0.171 55.623 0.357 14.643 HTR 6 Peak 110.540 0.092 32.260 -0.321 58.353 -0.108 -7.070 HTR 7 Peak 52.070 0.196 13.190 -0.375 26.150 -0.085 -2.433 HTR 8 Peak 81.450 0.680 31.100 0.386 47.883 0.539 16.763 DC 1 Peak 178.000 0.447 32.000 -0.256 80.667 0.158 11.000 DC 2 Peak 118.000 0.297 27.000 -0.289 57.333 0.030 1.667 DC 3 Peak 109.000 0.038 41.000 -0.241 63.667 -0.103 -7.333 DC 4 Peak 113.000 -0.242 30.000 -0.388 57.667 -0.300 -24.667 DC 5 Peak 127.000 0.058 17.000 -0.575 53.667 -0.195 -13.000 DC 6 Peak 147.000 0.235 23.000 -0.378 64.333 0.000 0.000 DC 7 Peak 134.000 0.072 26.000 -0.500 62.000 -0.188 -14.333      105     Pulsitility MAP     Index Systolic Relative Diastolic Relative Mean Relative       (mmHg) (%) (mmHg) (%) (mmHg) (%) AM 1 Base 0.725 120.000 0.000 80.000 0.000 99.000 0.000 AM 2 Base 0.918 129.000 0.000 87.000 0.000 101.000 0.000 AM 3 Base 1.615 183.000 0.000 92.000 0.000 122.333 0.000 AM 4 Base 1.270 104.000 0.000 62.000 0.000 76.000 0.000 AM 5 Base 1.084 117.000 0.000 70.000 0.000 85.667 0.000 AM 6 Base 0.859 140.000 0.000 95.000 0.000 110.000 0.000 AM 7 Base 0.890 143.000 0.000 87.000 0.000 105.667 0.000 HTR 1 Base 0.726 121.000 0.000 88.000 0.000 99.000 0.000 HTR 2 Base 0.961 120.000 0.000 97.000 0.000 104.667 0.000 HTR 3 Base 0.681 120.000 0.000 92.000 0.000 101.333 0.000 HTR 4 Base 1.381 129.000 0.000 76.000 0.000 93.667 0.000 HTR 6 Base 0.822 131.000 0.000 83.000 0.000 99.000 0.000 HTR 7 Base 0.785 120.000 0.000 90.000 0.000 100.000 0.000 HTR 8 Base 0.837 120.000 0.000 83.000 0.000 95.333 0.000 DC 1 Base 1.148 124.000 0.000 78.000 0.000 93.333 0.000 DC 2 Base 0.952 129.000 0.000 89.000 0.000 102.333 0.000 DC 3 Base 0.718 106.000 0.000 74.000 0.000 84.667 0.000 DC 4 Base 1.215 117.000 0.000 83.000 0.000 94.333 0.000 DC 5 Base 1.200 109.000 0.000 80.000 0.000 89.667 0.000 DC 6 Base 1.275 103.000 0.000 78.000 0.000 86.333 0.000 DC 7 Base 0.956 131.000 0.000 62.000 0.000 85.000 0.000 AM 1 50% 1.082 150.000 -0.351 82.000 0.025 104.667 -0.197 AM 2 50% 1.290 168.000 0.302 83.000 -0.046 111.333 0.102 AM 3 50% 1.847 195.000 0.066 78.000 -0.152 117.000 -0.044 AM 4 50% 1.472 136.000 0.308 72.000 0.161 93.333 0.228 AM 5 50% 1.362 171.000 0.462 70.000 0.000 103.667 0.210 AM 6 50% 1.196 191.000 0.364 101.000 0.063 131.000 0.191 AM 7 50% 1.153 170.000 0.189 80.000 -0.080 110.000 0.041 HTR 1 50% 0.839 154.000 0.273 93.000 0.057 113.333 0.145 HTR 2 50% 1.338 140.000 0.167 95.000 -0.021 110.000 0.051 HTR 3 50% 1.058 169.000 0.408 82.000 -0.109 111.000 0.095 HTR 4 50% 1.456 135.000 0.047 74.000 -0.026 94.333 0.007 HTR 6 50% 1.025 149.000 0.137 82.000 -0.012 104.333 0.054 HTR 7 50% 1.146 138.000 0.150 89.000 -0.011 105.333 0.053 HTR 8 50% 1.026 157.000 0.308 84.000 0.012 108.333 0.136 DC 1 50% 1.388 138.000 0.113 77.000 -0.013 97.333 0.043 DC 2 50% 1.412 159.000 0.233 86.000 -0.034 110.333 0.078 DC 3 50% 1.019 138.000 0.302 73.000 -0.014 94.667 0.118 DC 4 50% 1.403 143.000 0.222 84.000 0.012 103.667 0.099 DC 5 50% 1.143 137.000 0.257 79.000 -0.013 98.333 0.097 DC 6 50% 1.660 128.000 0.243 78.000 0.000 94.667 0.097 DC 7 50% 1.295 156.000 0.191 61.000 -0.016 92.667 0.090   106     Pulsitility MAP     Index Systolic Relative Diastolic Relative Mean Relative       (mmHg) (%) (mmHg) (%) (mmHg) (%) AM 1 70% 1.201 176.000 -0.238 85.000 0.063 115.333 -0.115 AM 2 70% 1.560 208.000 0.612 83.000 -0.046 124.667 0.234 AM 3 70% 2.013 205.000 0.120 72.000 -0.217 116.333 -0.049 AM 4 70% 1.591 156.000 0.500 69.000 0.113 98.000 0.289 AM 5 70% 1.535 241.000 1.060 83.000 0.186 135.667 0.584 AM 6 70% 1.355 211.000 0.507 98.000 0.032 135.667 0.233 AM 7 70% 1.311 182.000 0.273 79.000 -0.092 113.333 0.073 HTR 1 70% 0.939 170.000 0.405 94.000 0.068 119.333 0.205 HTR 2 70% 1.477 193.000 0.608 82.000 -0.155 119.000 0.137 HTR 3 70% 1.189 162.000 0.350 82.000 -0.109 108.667 0.072 HTR 4 70% 1.528 141.000 0.093 77.000 0.013 98.333 0.050 HTR 6 70% 1.178 213.000 0.626 87.000 0.048 129.000 0.303 HTR 7 70% 1.352 166.000 0.383 91.000 0.011 116.000 0.160 HTR 8 70% 1.036 189.000 0.575 106.000 0.277 133.667 0.402 DC 1 70% 1.309 167.000 0.347 75.000 -0.038 105.667 0.132 DC 2 70% 1.601 178.000 0.380 85.000 -0.045 116.000 0.134 DC 3 70% 1.159 152.000 0.434 72.000 -0.027 98.667 0.165 DC 4 70% 1.452 165.000 0.410 82.000 -0.012 109.667 0.163 DC 5 70% 1.357 145.000 0.330 78.000 -0.025 100.333 0.119 DC 6 70% 1.703 156.000 0.515 77.000 -0.013 103.333 0.197 DC 7 70% 1.284 179.000 0.366 60.000 -0.032 99.667 0.173 AM 1 90% 1.239 193.000 -0.165 86.000 0.075 121.667 -0.066 AM 2 90% 1.812 221.000 0.713 94.000 0.080 136.333 0.350 AM 3 90% 2.100 209.000 0.142 65.000 -0.293 113.000 -0.076 AM 4 90% 1.674 206.000 0.981 75.000 0.210 118.667 0.561 AM 5 90% 1.541 244.000 1.085 92.000 0.314 142.667 0.665 AM 6 90% 1.447 233.000 0.664 87.000 -0.084 135.667 0.233 AM 7 90% 1.490 190.000 0.329 83.000 -0.046 118.667 0.123 HTR 1 90% 1.110 185.000 0.529 97.000 0.102 126.333 0.276 HTR 2 90% 1.762 193.000 0.608 82.000 -0.155 119.000 0.137 HTR 3 90% 1.281 180.000 0.500 81.000 -0.120 114.000 0.125 HTR 4 90% 1.621 181.000 0.403 80.000 0.053 113.667 0.214 HTR 6 90% 1.309 226.000 0.725 87.000 0.048 133.333 0.347 HTR 7 90% 1.490 199.000 0.658 96.000 0.067 130.333 0.303 HTR 8 90% 1.041 211.000 0.758 112.000 0.349 145.000 0.521 DC 1 90% 1.602 183.000 0.476 76.000 -0.026 111.667 0.196 DC 2 90% 1.623 191.000 0.481 86.000 -0.034 121.000 0.182 DC 3 90% 1.103 167.000 0.575 70.000 -0.054 102.333 0.209 DC 4 90% 1.431 172.000 0.470 78.000 -0.060 109.333 0.159 DC 5 90% 1.466 169.000 0.550 76.000 -0.050 107.000 0.193 DC 6 90% 1.759 176.000 0.709 76.000 -0.026 109.333 0.266 DC 7 90% 1.360 187.000 0.427 59.000 -0.048 101.667 0.196   107     Pulsitility MAP     Index Systolic Relative Diastolic Relative Mean Relative       (mmHg) (%) (mmHg) (%) (mmHg) (%) AM 1 Peak 1.432 212.000 -0.082 83.000 0.038 126.000 -0.033 AM 2 Peak 1.704 220.000 0.705 89.500 0.029 133.000 0.317 AM 3 Peak 2.078 225.000 0.230 68.000 -0.261 120.333 -0.016 AM 4 Peak 1.617 206.000 0.981 80.000 0.290 122.000 0.605 AM 5 Peak 1.648 247.000 1.111 70.000 0.000 129.000 0.506 AM 6 Peak 1.538 235.000 0.679 119.000 0.253 157.667 0.433 AM 7 Peak 1.411 200.000 0.399 77.000 -0.115 118.000 0.117 HTR 1 Peak 1.172 183.000 0.512 97.000 0.102 125.667 0.269 HTR 2 Peak 1.981 199.000 0.658 76.000 -0.216 117.000 0.118 HTR 3 Peak 1.425 203.000 0.692 82.000 -0.109 122.333 0.207 HTR 4 Peak 1.603 190.000 0.473 88.000 0.158 122.000 0.302 HTR 6 Peak 1.341 234.000 0.786 83.000 0.000 133.333 0.347 HTR 7 Peak 1.487 204.000 0.700 99.000 0.100 134.000 0.340 HTR 8 Peak 1.052 203.000 0.692 114.000 0.373 143.667 0.507 DC 1 Peak 1.810 202.000 0.629 79.000 0.013 120.000 0.286 DC 2 Peak 1.587 200.000 0.550 82.000 -0.079 121.333 0.186 DC 3 Peak 1.068 172.000 0.623 71.000 -0.041 104.667 0.236 DC 4 Peak 1.439 189.000 0.615 76.000 -0.084 113.667 0.205 DC 5 Peak 2.050 173.000 0.587 72.000 -0.100 105.667 0.178 DC 6 Peak 1.927 180.000 0.748 74.000 -0.051 109.333 0.266 DC 7 Peak 1.742 194.000 0.481 58.000 -0.065 103.333 0.216      108     HR Et CO2 CVR Yrs Post  Age     Absolute Absol Relative Absolute Relative         (bpm) (mmHg) (%) (mmHg/cm/s)) (%)     AM 1 Base 75.500 30.700 0.000 2.784 0.000   59.00 AM 2 Base 65.100 30.700 0.000 2.289 0.000   53.00 AM 3 Base 83.500 26.200 0.000 2.999 0.000   74.00 AM 4 Base 90.580 32.500 0.000 2.031 0.000   69.00 AM 5 Base 71.920 25.200 0.000 2.850 0.000   64.00 AM 6 Base 62.050 29.000 0.000 2.952 0.000   53.00 AM 7 Base 67.500 29.700 0.000 2.052 0.000   58.00 HTR 1 Base 90.580 31.900 0.000 2.881 0.000 12.0 63.00 HTR 2 Base 90.580 31.300 0.000 2.992 0.000 23.0 50.00 HTR 3 Base 96.120 27.400 0.000 2.314 0.000 6.0 65.00 HTR 4 Base 105.700 23.000 0.000 2.286 0.000 1.0 61.00 HTR 6 Base 87.260 30.300 0.000 1.513 0.000 8.5 61.00 HTR 7 Base 100.240 31.200 0.000 3.499 0.000 10.0 78.00 HTR 8 Base 77.670 19.600 0.000 3.063 0.000 4.5 54.00 DC 1 Base 75.000 37.000 0.000 1.340 0.000   20.00 DC 2 Base 83.000 32.000 0.000 1.838 0.000   21.00 DC 3 Base 53.000 42.000 0.000 1.192 0.000   24.00 DC 4 Base 78.000 39.000 0.000 1.146 0.000   26.00 DC 5 Base 74.000 34.000 0.000 1.345 0.000   18.00 DC 6 Base 86.000 40.000 0.000 1.342 0.000   22.00 DC 7 Base 53.000 36.000 0.000 1.114 0.000   20.00 AM 1 50% 109.170 36.200 0.179 2.059 -0.260   59.00 AM 2 50% 109.170 36.400 0.186 2.337 0.021   53.00 AM 3 50% 93.550 32.400 0.237 2.527 -0.157   74.00 AM 4 50% 83.090 33.100 0.018 2.246 0.106   69.00 AM 5 50% 95.670 38.400 0.524 2.155 -0.244   64.00 AM 6 50% 105.070 39.900 0.376 2.418 -0.181   53.00 AM 7 50% 105.470 40.000 0.347 1.490 -0.274   58.00 HTR 1 50% 103.790 36.200 0.135 2.918 0.013 12.0 63.00 HTR 2 50% 111.060 36.100 0.153 2.393 -0.200 23.0 50.00 HTR 3 50% 115.420 32.900 0.201 2.496 0.079 6.0 65.00 HTR 4 50% 114.340 28.500 0.239 1.891 -0.173 1.0 61.00 HTR 6 50% 95.300 32.100 0.059 1.662 0.098 8.5 61.00 HTR 7 50% 111.490 33.500 0.074 3.590 0.026 10.0 78.00 HTR 8 50% 97.620 34.900 0.781 2.262 -0.262 4.5 54.00 DC 1 50% 125.000 45.000 0.216 1.090 -0.187   20.00 DC 2 50% 142.000 38.000 0.188 1.623 -0.117   21.00 DC 3 50% 97.000 49.000 0.167 1.097 -0.080   24.00 DC 4 50% 135.000 46.000 0.179 1.061 -0.074   26.00 DC 5 50% 129.000 39.000 0.147 1.323 -0.016   18.00 DC 6 50% 134.000 46.000 0.150 1.209 -0.099   22.00 DC 7 50% 94.000 41.000 0.139 0.975 -0.124   20.00   109     HR Et CO2 CVR Yrs Post Age      Abs Abs Rel Abs Rel     (bpm) (mmHg) (%) (mmHg/cm/s)) (%)     AM 1 70% 122.50 37.200 0.212 2.116 -0.240   59.00 AM 2 70% 151.54 34.900 0.137 2.437 0.065   53.00 AM 3 70% 100.35 34.000 0.298 2.450 -0.183   74.00 AM 4 70% 100.80 36.500 0.123 2.110 0.039   69.00 AM 5 70% 128.48 39.000 0.548 2.656 -0.068   64.00 AM 6 70% 132.20 39.500 0.362 2.440 -0.174   53.00 AM 7 70% 136.01 40.100 0.350 1.604 -0.218   58.00 HTR 1 70% 126.48 36.600 0.147 3.038 0.054 12.0 63.00 HTR 2 70% 122.41 36.300 0.160 2.360 -0.211 23.0 50.00 HTR 3 70% 124.03 32.000 0.168 2.378 0.028 6.0 65.00 HTR 4 70% 124.80 33.600 0.461 1.755 -0.232 1.0 61.00 HTR 6 70% 123.21 32.000 0.056 2.102 0.389 8.5 61.00 HTR 7 70% 122.69 32.800 0.051 4.069 0.163 10.0 78.00 HTR 8 70% 130.49 33.500 0.709 2.628 -0.142 4.5 54.00 DC 1 70% 171.00 46.000 0.243 1.124 -0.161   20.00 DC 2 70% 181.00 41.000 0.281 1.803 -0.019   21.00 DC 3 70% 154.00 47.000 0.119 1.121 -0.060   24.00 DC 4 70% 174.00 46.000 0.179 1.179 0.029   26.00 DC 5 70% 160.00 38.000 0.118 1.309 -0.027   18.00 DC 6 70% 167.00 48.000 0.200 1.314 -0.021   22.00 DC 7 70% 142.00 40.000 0.111 1.076 -0.034   20.00 AM 1 90% 138.00 33.500 0.091 1.915 -0.312   59.00 AM 2 90% 167.23 25.700 -0.163 2.776 0.213   53.00 AM 3 90% 111.31 33.300 0.271 2.304 -0.232   74.00 AM 4 90% 130.43 35.600 0.095 2.336 0.150   69.00 AM 5 90% 140.05 39.000 0.548 2.765 -0.030   64.00 AM 6 90% 147.80 36.700 0.266 2.602 -0.119   53.00 AM 7 90% 164.54 38.800 0.306 1.877 -0.085   58.00 HTR 1 90% 155.30 34.200 0.072 3.546 0.231 12.0 63.00 HTR 2 90% 142.39 32.200 0.029 2.651 -0.114 23.0 50.00 HTR 3 90% 134.31 30.800 0.124 2.418 0.045 6.0 65.00 HTR 4 90% 138.97 32.300 0.404 2.012 -0.120 1.0 61.00 HTR 6 90% 137.37 30.500 0.007 2.224 0.470 8.5 61.00 HTR 7 90% 146.98 28.600 -0.083 4.860 0.389 10.0 78.00 HTR 8 90% 138.84 33.500 0.709 2.909 -0.050 4.5 54.00 DC 1 90% 189.00 36.000 -0.027 1.269 -0.053   20.00 DC 2 90% 192.00 33.000 0.031 1.984 0.079   21.00 DC 3 90% 194.00 40.000 -0.048 1.377 0.154   24.00 DC 4 90% 182.00 38.000 -0.026 1.505 0.313   26.00 DC 5 90% 187.00 32.000 -0.059 1.439 0.070   18.00 DC 6 90% 180.00 42.000 0.050 1.414 0.054   22.00 DC 7 90% 171.00 32.000 -0.111 1.356 0.217   20.00   110              HR Et CO2 CVR Years Post Transplant Age     Absolute Absolute Relative Absolute Relative         (bpm) (mmHg) (%) (mmHg/cm/s)) (%)     AM 1 Peak 146.000 32.000 0.042 2.278 -0.182   59.00 AM 2 Peak 171.750 23.400 -0.238 2.646 0.156   53.00 AM 3 Peak 122.850 31.100 0.187 2.379 -0.207   74.00 AM 4 Peak 138.900 35.100 0.080 2.504 0.233   69.00 AM 5 Peak 159.180 31.700 0.258 2.834 -0.006   64.00 AM 6 Peak 159.550 32.900 0.134 3.220 0.091   53.00 AM 7 Peak 176.300 36.600 0.232 1.721 -0.161   58.00 HTR 1 Peak 163.140 31.400 -0.016 3.694 0.282 12.0 63.00 HTR 2 Peak 159.000 25.400 -0.188 2.582 -0.137 23.0 50.00 HTR 3 Peak 142.630 28.500 0.040 2.619 0.132 6.0 65.00 HTR 4 Peak 144.120 27.800 0.209 2.193 -0.040 1.0 61.00 HTR 6 Peak 139.260 29.400 -0.030 2.285 0.510 8.5 61.00 HTR 7 Peak 158.900 24.200 -0.224 5.124 0.465 10.0 78.00 HTR 8 Peak 159.100 27.800 0.418 3.000 -0.021 4.5 54.00 DC 1 Peak 200.000 23.000 -0.378 1.488 0.110   20.00 DC 2 Peak 204.000 26.000 -0.188 2.116 0.151   21.00 DC 3 Peak 195.000 35.000 -0.167 1.644 0.379   24.00 DC 4 Peak 199.000 27.000 -0.308 1.971 0.720   26.00 DC 5 Peak 192.000 27.000 -0.206 1.969 0.464   18.00 DC 6 Peak 192.000 35.000 -0.125 1.699 0.266   22.00 DC 7 Peak 189.000 25.000 -0.306 1.667 0.497   20.00   AM – Age Matched, HTR – Heart Transplant Recipient, DC – Donor Population Control, MCAv – Middle Cerebral Artery Velocity, MAP - Blood Pressure, Et CO2 – End Tidal Carbon Dioxide, CVR – Cerebrovascular Resistance, YRS Post – Years Post Transplant  Data is presented as raw data output from LabChart and recorded in Excel, subsequent analysis was preformed in PASW 18.0 and graphs were produced in Sigma Plot. 

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