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MR-Based Mapping of Cerebral Hemodynamics in Children with Sickle Cell Disease
by
Przemyslaw D. Kosinski
A thesis submitted in conformity with the requirements for the degree of Master of Science
Institute of Medical Science University of Toronto
© Copyright by Przemyslaw D. Kosinski 2016
ii
MR-Based Mapping of Cerebral Hemodynamics in Children with Sickle Cell Disease
Przemyslaw D. Kosinski
Master of Science
Institute of Medical Science
University of Toronto
2016
Abstract
Sickle-cell disease (SCD) is the most common cause of stroke in children, which follows the
exhaustion of vasodilatory capacity known as cerebrovascular reserve. Cerebrovascular reserve
may be impaired in SCD due to chronic anemia and endothelial dysfunction, which may place
SCD children at an increased risk for stroke. Therefore, we set out to assess cerebrovascular
reserve in children with SCD using MR-based measures of cerebrovascular reactivity (CVR).
We demonstrated that SCD children with a greater severity of anemia have higher cerebral blood
flow and lower CVR. In addition, we have shown that SCD children on hydroxyurea or
transfusion treatments have reduced severity of anemia and greater CVR than untreated patients.
These results suggest that SCD children with greater severity of anemia access more of their
cerebrovascular reserve in an attempt to maintain adequate oxygen supply and hydroxyurea or
transfusion-treated patients have increased reserve, which may be beneficial in stroke prevention.
iii
Acknowledgments
I'd like to acknowledge several individuals who have made my time as a Masters student
the most unforgettable and rewarding experience of my academic career. First and foremost, I'd
like to thank Dr. Andrea Kassner for providing me with the opportunity to undertake an elegantly
complex and intellectually stimulating research project, the autonomy to forge my own path, and
the greatly appreciated guidance to ensure I didn't stray too far along the way. I would also like
to extend a very special thanks to the members of my advisory committee Drs. Mikulis and
Grasemann, who have provided several ideas and insightful discussions that helped guide the
course of my research. I'd also like to sincerely thank my labmates Jackie, Junseok, and Paula
who after more than two years have become my second family and without whom this work
would not have been possible. I'd like to thank the staff at the Sickle Cell Clinic, Marcia and Drs.
Williams, Odame, and Kirby who've fielded countless numbers of questions pertaining to sickle
cell disease and who have helped make recruitment efforts successful. I would also like to thank
the MRI technologists; Tammy, Ruth, and Garry for their contributions on those early and long
Saturday scans and Dr. Shroff for reviewing our images. Last but definitely not least I'd like to
thank Dr. Amid, who showed me first-hand the clinical management of children with sickle cell
disease, which served as a constant reminder of why we perform research in the first place and
whose lessons will serve me well in medical school.
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My contributions
As this work presented in my thesis was of multidisciplinary nature, a number of people
contributed to the experimental work. Dr. Andrea Kassner was responsible for study design and
the supervision of the work presented here. She directed the focus of the work, and reviewed the
work as well as related publications. Her team member, Jackie Leung liaised with the
hematology clinic for subject recruitment and consent according to our study inclusion and
exclusion criteria, acquired the MR data, and processed the raw anonymized MRI data. Dr.
Shroff, neuroradiologist at Sickkids, evaluated the anatomical MRI images of both healthy
volunteers and patients to assess for stenosis and white matter lesions. Paula Croal, postdoctoral
fellow in Dr. Kassner's lab, provided helpful discussions and some statistical support. In addition
to writing the thesis, I (P.D.K) was involved in collecting clinical measures from study subjects
via existing databases, analyzed all data, performed statistical tests and wrote a draft for a
manuscript. My position was funded by funds from my supervisor Dr Kassner (in part from the
Canada Research Chair (CRC) program and trainee fund from the Hospital for Sick Children.
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Table of Contents
Acknowledgments .......................................................................................................................... iii
My contributions ............................................................................................................................ iv
List of Abbreviations ................................................................................................................... viii
List of Tables ................................................................................................................................ xii
List of Figures .............................................................................................................................. xiii
1 Introduction & Literature Review .............................................................................................. 1
1.1 Motivation and Introduction ............................................................................................... 1
1.2 Outline ................................................................................................................................. 2
1.3 Normal Cerebrovascular Physiology .................................................................................. 5
1.3.1 CBF Physiology ...................................................................................................... 5
1.3.2 Hemodynamic Compromise during Stroke .......................................................... 19
1.4 Sickle Cell Disease ........................................................................................................... 23
1.4.1 Introduction ........................................................................................................... 23
1.4.2 SCD Pathophysiology ........................................................................................... 23
1.4.3 Stroke Epidemiology and Risk Factors ................................................................. 29
1.4.4 Prevention of Primary and Recurrent Stroke ........................................................ 31
1.5 Magnetic Resonance Imaging ........................................................................................... 35
1.5.1 Basic MRI Principles and Contrast Mechanisms .................................................. 35
1.5.2 Cerebrovascular Imaging ...................................................................................... 46
2 Study 1: The impact of anemia on cerebrovascular reserve and cerebral blood flow in
children with sickle cell disease: a quantitative MRI Study .................................................... 58
2.1 Introduction ....................................................................................................................... 58
2.2 Purpose and Hypothesis .................................................................................................... 60
2.3 Materials & Methods ........................................................................................................ 61
vi
2.3.1 Subject recruitment ............................................................................................... 61
2.3.2 CO2 Challenge ...................................................................................................... 61
2.3.3 Magnetic Resonance Imaging ............................................................................... 62
2.3.4 MRI Review – Anatomical ................................................................................... 63
2.3.5 Data processing (CVR and CBF) .......................................................................... 63
2.3.6 Hematological and Clinical Measures .................................................................. 64
2.3.7 Statistical analysis ................................................................................................. 64
2.4 Results ............................................................................................................................... 65
2.5 Discussion ......................................................................................................................... 74
3 Study 2: Transfusion and Hydroxyurea Increases Cerebrovascular Reserve in Children
with Sickle Cell Disease: A Quantitative MRI Study .............................................................. 77
3.1 Introduction ....................................................................................................................... 77
3.2 Purpose and Hypothesis .................................................................................................... 79
3.3 Methods ............................................................................................................................. 80
3.3.1 Subject Recruitment .............................................................................................. 80
3.3.2 MRI ....................................................................................................................... 80
3.3.3 CO2 challenge ........................................................................................................ 81
3.3.4 Data Processing ..................................................................................................... 82
3.3.5 Statistical analysis ................................................................................................. 82
3.4 Results ............................................................................................................................... 83
3.5 Discussion ......................................................................................................................... 89
4 Overall Discussion and Future Directions ............................................................................... 95
4.1 Overall Discussion ............................................................................................................ 95
4.2 Limitations ........................................................................................................................ 99
4.3 Future Directions ............................................................................................................ 103
4.4 Conclusion ...................................................................................................................... 106
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5 References .............................................................................................................................. 109
viii
List of Abbreviations
SCD Sickle Cell Disease
CBF Cerebral Blood Flow
MRI Magnetic Resonance Imaging
cTx Chronic Transfusion Therapy
HU Hydroxyurea
GM Gray Matter
WM White Matter
CMRO2 Cerebral Metabolic Rate of Oxygen
CVR Cerebrovascular Reactivity
CPP Cerebral Perfusion Pressure
MAP Mean Arterial Pressure
CeVP Cerebral Venous Pressure
ICP Intracranial Pressure
CO Cardiac Output
SVR Systemic Vascular Resistance
CVP Central Venous Pressure
Hct Hematocrit
RBC Red Blood Cell
SMC Smooth Muscle Cells
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PaCO2 Arterial Partial Pressure of Carbon Dioxide
PETCO2 End-Tidal Partial Pressure of Carbon Dioxide
PaO2 Arterial Partial Pressure of Oxygen
PETO2 End-Tidal Partial Pressure of Oxygen
CaO2 Arterial Oxygen Content
Hb Hemoglobin
SaO2 Arterial Hemoglobin Oxygen Saturation
PvO2 Venous Partial Pressure of Oxygen
SvO2 Venous Hemoglobin Oxygen Saturation
OEF Oxygen Extraction Fraction
HbS Hemoglobin S
SCA Sickle Cell Anemia
HbA Adult Hemoglobin
Ts Time to Sickle
Tc Capillary Transit Time
NO Nitric Oxide
NOS Nitric Oxide Synthase
cGMP Guanosine-3',5'-monophosphate
TCD Transcranial Doppler Ultrasound
MCA Middle Cerebral Artery
x
SCI Silent Cerebral Infarcts
ACS Acute Chest Syndrome
ICA Intracranial Carotid Artery
ACA Anterior Cerebral Artery
PCA Posterior Cerebral Artery
TCDv TCD velocities
CSSCD Cooperative Study in Sickle Cell Disease
SIT Silent Cerebral Infarct MultiCenter Clinical Trial
STOP Stroke Prevention Trial in Sickle Cell Anemia
STOP II Optimizing Stroke Prevention in Sickle Cell Anemia
HbF Fetal Hemoglobin
SWiTCH Stroke With Transfusions Changing to Hydroxyurea
TWiTCH Transfusions Changing to Hydroxyurea
Bo Main Magnetic Field
Gx, Gy, Gz Gradient Field in the x, y, and z-axis
M Net Magnetization
Mz, Mxy Magnetization Components in the z and xy planes
RF Radiofrequency
T1 Spin-Lattice Relaxation
SDP Spectral Density Function
xi
T2 Spin-Spin Relaxation
CSF Cerebral Spinal Fluid
SE Spin Echo
GE Gradient Echo
TE Echo Time
TR Repetition Time
TI Inversion Time
FLAIR Fluid Attenuated Inversion Recovery
TOF Time of Flight
MRA Magnetic Resonance Angiography
CT Computed Tomography
PET Positron Emission Tomography
SNR Signal-to-Noise Ratio
ASL Arterial Spin Labeling
DSC Dynamic Susceptibility Contrast
PLD Post Label Delay
CASL, pASL, pCASL Continuous, Pulsed, Pseudo-Continuous ASL
BOLD Blood Oxygen Level Dependent
CBV Cerebral Blood Volume
xii
List of Tables
Table 2-1. Patient Demographics and Hematological Parameters ................................................ 65
Table 2-2. CO2 Challenge Results ................................................................................................ 66
Table 3-1 Patient Demographics ................................................................................................... 84
xiii
List of Figures
Figure 1-1. Coupling between CBF and CMRO2 in healthy cerebrovasculature. Adapted from
Hill L, 2007. .................................................................................................................................. 11
Figure 1-2 Relationship between CBF and PaCO2 in healthy cerebrovasculature. Adapted from
Hill L, 2007. .................................................................................................................................. 13
Figure 1-3. Relationship between PaO2 and CBF in healthy cerebrovasculature. Adapted from
Hill L, 2007. .................................................................................................................................. 14
Figure 1-4. Relationship between CPP and CBF in healthy cerebrovasculature. Adapted fom Hill
L, 2007. ......................................................................................................................................... 16
Figure 1-5. Cerebral hemodynamic responses to decreasing perfusion pressure following a
stroke. Adapted from Powers W, 1991 and Hulbert M, 2014. ..................................................... 22
Figure 1-6 NO interaction with iron pigment in hemoglobin in the circulation. .......................... 28
Figure 1-7. Vector representation of hydrogen nuclei magnetization. Obtained with permission
from Dr. Kassner's Lab. ................................................................................................................ 38
Figure 1-8 Graph depicting T1 recovery following a 90o excitation pulse. Adapted from
http://www.revisemri.com/tools/timeconst/images/90recovery.gif .............................................. 40
Figure 1-9. Graph depicting T2 decay following a 90o excitation pulse. Adapted from
http://www.revisemri.com/tools/timeconst/images/90decay.gif. ................................................. 42
Figure 1-10 Generating T1 contrast with changing Repetition and Echo Time in Gray Matter,
White Matter, and Cerebrospinal Fluid. Obtained with permifsion from Dr. Kassner's lecture
slides. ............................................................................................................................................ 45
Figure 1-11 Time of Flight Concept. Obtained with permission from Dr. Kassner's Lecture
Slides. ............................................................................................................................................ 47
xiv
Figure 1-12. Basic representation of labeling and Imaging planes in ASL. Obtained with
permission from Dr. Kassner's slides. ........................................................................................... 49
Figure 1-13 Representative arterial spin labeling image of cerebral blood flow. Obtained with
permission from Dr. Kassner's lab. ............................................................................................... 49
Figure 1-14. Computer-controlled gas blender and delivery apparatus. Obtained with permission
from Dr. Kassner's Lab. ................................................................................................................ 54
Figure 1-15 Blood-Oxygen Level-Dependent Schematic adapted with permission from Dr.
Kassner Neuroimaging Course Slides. ......................................................................................... 56
Figure 1-16 Blood-Oxygen Level-Dependent Image obtained with permission from Dr. Kassner's
Lab. ............................................................................................................................................... 57
Figure 1-17 Relationship Between Blood-Oxygen Level-Dependent Signal and Carbon Dioxide.
Obtained with permission from Dr. Kassner's Lab. ...................................................................... 57
Figure 2-1. Patient Demographics and Hematological Parameters .............................................. 65
Figure 2-2 CO2 Challenge Results ................................................................................................ 66
Figure 2-3. Representative Cerebrovascular Reactivity (CVR)(A) and Cerebral Blood Flow
(CBF)(B) maps in both SCD patients (Bottom) and Healthy Controls (Top). ............................. 67
Figure 2-4 Cerebrovascular reactivity (CVR) in patients with sickle cell disease (SCD) and
healthy controls. CVR, expressed as a relative change in blood-oxygen level-dependent magnetic
resonance signal per millimeter of mercury change in end-tidal partial pressure of carbon dioxide
was significantly decreased in children with SCD (red, n=26) when compared to healthy controls
(green, n=22) in the gray matter (A) and white matter (B). Bars represent mean ± standard error
of the mean. ................................................................................................................................... 68
Figure 2-5. Cerebral blood flow in patients with sickle cell disease (SCD) and healthy controls.
Cerebral perfusion, expressed as the volume of blood signal in milliliters per minute in 100g of
tissue, was significantly increased in children with SCD (red, n=26) when compared to healthy
xv
controls (green, n=22) in the gray matter (A) and white matter (B). Bars represent mean ±
standard error of the mean. ........................................................................................................... 69
Figure 2-6. Relationship between cerebrovascular reactivity (CVR) and the degree of anemia
(Hct) in patients with sickle cell disease (SCD). Cerebrovascular reactivity expressed as a
relative change in blood-oxygen level-dependent magnetic resonance signal per millimeter of
mercury change in end-tidal partial pressure of carbon dioxide (PETCO2) was significantly
associated with the degree of anemia in children with SCD (n=26) in the gray matter (A) and
white matter (B). ........................................................................................................................... 71
Figure 2-7. Relationship between cerebral blood flow and the degree of anemia (Hct) in patients
with sickle cell disease (SCD). Cerebral blood flow, expressed as the volume of blood signal in
milliliters per minute in 100g of tissue, was significantly associated with Hct in children with
SCD (n=26) in the gray matter (A) and white matter (B). ............................................................ 72
Figure 2-8. Relationship between cerebrovascular reactivity (CVR) and cerebral blood flow in
patients with sickle cell disease (SCD) and healthy controls. CVR expressed as a relative change
in blood-oxygen level-dependent magnetic resonance signal per millimeter of mercury change in
end-tidal partial pressure of carbon dioxide was significantly associated with cerebral blood flow,
expressed as the volume of blood signal in milliliters per minute in 100g of tissue in children
with SCD (n=26) and healthy controls (n=22) in the gray matter (A) and white matter (B). ...... 73
Figure 3-1 Patient Demographics ................................................................................................. 84
Figure 3-2. Hematocrit is significantly increased in Hydroxyurea-treated and Transfusion-treated
children with sickle cell anemia compared to no treatment children with SCA. Error bars depict
standard error of the mean. ........................................................................................................... 85
Figure 3-3 Cerebrovascular Reactivity is significantly increased in the gray matter (A) but not
the white matter (B) of hydroxyurea-treated and transfusion-treated children with sickle cell
anemia compared to non treated children with sickle cell anemia. Error bars denote standard
error of the mean. .......................................................................................................................... 86
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Figure 3-4 Cerebral blood flow (CBF) was signficantly decreased in the gray matter (A) and
white matter (B) of transfusion-treated children with sickle cell anemia (SCA) compared to non
treated SCA children. HU-treated SCA children showed no signficantly different CBF changes
in either gray matter or white matter compared to non treated SCA children. Error bars represent
standard error of the mean. ........................................................................................................... 88
1
1 Introduction & Literature Review
1.1 Motivation and Introduction
Sickle cell disease (SCD) is a life-long genetic disorder that causes erythrocytes to take
on the characteristic 'sickled' morphology upon deoxygenation. The most devastating
complication in SCD is an overt stroke, which occurs in 11% of children by the age of 20. The
pathophysiology of stroke is not well understood, however currently it is believed to arise from a
critical narrowing in the large intracranial vessels. However, 1 in 3 SCD children who suffer a
stroke have no visible narrowing on conventional imaging. Currently, it is hypothesized that
altered cerebral hemodynamics, such as cerebrovascular reactivity plays a critical role in the
pathophysiology of stroke in SCD, which can arise with or without vessel narrowing. Cerebral
hemodynamics include physical factors that maintain tissue viability through governing cerebral
blood flow (CBF). The capacity to moderate CBF through vasodilation is referred to as
cerebrovascular reserve, which is utilized during changes in perfusion pressure and oxygen
content. Failure in providing the highly metabolically active cerebral tissue with sufficient CBF
can result in infarction, therefore understanding cerebrovascular reserve in children with SCD
may be important to understanding the pathophysiology of neurologic injury such as stroke.
SCD is clinically characterized by chronic hemolytic anemia, vaso-occlusion, which
work synergistically to cause endothelial injury and dysfunction. Previous studies by Prohovnik
and Nur have shown that the cerebral vessels in adults with SCD are less responsive to a dilatory
stimulus when compared to healthy controls. If the cerebrovasculature is less responsive to a
dilatory stimuli , such as CO2, then it is argued that this response will also be blunted when the
children face acute reductions in perfusion pressure from vasculopathy or reductions in oxygen
2
content from acute anemic events. If the cerebrovascular response to such complications is
insufficient then the mismatch between metabolic demand and ability to deliver may increase the
risk of brain injury. However, in addition to endothelial injury and dysfunction, children with
SCD also face chronic reductions in oxygen carrying capacity as a result of hemolysis of red
blood cells, which may utilize this reserve. Considering the severity of anemia is a risk factor for
overt stroke, it therefore may be useful to assess the impact of anemia on cerebrovascular reserve
in children with sickle cell disease, which is the aim of the first experimental study. In addition,
the second experimental study assessed the effect of chronic transfusion therapy and
hydroxyurea, which are two of key therapies for stroke prevention, on cerebrovascular reserve
and CBF.
1.2 Outline
This thesis describes how MRI can be used in order to describe the altered cerebral
hemodynamic environment in children with sickle cell disease. Chapter 1.3-1.5 provides the
relevant background information on cerebral physiology, sickle cell disease, and magnetic
resonance imaging, respectively. The experimental findings are then presented in chapters 2 and
3, which were carried out in The Hospital for Sick Children between September 2013 and
September 2015. A brief summary of the chapters is described below;
Chapter 1.3 describes the normal cerebrovascular physiology and hemodynamic principles
governing the control of cerebral blood flow (CBF) in healthy adults and children. The cerebral
blood flow section contains a focus on factors that modulate the radius of the cerebral vessels as it
is the means in which CBF is manipulated within the experimental studies. In addition, the reader
is introduced to healthy oxygen content and cerebrovascular reserve as they are two parameters of
particular importance in the experimental work.
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Chapter 1.4 provides an introduction into SCD, in particular, the pathophysiology behind vaso-
occlusion and hemolytic anemia as these processes contribute to alterations in the vasodilatory
response of cerebral vessels and CBF. Then the reader is introduced to the impact of stroke in
children with SCD, as well as the major stroke clinical trials, which provided the clinical standard
of stroke prevention. Finally, an introduction to red blood cell transfusions and hydroxyurea
treatment in the context of stroke prevention is provided.
Chapter 1.5 demonstrates how magnetic resonance imaging (MRI) is used to obtain hemodynamic
parameters such as cerebrovascular reactivity (CVR) and CBF. In addition, a basic introduction to
MRI architecture and image contrast mechanism is provided to orient the reader.
Chapter 2 contains the first experimental study of this thesis, which was to determine the effect of
anemia on CVR and CBF in children with sickle cell disease. This chapter includes an introduction
to anemia and the cerebral hemodynamics in SCD, followed by the methods used to obtain these
measurements and the results of the experiment, and a discussion which provides an interpretation
of the findings into the context of what is currently known about cerebrovascular reserve in SCD.
Chapter 3 contains the second experimental study of this thesis, which set out to determine and
compare the effects of cTx and HU treatment on CVR and CBF in children with SCD. This chapter
includes a detailed introduction to cTx and HU treatment and how these treatments may impact
CVR and CBF. The introduction is followed by the methods that were used to carry out the
experiment and the results, which revealed the effects of these treatments on CVR and CBF
compared to healthy pediatric controls. Finally, a discussion is provided in order to interpret the
results and shed light on the possible implications of the treatments on cerebrovascular
hemodynamics, as well as the technical limitations within the study.
4
Chapter 4 contains the general, overarching discussion, which connects the results of the study in
the context of anemia, cerebral hemodynamics, and stroke pathophysiology in SCD. In addition,
this chapter provides the limitations found in the studies, as well as the future directions of cerebral
physiology research in SCD.
Chapter 5 contains the list of references.
5
1.3 Normal Cerebrovascular Physiology
1.3.1 CBF Physiology
Regulation of blood flow is critical to maintain viability of all tissue-types, however the
cerebral parenchyma is particularly sensitive to fluctuations in CBF for two main reasons; firstly
the primary form of metabolism in the brain is aerobic respiration, which uses oxygen as a substrate
(Bor-Seng-Shu et al., 2012). Considering that virtually all of oxygen is transported through the
blood, the brain therefore relies solely on CBF to supply its oxygen demand (Hill et al., 2007).
Secondly, the cerebral tissue has a disproportionately high demand for oxygen due to its significant
metabolic activity. The average adult brain comprises 2% of total body weight but consumes
roughly 20% of all available oxygen and 15-20% of cardiac output to fuel its metabolism (Bor-
Seng-Shu et al., 2012; Khurana, 2013). The average CBF in a healthy adult is approximately
50mL/100g/min (Bor-Seng-Shu et al., 2012). However, there exists regional variability, where the
CBF in grey matter (GM) and white matter (WM) is approximately 70-80 mL/100g/min and
20mL/100g/min, respectively (Vavilala, Lee, & Lam, 2002). In children, CBF is significantly
increased where the GM and WM receives approximately 97.5 mL/100g/min and
26.1mL/100g/min of blood, respectively (Biagi et al., 2007). The discrepancy between GM and
WM CBF is accounted for by that fact that the GM has a much greater vascularization than the
white matter (WM) and has greater metabolic activity therefore greater oxygen demand (Hill et
al., 2007). The oxygen consumption of the adult brain averages out to 3.3mL/100g/min and is
referred to as the cerebral metabolic rate for oxygen (CMRO2). In children, brain activity is
significantly increased as demonstrated by increased CMRO2 compared to healthy adults
(Takahashi, Shirane, Sato, & Yoshimoto, 1999). CMRO2 is much greater in the adult grey matter
(6mL/100g/min) than in the white (2mL/100g/min) due to differences in metabolic activity and
6
therefore demand (Vavilala et al., 2002). Considering that the cerebral parenchyma is so dependent
on CBF, the regulators of CBF will be detailed next. In addition, in the experimental work of this
thesis, we obtain baseline CBF measures and cerebrovascular reactivity, which is defined as the
change in CBF in response to a vasodilator stimulus, therefore understanding CBF regulators is
critical to the appreciation of the experimental work.
Blood flow to the brain is regulated by a series of mechanisms, which can broadly be
subdivided into those that effect the radius of the cerebral blood vessels/resistance or the cerebral
perfusion pressure. This relationship can be described by the Hagen-Poiseuille's law as shown;
𝑪𝑩𝑭 = ∆𝑷𝝅𝑹𝟒
𝟖𝒏𝑳 Equation 1
Where CBF is expressed in mL/100g/min (depicted as /second here), ∆𝑃 is the cerebral perfusion
pressure in Pascals, R is the radius of a cerebral blood vessel in meters, n is the viscosity of the
blood in Pascals x second, and L is the length of the blood vessel in meters.
1.3.1.1 Cerebral Perfusion Pressure
As can be seen from Equation 1, various factors can alter CBF. Beginning with cerebral
perfusion pressure (∆𝑃 𝑜𝑟 𝐶𝑃𝑃), which can be approximated from the following equation;
𝑪𝑷𝑷 = 𝑴𝑨𝑷 − 𝑪𝒆𝑽𝑷 Equation 2
Where CPP is the cerebral perfusion pressure in millimeters of mercury (mmHg), MAP is the mean
arterial pressure in mmHg, and CeVP is the cerebral venous pressure in mmHg.
The pressure gradient that drives flow to the brain is the difference between the pressure provided
by the arteries (MAP) and the pressure originating from the cerebral venous system (CeVP).
7
Because CeVP is not easily measured, intracranial pressure (ICP) is used as an approximate
surrogate. Typically, MAP is roughly 90mmHg and ICP is normally less than 13 mmHg therefore
it is mostly negligible. As such, MAP is the driving force for CPP and is defined as the average
pressure existing within the systemic circulate and can be approximated by the following
relationships;
𝑴𝑨𝑷 = (𝑪𝑶 × 𝑺𝑽𝑹) + 𝑪𝑽𝑷 Equation 3
Where MAP is the mean arterial pressure in mmHg, CO is the cardiac output typically expressed
in L/min, SVR is the systemic vascular resistance and CVP is the central venous pressure in
mmHg.
CVP is typically approximately 0mmHg, which simplifies Equation 3 to;
𝑴𝑨𝑷 = 𝑪𝑶 × 𝑺𝑽𝑹 Equation 4
Therefore, here we see how the output of the heart (CO) and the vessel radius/tone of the systemic
vessels (SVR) come together to produce a MAP which acts as the main driving force of CPP.
Taken together the equation can be written as follows;
CPP = [(𝑪𝑶 × 𝑺𝑽𝑹)] – ICP. Equation 5
When substituting Equation 5 into equation 1, we obtain the following CBF relationship;
𝑪𝑩𝑭 = [(𝑪𝑶×𝑺𝑽𝑹) – 𝐈𝐂𝐏]𝝅𝑹𝟒
𝟖𝒏𝑳 Equation 6
8
The purpose of this exercise was to show that CBF can physically be related to many variables,
which not only includes the vessels of the brain, but also the vessels in the systemic circulatory
system, the heart, and the fluid itself
1.3.1.2 Viscosity
The viscosity of blood is dependent upon the hematocrit (Hct) as well as the deformability
of cells (Schmalzer, Lee, Brown, Usami, & Chien, 1987). The blood can be broken down into its
components of red blood cells (RBCs) and plasma. The Hct is the proportion of RBCs in a
centrifuged volume of blood and is typically used as a marker for oxygen content. Healthy Hct
ranges from 0.36 – 0.50 and is affected by gender where average male Hct is 0.45 ± 0.05 and
average female Hct is 0.41 ± 0.05 (Lewis, Brain, & Bates, 2001). The Hct can be altered by
changing either the amount of RBCs or plasma (Conrad, 1990). Therefore patients that are
dehydrated have less plasma resulting in their Hct to be inflated above its baseline normal value.
The amount of circulating red blood cells is the net difference between RBC production by the
bone marrow and the degree of breakdown. Pathologies can affect either the production of RBCs
such as is seen in polycythemia patients, who have a Hct greater than 0.56 (Tefferi, 2003). The
result of the RBC overproduction is a dramatically increased blood viscosity increases, which
impedes blood flow thus requiring compensatory mechanisms. Alternatively, a reduction in
circulating RBCs and therefore Hct could be afforded by a pathological increase in RBC
breakdown such as is seen in sickle cell disease (SCD). The World Health Organization defines
red blood cells below 13g/dl (0.39 Hct) in males and 12g/dl (~0.36Hct) in females is defined as
anemia. In children aged 6 months to 6 years, the criteria for anemia is considered to be 11g/dl
(0.33Hct). Between 6 and 14 years of age, the anemia criteria is below 12g/dl (0.36 Hct) (Conrad,
9
1990). In hemolytic anemias, the rate of RBC destruction is increased such as in paroxysmal
nocturnal hemoglobinuria or sickle cell disease (SCD), which will be detailed in Chapter 1.4.
1.3.1.3 Cerebral Vessel Radius
In the experimental work of this thesis, CO2 inhalation was utilized in order to increase
CBF by causing the cerebral resistance vessels to dilate, which increases the radius of the
vessels. Hence, it is important to introduce how the radius of the vessel affects CBF and
acknowledge other physiological factors that can affect the radius of cerebral vessels.
Equation 1 can be rewritten to express CBF as;
𝑪𝑩𝑭 = ∆𝑷
𝑹 Equation 7
Where the ∆𝑃 is the change in pressure in Pascals, or CPP in mmHg and 𝑅 is the resistance to
flow. The resistance is a function of vessel length, viscosity, and vessel radius as shown below;
𝑹 =𝒏𝑳
𝒓𝟒 Equation 8
Where n is the viscosity of the fluids in Pascals*second, L is the length of the vessel in meters, and
r is the vessel radius in meters.
Viscosity was just detailed as a factor contributing to a fluid's resistance to flow. In addition to the
length of the vessel, the remaining component to understanding resistance is the vessel radius. As
can be seen from Equation 8, the relation between resistance and the radius is quadratic in nature
meaning that small changes in vessel radius leads to dramatic reductions in resistance.
Furthermore, according to Equation 1, this relationship carries through where small changes in
vessel tone result in dramatic changes in CBF. Unlike viscosity or the length of the vessel, the
10
radius of the vessel can be actively modulated thereby providing the body with a way to actively
change CBF. Changing vessel radius is a critical aspect of the protocol in this thesis as we utilize
a vasodilator in order to cause a change in CBF. Due to this importance, the general factors
regulating vessel radius will be introduced next.
The radius of cerebral vessels is regulated by four main mechanisms; cerebral metabolism,
carbon dioxide and oxygen blood gas concentrations, cerebral autoregulation, neurohumeral
factors. (Hill et al., 2007)
1.3.1.3.1 Cerebral Metabolism
Increases in neural activity, e.g. due to a visual stimulus, increases CMRO2. The increase
in oxygen demand is met by an increase in CBF through vasodilation and is referred to as
neurovascular coupling. The mediators of this vasodilation is believed to include; hydrogen ions,
potassium, CO2, adenosine, glycolytic intermediates, phospholipid metabolites, and nitric oxide
(NO). (Bor-Seng-Shu et al., 2012).
The relationship between CBF and CMRO2 is presented below in Figure 1.1, which depicts
that changes in CMRO2 are met linearly (for simplistic purposes) with changes in CBF.
11
1.3.1.3.2 Carbon dioxide and oxygen partial pressures
Gas exchange occurs at the capillaries, where the tissue consumes O2 and produces CO2,
therefore relative to the arterial blood gas concentrations, the blood gas concentrations of O2 and
CO2 in the venous system decreases and increases respectively. These respective concentrations
are relatively maintained until gas exchange occurs at the alveoli within the lungs, where the
pressure gradient drives CO2 out of the blood stream and O2 into the blood stream. This is a
simplistic interpretation of gas exchange, the details include complex blood gas chemistry with the
Bohr and Haldane effect and are not in the scope of this thesis. The end result is that the arterial
partial pressure of oxygen (PaO2) and carbon dioxide (PaCO2) is approximately 100 and 40 mmHg,
respectively. Whereas in mixed venous blood, the venous partial pressure of oxygen and carbon
dioxide is approximately 40 and 46mmHg, respectively (Pittman, 2011).
Figure 1-1. Coupling between CBF and CMRO2 in healthy cerebrovasculature. Adapted from
Hill L, 2007.
12
Resistance arterioles are characterized by smooth muscle cells (SMC), which circumscribe
the vessel. The relaxation of the SMC causes vasodilation of the vessel whereas the constriction
of SMCs causes vasoconstriction. CO2 is a potent vasodilator, which is thought to act by decreasing
pH, which reflexively results in vasodilation through specific potassium-channel activation
(Horiuchi, Dietrich, Hongo, Goto, & Dacey, 2002). However, this mechanism isn't well elucidated
and nitric oxide, prostanoids, cyclic nucleotides, potassium channels are all implicated in the
mechanism. These regulators all come together to affect SMC intracellular calcium concentrations,
which is responsible for causing SMC relaxation or tension (Iadecola, 1992). Vasodilation
increases the radius of the vessel, which dramatically reduces the resistance to flow thereby
increasing CBF. The relationship between the partial pressure of arterial CO2 (PaCO2) and CBF
can be seen below in Figure 1.2, which contains a roughly linear range from approximately 20-80
mmHg PaCO2. Normal values for PaCO2 is between 35-45 mmHg, which is well situated within
the linear range, thereby facilitating relatively large changes in CBF for small changes in PaCO2.
This is demonstrated by the fact that inhaling 5% CO2 has been shown to cause a 50% increase in
CBF (Kety & Schmidt, 1948). Above 80 mmHg, any further increase in PaCO2 is generally met
without an increase in CBF because the vessels have reached their vasodilatory capacity.
Conversely, below 20 mmHg PaCO2, any reductions in PaCO2 will not reduce CBF because the
vessels have reached maximum constriction.
13
With respect to arterial partial pressure of oxygen (PaO2), the normal range of PaO2 is above
80mmHg/10.6kPa (mellengard K, 1966, Sorbini CA et al, 1968). PaO2 has approximately no effect
on CBF in clinically relevant ranges, unless PaO2 drops below the threshold of approximately
50mmHg where it dramatically increases in order to maintain constant delivery of oxygen as seen
below in Figure 1.3 (Masamoto & Tanishita, 2009). The mechanism is hypoxia driven, which
causes the releases of adenosine to promote vasodilation. In addition, hypoxia causes
hyperpolarisation of the SMCs which reduces calcium update, which also contributes to
vasodilation (Taguchi, Heistad, Kitazono, & Faraci, 1994).
Figure 1-2 Relationship between CBF and PaCO2 in healthy cerebrovasculature.
Adapted from Hill L, 2007.
14
1.3.1.3.3 Cerebral Autoregulation
Up to now, we've discussed how changes in CMRO2, PaCO2, PaO2 effect the radius/tone of
the cerebral vessels. Another stimulus that effects vessel tone are changes in CPP. As seen in
Equation 1, CPP is the difference between MAP and ICP. As previously noted, ICP under normal
circumstances is <13mmHg, therefore changes in MAP such as during acute hypertension or
hypotension have significant impact on CPP. In situations such as major bleeding, MAP can drop,
which if no compensatory mechanisms existed, would result in a reduction in CBF. However, as
seen below in Figure 1.4, within the CPP range of approximately 60-160mmHg, CBF is
approximately held constant through vasodilation/constriction in a compensatory process called
cerebral autoregulation (Paulson, Strandgaard, & Edvinsson, 1990). As CPP drops, such as during
the major bleeding event, cerebral vessels dilate, the effect is that CBF is maintained. Conversely,
if CPP increases then cerebral vessels constrict in order to increase resistance to flow thereby
Figure 1-3. Relationship between PaO2 and CBF in healthy
cerebrovasculature. Adapted from Hill L, 2007.
15
maintaining CBF. When CPP falls below 60 mmHg, CBF begins to drop as the vessels have
reached their dilatory capacity, which is known as autoregulatory failure/exhaustion (Cipolla &
Osol, 1998; Harder, Gross, Nasjletti, Bonham, & Crystal, 2004). Further decreases in CPP can
cause the vessels to collapse because of the lack of pressure which increases the resistance to flow
thereby facilitating a dramatic decrease in CBF which can cause ischemia (BYROM, 1954).
Alternatively, increases in CPP above approximately 160, CBF starts to increase as the vessels
cannot constrict any further (Cipolla & Osol, 1998). In addition, there exists a point with increasing
CPP where the constrictive vessels are forced open by the pressure which causes a dramatic
increase in CBF as both the increase in CPP and reductions in resistance facilitates increasing CBF.
The increase in CBF can cause damage to the blood brain barrier (BBB) (Skinhoj & Strandgaard,
1973). Cerebral autoregulation is assumed to have no age or gender related differences, however,
there is data showing that the lower limit of cerebral autoregulation is lower compared to healthy
adults. This suggests that children can better tolerate hypotension.
16
1.3.1.3.4 Neurohumeral Factors
Several circulating factors in the peripheral circulation such as catecholamines and
vasoactive peptides have effects on peripheral vascular tone. However, the BBB prevents these
peripheral factors for entering the cerebral tissue, therefore the effect of humoral or autonomic
control on cerebrovascular tone is relatively less active in the brain. With reference to the
autonomic system, the sympathetic nerve stimulation causes vasoconstriction whereas the
parasympathetic nervous system helps to contribute to vasodilation (Hill et al., 2007).
Figure 1-4. Relationship between CPP and CBF in healthy cerebrovasculature. Adapted fom
Hill L, 2007.
17
1.3.1.4 Oxygen Content
As mentioned previously, the brain is dependent entirely on perfusion for oxygen and
nutrients. The importance of understanding arterial oxygen content (CaO2) is twofold; first, patients
with CaO2 rely on an increase in CBF to maintain the delivery of oxygen (Kuwabara et al., 1990)
and the study population in this thesis have reduced CaO2 due to their anemia as will be outlined
below and in Chapter 1.4. The CaO2 is a function of the oxygen bound to hemoglobin (Hb) and the
oxygen dissolved in the plasma, which is demonstrated by the following equation.
𝑪𝒂𝑶𝟐 = (𝟏. 𝟑𝟒 × [𝑯𝒃] × 𝑺𝑨𝑶𝟐) + (𝑷𝑨𝑶𝟐 × 𝟎. 𝟎𝟎𝟑) Equation 9
Where CaO2 is the arterial oxygen content in mL O2/dL, 1.34 is the volume of O2 held by
1 gm of Hb in units of mL O2/g Hb, SaO2 is the percent hemoglobin saturation, PaO2 is the partial
pressure of oxygen in blood plasma in mmHg, and 0.003 is the volume of O2 dissolved in blood
plasma at different PaO2 pressures in units of mL O2/mmHg/dL.
In Equation 9, the Hb contribution to arterial oxygen content is on the left of the "+" sign
and the plasma contribution to arterial oxygen content is on the right. To put it into perspective,
the amount of oxygen dissolved in blood comprises only 2% of the total oxygen content, whereas
the reminder 98% is entirely dependent on Hb. Because of the relatively insignificant contribution
of dissolved O2 to CaO2 and that the average SaO2 in arterial blood is 98-100% in healthy controls,
Hb measures are used as a proxy for total CaO2. Hematocrit (hct) is an alternative measure to Hb,
which describes the proportion of red cells in a centrifuged vial of blood and is well associated
with haemoglobin (Hct% = Hbg (g/dl) X 3) (Bain & Bates, 2001). Hemoglobin’s contribution to
CaO2 is a function of the amount of Hb, and the percent oxygen saturation (SaO2). Each Hb
molecule is comprised of two alpha and two beta globins, which each have a iron-porphyrin ring
18
with the ability to bind bimolecular oxygen, therefore each Hb can bind 4 O2 molecules. The
amount of haemoglobin can be expressed as either haemoglobin or hematocrit and is generally
assumed to be 120g/L or 0.4 Hct. There are several issues with assuming this level of Hct; first,
Hb and Hct is gender-dependent, where the average Hb and Hct for men is 120-130 g/l or 0.40-
0.50 Hct whereas for women it is 119-129 g/L or 0.33-0.43. Secondly, many diseases or treatments
that affect the bone marrow or erythropoeisis can have a dramatic effect on Hb/Hct levels. For
example polycythemia results in significantly increased Hct levels whereas anemias are marked
by a reduction in Hct levels. As just mentioned, the Hb/Hct accounts for 98% of all oxygen content
therefore not only is the amount of Hb/Hct critical, but also the saturation of the hemoglobin
molecules. SaO2 is a result of the PaO2 in the circulation and the oxyhaemoglobin dissociation
curve. Under normal conditions, PaO2 in the arterial system (PaO2) is roughly 100mmHg (Pittman,
2011). At this pressure, oxygen is able to completely saturates the Hb molecules, which results in
SaO2 to be approximately 98-100%. As Hb traverses the capillaries, it experiences lower PaO2 set
by the consumption of oxygen by cells fueling their aerobic respiration. As PaO2 begin to drop, the
pressure is no longer high enough to maintain 100% SaO2, which causes some of the oxygen to
unbind from the hemoglobin thereby decreasing SaO2. In addition, the PaCO2 begins to increase as
the cells continuously produce CO2 from aerobic respiration, which interacts with Hb to facilitate
the offloading of O2. As the Hb drain into the venous system, the venous partial pressure of oxygen
(PvO2) results in a venous hemoglobin saturation (SvO2) of approximately 60%. The difference
between SaO2 and SvO2 divided by the SaO2 is the oxygen extraction fraction (OEF). The
importance of introducing OEF is to demonstrate that CBF is not the only way to provide tissue
with oxygen and even though OEF is approximately 40% in healthy, resting brains, it may vary in
disease states. Especially in diseases in which the hemoglobin is altered such as SCD.
19
1.3.1.5 Vascular Reserve
As has been detailed in Chapter 1.3, differences in CMRO2, PaCO2, PaO2, CaO2, and
MAP can induce changes in CBF through vasodilation. However, there is a finite capacity as
shown in Figure 1.2 where increasing PaCO2 past a certain point does not produce a change in
CBF due to the vasodilatory limit of vessels. The collective term for the dilatory capacity of
cerebral arterials is called cerebrovascular reserve (Ozgur et al., 2001). Considering that vessel
dilation seems to be a common pathway shared by several key factors and that there is a finite
reserve to dilate, therefore being able to assess this reserve may provide additional information to
the cerebral hemodynamics of patients with cerebrovascular disorders. As previously mentioned,
both CMRO2 and CBF is significantly increased in the pediatric brain (Biagi et al., 2007;
Takahashi et al., 1999). Work in our lab suggests that children utilize vascular reserve in order to
maintain an elevated CBF (Leung J, works submitted). Assessing cerebrovascular reserve in
cerebrovascular disorders is useful to determine the capacity to accommodate an increased
demand for blood flow. If the cerebrovasculature cannot accommodate an increased demand for
blood flow or the response is insufficient, then this may pose a risk for the cerebral tissue during
acute events that demand an increased blood delivery. Cerebrovascular reserve in this case would
then act to stratify patients who may appear asymptomatic due to their compensatory capacities
but are in reality at risk for insufficient compensation when the demand or supply of blood flow
changes for whatever circumstance. One circumstance that can acutely compromise the supply of
blood is an ischemic stroke.
1.3.2 Hemodynamic Compromise during Stroke
Generally speaking, a stroke occurs when the supply of oxygen cannot meet the tissue’s
oxygen demand. As explained in Chapter 1.3, the brain is particularly susceptible to perturbations
20
in this supply-demand balance primarily due to two reasons; the first being that the brain basically
has no energy or oxygen storage mechanisms and is therefore entirely reliant on CBF. The second
reason is that cerebral tissue is the most metabolically active tissue in the body and therefore
requires a disproportionally high amount of oxygen and glucose. Although the demand for oxygen
can increase, such as during febrile illnesses or increased brain activity, the body has particular
and sufficient compensatory mechanisms in place to maintain an appropriate supply of blood flow.
When the supply is critically reduced, such as during a stroke, the brain tissue can undergo
infarction.
This section will focus on ischemic stroke as it is the major type of stroke in children with
sickle cell disease. Cerebral ischemia is defined as insufficient blood supply to the cerebral tissue
and is occlusive in nature (Gusev & Skvortsova, 2003). An ischemic stroke can generally be
subdivided to two general etiologies; the first is the formation of a thrombus at the site of occlusion
or in another region of the body (embolis) such as in the heart that occludes a vessel in the brain
(Sobieszczyk, 2006). The second is of a hemodynamic etiology that results a critical narrowing of
the diseased vessel itself, where atherosclerosis and cholesterol plaques cause narrowing of the
vessel lumen, known as stenosis, which can slowly result in overt occlusion. As the vessel begins
to narrow, a particular set of hemodynamic responses are set in motion in an attempt to maintain
the supply of oxygen in order to preserve cerebral tissue. The extent of the compensatory responses
depend primarily on two factors; the degree vessel narrowing and the extent of collateral flow
(Powers, 1991). Initially, small reductions in vessel diameter does not change CBF due to the
participation of collateral flow. However, if the diameter of the vessel is reduced to a critical value,
then the collateral circulation will not be adequate, which results in a decrease in mean arterial
pressure (MAP) in the distal circulation (Powers, Fox, & Raichle, 1988). Because MAP decreases,
21
as does CPP, which would reduce CBF but vasculature responds by dilating the vessels
downstream of the stenotic lesion. Through vasodilation, the resistance to flow is reduced and CBF
is maintained despite the decrease in CPP. The critical value prior to initiating the vasodilatory
response has been shown to be approximately 50-75% of the original vessel diameter (Powers,
1991). However, there is a limit to which a vessel can dilate. As the perfusion pressure continues
to drop, the vasodilatory capacity is reached and the CBF can no longer be maintained and begins
to drop as depicted below in Figure 1.5 below. This stage is marked as stage 1 hemodynamic
compromise. Despite decreasing CBF, the tissue is able to maintain viability by increasing the
extraction of oxygen (OEF). If the perfusion pressure continues to decrease, CBF will continue to
decrease, while OEF increases until a critical point where CMRO2 can no longer be maintained
and begins to decrease. This is known as stage 2 hemodynamic compromise. Unless blood flow is
restored, reductions in CMRO2 can lead to reversible or irreversible tissue damage and death,
known as tissue infarction.
22
Figure 1-5. Cerebral hemodynamic responses to decreasing perfusion pressure following a stroke.
Adapted from Powers W, 1991 and Hulbert M, 2014.
23
1.4 Sickle Cell Disease
1.4.1 Introduction
Sickle cell disease (SCD) is an inherited blood disorder associated with a lifetime of acute
complications and progressive organ damage. The characteristic feature of SCD is the presence of
sickle shaped erythrocytes, which was initially described by Herrick in 1910 (Savitt & Goldberg,
1989). SCD is an umbrella term used to describe several genotypes that share in the potential for
cellular sickling and have similar clinical syndromes albeit encompassing a spectrum of severities.
The genotypes represent a family of mutations in the beta globin gene that manifest as abnormal
beta-globins (β). These genotypes include β; S, C, D, E, etc. Homozygous βS is the most severe
and prevalent genotypic variant of SCD and is called sickle cell anemia (SCA). The second most
common SCD is hemoglobin S/C (HbSC) disease, which is a result of a coinheritance of a βS and
βC mutations. The third major type of SCD occurs when βS is coinherited with β-thalassemia
(HbS/β-thalassemia), which can vary in severity depending on the type of β-thallassemia.
1.4.2 SCD Pathophysiology
1.4.2.1 Cellular sickling
Although SCD encompasses various hemoglobinopathies, this thesis will primarily focus
on SCA because it represents the vast majority of the patient population in the experimental work
of this thesis. The devastating impacts of SCA originate from the effect of the βS on Hb. As
previously mentioned, adult hemoglobin (HbA) is a tetramer comprised of 2 α and 2 β globins
(α2β2), which accounts for approximately 98% of a healthy human's oxygen carrying capacity.
More specifically, SCA is caused by a coinheritance of an adenine-thymine point mutation in the
24
first exon of the β-globin gene, which substitutes valine for a glutamic acid at the sixth position of
the β-globin (INGRAM, 1956; Schechter & Dc, 2008) . This particular mutation in the β-globin is
termed βS, where S stands for sickle. When the βS globins are inherited from both the mother and
father, they package alongside the α globins to form hemoglobin S (HbSS, α2β2S). The introduction
of valine imbues a hydrophobic motif into a critical position of the beta globin, which under
deoxygenation allows HbS molecules to polyermize via their βS-globin hydrophobic sites
(Schechter & Dc, 2008). As the HbS polymer grows, it reaches a critical mass where the
surrounding aqueous environment can no longer keep it in solution, thereby resulting in the
formation of an insoluble gel (Ferrone, 2015). For simplistic purposes, The gel-polymer grows
exponentially and pushes against the cell wall, which causes mechanical stress and oxidative
cellular stress while disrupting the cellular architecture. The process causes significant cellular
dehydration and increased rigidity and culminates in the characteristic sickle morphology. The
intricacies of cellular sickling is a whole field in it itself and won't be detailed in this thesis as it is
not the focus.
The rate of HbS polyermization and therefore sickling is dependent upon the concentration
of HbS within the erythrocyte, the duration and extent of deoxygenation, and the activity
coefficient (Ferrone, 2015). Therefore as soon as the erythrocyte begins offloading oxygen at the
level of the capillaries, a race against the clock begins as the cells circulate out of the smaller
capillaries and into the venous circulation before sickling occurs. The time it takes for the HbS to
polyermize and sickle is referred to as the time to sickle (Ts), which is a function of the
concentration of deoxy sickle haemoglobin and an activity coefficient that takes into account the
space-filling properties of hemoglobin and other molecules within the RBC (Ferrone, 2015). The
capillary transit time (Tc) is the time it takes for the erythrocyte to traverse the capillaries/venules
25
and is a function of perfusion and rheological properties of blood. If the time to sickle is less than
the time it takes to circulate through the microvasculature then the cells will sickle and through a
complex interaction between adherence with other cells, endothelia, and recruitment of immune
cells, may cause an occlusion which is referred to as a vaso-occlusive crisis (Ferrone, 2015) The
diameter of a capillary is smaller than the diameter of a typical healthy red blood cell, which
requires the RBC to squeeze through the capillaries thereby requiring a high degree of flexibility.
The large size of the RBC is required in order to maximize surface area for gas exchange and
reduce the distance for gas exchange. Erythrocytes in SCA have increased viscosity for their
respective hematocrit, are less flexible due to dehydration and morphological changes, which
increases Tc (Connes et al., 2015). Other factors. which increase Tc are cold temperatures and
dehydration, which act to reduce perfusion. Therefore precautions such as avoiding cold
temperatures and remaining well hydrated are suggested in order to help prevent such events. The
goal of therapy in SCD to prevent these crises and their downstream effects is to prolong Ts so that
the cells never sickle in the microvasculature. To that end, the ultimate goal is to prolong Ts so
long that the cell is reoxygenated by the lungs before sickling has occurred because once a sickled
erythrocyte becomes oxygenated, the polyermizing sickle Hb will dissolve back into solution and
the cell will revert back to its original morphology. To note, cells in SCD go through constant
cycles of sickling and unsickling upon oxygenation and after an unknown amount of cycles some
cells become irreversible sickled. Having introduced the dependency of [HbS] on intracellular
sickling and its implication with respect to Ts and Tc, the therapeutic effect of hydroxyurea (HU)
will become relevant within this chapter, as well as, the experimental work in Chapter 3.
26
1.4.2.2 Cellular Hemolysis
The average lifespan of a healthy erythrocyte is 115 days, ranging from 80-130 days (Mock
et al., 2014), however sickled cells have a survival estimate of ~2-20 days due to chronic
hemolysis, which causes a chronic state of reduced CaO2 (Uthman, 1995). The sickled cell has
undergone extensive oxidative damage and the cell displays the damaged proteins causing
circulating macrophages to phagocytose the damaged cells. Upon rupture, the cell releases various
cargo into the blood stream such as the hemoglobin, the heme-iron group, and arginase, which all
have significant roles in causing endothelial dysfunction as detailed below. The bone marrow
responds to the increased destruction of erythrocytes by increasing production of red blood cells,
therefore patients with SCD have an increased reticulocyte count. However, even with the
increased reticulocyte count, the average hematocrit (the fraction of whole blood comprised of red
blood cells) of a patient with SCD ranges from ~15-30% whereas in the healthy adult population
it is 0.36-0.44 for women and 0.41-0.50 for men (Steinberg, 1999). The state of reduced oxygen
carrying capacity is called anemia meaning "without blood"(Conrad, 1990). Because tissues
require oxygen to function normally, in particular the brain which consumes 20% of total oxygen
whereas only weighing 2% of total body mass, the body adapts to maintain adequate oxygen
supply. The primary adaption is to increase CBF in order to meet the oxygen demand (I. Prohovnik
et al., 1989). An increase in CBF is accomplished by dilation of the cerebral vessels, which reduces
the resistance to flow thereby increasing CBF. In addition, the cardiac stroke volume increases,
which increases CO thereby maintaining blood pressure within respectable limits in a state of
vasodilation (Debaun, Derdeyn, & McKinstry, 2006).
Hemolysis of red blood cells causes anemia. In addition, the hemolysis releases
hemoglobin and arginase into the circulation which affects the bioavailability of nitric oxide (NO)
27
(Wood, Hsu, & Gladwin, 2008). Nitric oxide (NO) is a gaseous free-radical with a half-life of ~6-
30s, which is produced by various cellular subtypes including; muscle, endothelial, neuronal and
glial cells (Palmer, Ashton, & Moncada, 1988). NO is a potent vaso-dilator and is synthesized
from L-arginine in the presence of several cofactors, by the following enzymatic iso-forms of nitric
oxide synthase (NOS); the constitutively expressed neuronal and endothelial NOS as well as the
inducible NOS, which is activated during acute inflammation(Schulz, Nava, & Moncada, 1992).
NO derives its vasodilatory activity through activation of guanylate cyclase resulting in cyclic
guanosine-3',5'-monophosphate (cGMP) production, which activates GMP-dependent kinases that
decrease intracellular calcium thus resulting in relaxation of smooth muscle cells and vessel
dilation (Moncada, Palmer, & Higgs, 1991). In addition, NO has anti-inflammatory properties by
inhibiting the synthesis of cytokines as well as cell adhesion molecules, thereby limiting the
recruitment of inflammatory cells and preventing their extravasation into the vessel wall (Bath,
Hassall, Gladwin, Palmer, & Martin, n.d.). Finally, NO is an important anti-oxidant where it has
been shown to scavenge radicals in vitro and in vivo (Kanner, Harel, & Granit, 1991).
The vasodilatory, anti-inflammatory, and anti-oxidant properties of NO are thought to be
executed in a para/autocrine fashion, where the NO is produced in the endothelium, diffuses across
the membrane, and directly acts on smooth muscle cells and/or endothelial cells. The fundamental
reason for NO's limited reach is based on its interaction with iron in RBCs. As NO traverses into
the circulation, it's solubility grants it the ability to diffuse across the RBC membrane and interact
with the iron pigment of hemoglobin. NO reacts at diffusion-limited rates with oxyhaemoglobin
to produce nitrate and methemoglobin, therefore effectively quenching its activity as seen in Figure
1.6 below (Ishiguro, Miya, & Nishida, 2003). It is in this light that RBCs are thought to be an NO
'sink'.
28
SCD is characterized by a state of limited NO bioavailability (Wood et al., 2008). The
release of hemoglobin into the circulation quenches the NO without the need of NO to cross the
RBC. Furthermore, the heme-iron can cause oxidative damage to the endothelia thus
compromising its function. In addition, upon hemolysis, arginase is also released into the
circulation. As mentioned above, L-arginine is necessary to produce NO in the endothelial cells.
Typically the arginase is sequestered within the cell but once it becomes released it will readily
consume all available L-arginine thereby limiting the production of NO. These two processes
reduce NO bioavailability, thereby affecting the endothelial vasodilatory, anti-inflammatory, and
anti-oxidant properties (Reiter et al., 2002). Because NO is a potent vasodilator and SCD patients
have reduced NO bioavailability, its role in the cerebral hemodynamics of children with sickle cell
disease seems to be of importance and will be further discussed in the experimental work of this
thesis.
Figure 1-6 NO interaction with iron pigment in hemoglobin in the
circulation.
29
1.4.3 Stroke Epidemiology and Risk Factors
The prevalence of SCA is 1 in 500-600 newborn African Americans infants and it is
estimated that 70,000 – 100,000 people in the US are living with the disease (Singh & Ballas,
2015). Furthermore, HbS trait occurs in 6.7-10.1% of all African Americans (Castro, Rana, Bang,
& Scott, 1987; Goldsmith et al., 2012). The most devastating complication in children with SCA
is overt ischemic stroke. The Cooperative Study of Sickle Cell Disease reported that the overall
prevalence of stroke is 3.75% in all patients with SCD (Ohene-frempong et al., 1998). However,
the prevalence was highest in the pediatric population where the prevalence of stroke was 11% in
patients by the age of 20 with HbSS disease (Ohene-frempong et al., 1998). The highest incidence
of stroke occurs in children between the age of 2 and 5 with HbSS, which is approximately 1.02
per 100 person-years (Webb & Kwiatkowski, 2013). The risk of stroke varies by SCD genotype
where the age adjusted incidence of stroke in SCD SS, SC, or HbS/β+/o is 0.61/100 patient years,
0.15/100 person-years, and 0.08 person-years, respectively (Kassim, Airewele, & Debaun, 2014;
Webb & Kwiatkowski, 2013).
Children with transcranial Doppler ultrasound (TCD) velocities greater than 200cm/s in
the middle cerebral artery (MCA) are at the highest risk for overt stroke. High TCD velocities are
due to two contributing factors; large-vessel vasculopathy and hyperemia as a compensatory
mechanism for anemia (Doepp, Kebelmann-Betzing, Kivi, & Schreiber, 2012). Large vessel
vasculopathy involves a progressive narrowing or stenosis of the major intracranial vessels where
the velocity of blood is highest at the most narrow portion of the stenotic vessel. Anemia is a result
of chronic hemolysis and reduced lifespan of sickled cells and encompasses a range of severities,
which requires a compensatory increase in CBF through vasodilation in order to attempt to
maintain CMRO2. Both vasculopathy and severe anemia are independent risk factors for overt
30
stroke. However, 79% of patients with abnormal TCD velocities do not have any narrowing of the
major intracranial arteries(Abboud et al., 2004). Furthermore, 1/3 patients suffering a stroke do
not present with any vasculopathy(Helton et al., 2014). Hence, a different stroke mechanism may
be related to anemia-related hyperemia. In addition to vasculopathy and anemia, silent cerebral
infarcts (SCI), prior transient ischemic attacks, frequency of acute chest syndrome (ACS),
increased systolic blood pressure, and nocturnal hypoxemia are all independent risk factors for
overt stroke and may play an important role in its pathophysiology (Kugler et al., 1993; Miller et
al., 2001; Ohene-frempong et al., 1998). SCI are a particularly important risk factor not only
because they share most of the risk factors with overt stroke, but also, they are currently believed
to arise from hemodynamic compromise following from vasculopathy or critically low CaO2 in
regions of the brain that are sensitive to hemodynamic impairment. In addition, they affect up to
40% of children with SCD by the age of 18 and present in a significant proportion of the patients
who underwent scanning. Due to their importance, an introduction to SCI will be provided next.
SCIs are defined as in increase signal/hyperintensity on conventional magnetic resonance
imaging, without corresponding neurologic deficits (Pegelow et al., 2002). Like overt strokes,
SCIs begin early on in life, showing up 13% of HbSS infants with an average age of 13.7 months
(Wang et al., 2008). By the age of 6, it's been reported that 27.7% of HbSS children demonstrate
SCI. The Cooperative Study in SCD reported an incidence of 21.8% of children between the ages
of 6 and 19 (Pegelow et al., 2002). Recently, Bernaudin's study reported a cumulative incidence
of SCI to be 37% in SCD patients by the age of 14 and 39.5% by the age of 18 (Bernaudin et al.,
2014). Therefore SCIs begin early on and continue to increase without plateau. The pathogenesis
of SCI is not currently well understood, however the watershed distribution implies a
hemodynamic rationale; either impaired regulation of CPP or diminished vasodilatory reserve,
31
however, this is still an active area of research (Quinn et al., 2013; Waterston, Brown, Butler, &
Swash, 1990)
1.4.4 Prevention of Primary and Recurrent Stroke
Until 1991, detection and prevention of overt stroke at its infancy. Adam’s longitudinal
observational study used transcranial Doppler ultrasound to assess the velocity (TCDv) of flowing
blood through the major intracranial arteries, which include the MCA, ICA, anterior cerebral artery
(ACA), posterior cerebral artery (PCA) (Weiner et al., 1997). The results showed that higher TCDv
were associated with a higher risk of stroke, where children with TCDv greater than 200cm/s in
the MCA had a 40% chance to stroke within 3 years and constituted an ‘abnormal’ TCDv. In
addition, children with TCDv who had TCDv>175 but <200cm/s were designated as conditional,
where the risk of stroke was not high enough to warrant the chronic transfusion protocol. The
threshold of 200cm/s allows for 94-100% sensitivity, meaning that TCD is able to detect virtually
almost every child that will suffer a stroke. However, the specificity of TCD is ‘no better than a
flip of the coin’, at 51%, which means that a significant proportion of children with abnormal
TCDv will not suffer a stroke (Seibert et al., 1998).
This observational study led to the Stroke Prevention Trial in Sickle Cell Anemia (STOP
trial) where children with over 200 cm/s TCDv were randomized into a chronic transfusion arm
(cTx) with the goal of keeping the HbS% less than 30% or the standard of care arm, which was
effectively observational. The trial set out to determine whether chronic transfusion in SCA
children at risk for stroke could reduce stroke events. The trial was terminated early due to
overwhelming success as the transfusion-arm of the trial showed a 92% relative risk reduction of
overt stroke compared to the observational arm (R. J. Adams, 1998). Although a controlled clinical
trial is lacking, standard care for preventing recurrent stroke is through cTx. Without cTx, recurrent
32
stroke occurs in 60% of stroke patients (Powars, Wilson, Imbus, Pegelow, & Allen, 1978). With
cTx, recurrent stroke occurs in approximately 20% of stroke patients (Hulbert, McKinstry, Lacey,
Moran, Panepinto, Thompson, Sarnaik, Woods, Casella, Inusa, Howard, Kirkham, Anie, Mullin,
Ichord, Noetzel, Yan, Rodeghier, & Debaun, 2011; Scothorn et al., 2002). Transfusion of red blood
cells can be performed either as a simple transfusion, also known as a top-up transfusion, or
exchange transfusion. It isn't entirely clear how cTx reduces the risk of stroke however, performing
exchange transfusions essentially allows for the removal of red blood cells containing sickle
hemoglobin and exchange them for healthy donor adult blood cells. In cTx programs, the HbS%
is typically kept below 30% meaning that the majority of blood contains healthy adult red blood
cells, which can properly oxygenate the tissue. In addition to primary and secondary stroke
prevention, cTx therapy was also shown to reduce the relative risk of SCI by 58% in the Silent
Cerebral Infarct MultiCenter Clinical Trial (SIT) (DeBaun et al., 2014).
Although cTx is an effective means of reducing the risk of both primary and secondary
stroke, as well as SCI, cTx is associated with several risks and complications such as; iron
overload, alloimmunizations, delayed hyperhemolysis reactions, and risk of bloodborne infection
(Chou, 2013). Furthermore, cTx poses a considerable financial burden on the health care system
and to the family. In addition, transfusion therapy is not readily available in all health centres
therefore requiring patients to travel to major institutions. To that end, when considering sickle
cell disease as a global issue, the major burden exists in sub-Saharan Africa where the resources
required to prevent stroke are virtually unavailable. In summary, the significant cost and negative
health effects of chronic transfusion led to the STOP II trial (optimizing Stroke Prevention in
Sickle Cell Anemia). The STOP II trial ran in 2000 to determine if transfusion could safely be
withdrawn after 30 months of cTx treatment in patients who had converted from abnormal to
33
normal TCDv. The STOP II trial halted early due to the advent of adverse events (stroke).
Therefore it is not safe to end transfusion after 30 months of treatment in those who had converted
to normal TCDv.
A major dilemma in the care and treatment of children with SCD is finding a therapy to
replace transfusion for stroke prevention due to its significant burden. Present efforts are being
directed at assessing how hydroxyurea (HU) compares to transfusion for stroke prevention. HU is
a myelosuppressive agent, which is also used in HIV and other blood cancers due to its cytostatic
capabilities (McGann & Ware, 2015). HU is able to abolish cell division by inhibition
ribonucleotide reductase activity therefore freezing the cell in S-phase of mitosis. Incidentally,
patients treated with HU showed higher fetal haemoglobin F (HbF) expression (Charache et al.,
1992; Letvin, Linch, Beardsley, McIntyre, & Nathan, 1984; Platt et al., 1984, 1994). Humans
solely express HbF in the fetal stage. HbF is comprised of two alpha and two gamma globins
whereas adult haemoglobin (HbA) is comprised of two alpha and two beta globins. Because the
sickle mutation codes for a sickle beta globin, by increasing fetal haemoglobin you introduce an
oxygen carrier that does not use beta globins and is less likely to undergo cellular sickling.
Introducing HbF essentially dilutes the red cell of HbS, this is critical as when a cell gives off
oxygen in the capillaries, it will be producing both deoxy HbF and deoxy HbS as opposed to just
HbS. By decreasing the number of deoxy HbS you limit the rate and time to sickle by a significant
margin. HbS and time to sickle are proportional to the power of 30 therefore any reduction in HbS
has a drastic affect on when the cell sickles (Alexy et al., 2010).
HU has been clinically proven to increase HbF in patients with SCD. The clinical indication
for offering HU therapy was vasooclusive crises or events of acute chest syndrome as the landmark
trial showed that HU significantly reduced the number of vasooclusive crises and ACS events
34
(Platt et al., 1994) .Subsequent studies showed that HU is safe both in pediatric and the infant
population (BABYHUG) (Pavlakis et al., 2010). The next step was to determine how HU compares
to cTx for stroke prevention. The Stoke With Transfusions Changing to Hydroxyurea (SWiTCH)
was a non-inferiority trial that compared cTx and chelation therapy to HU and phlebotomy for
secondary stroke prevention and iron management in children with SCD(Russell E. Ware &
Helms, 2012). The trial was not designed to test whether HU is superior to cTx for secondary
stroke prevention because it was already well accepted that it was not. However, because cTx
causes such significant complications with iron overload, the relative increased risk of secondary
stroke of patients on HU compared to cTx could be accepted if the iron levels were managed. The
outcome of the SWiTCH showed that transfusion and chelation therapy remain a more appropriate
way to manage stroke and iron overload than hydroxyurea and phlebotomy. Currently, the
Transfusions Changing to Hydroxyurea (TWiTCH) clinical trial is ongoing, which sought to
determine whether HU lowers TCDv to a similar degree as cTx for primary stroke prevention.
Early results have shown that HU is not inferior to cTx with respect to reducing TCDv in children
who are at high risk for stroke and therefore may possibly serve a place in patient management of
primary stroke (http://www.nih.gov/news/health/nov2014/nhlbi-19.htm). Considering the
importance of HU in stroke prevention and that the experimental work in Chapter 3 assessed its
effect on cerebral hemodynamics, an introduction to the mechanism and implications of HU on
the cerebrovasculature is warranted.
HU increases HbF, which reduces the sickling rate and therefore all of the downstream
effects of the disease such as hemolysis and the previously mentioned vassoclusive crises. HU
reducing the rate of hemolysis is supported by changes in haemolytic parameters such as a decrease
in reticulocytes, liver dehydrogenase, and plasma arginase and plasma free hemoglobin levels
35
(Ballas, Marcolina, Dover, & Barton, 1999a). The beneficial effect of reducing hemolysis is
multifaceted. Firstly, by reducing hemolysis the Hct and hemoglobin levels increase which
ameliorates the degree of anemia whose importance will be provided in Chapter 2. Secondly,
hemolysis releases arginase and Hb, which is typically sequestered within the erythrocyte, into the
circulation which has a dramatic effect on NO bioavailability whose activities include maintain
vascular tone, anti-inflammatory and antioxidant, etc. By reducing hemolysis and thereby reducing
its NO quenching abilities, HU has shown to improve NO bioavailability (Gladwin et al., 2002;
Wood et al., 2008). Lastly, the haem iron can oxidize surrounding materials and promote the
production of free radicals which can damage the endothelia therefore reducing the rate of sickling
has a general and overwhelmingly positive effect in this disease.
1.5 Magnetic Resonance Imaging
In Chapter 1.3, the role of CBF and cerebrovascular reserve in maintaining oxygen supply
to the cerebral tissue was described. Following that, SCD was characterized as a disease of
chronically reduced oxygen content and dysfunctional endothelia, which is implicated in the
pathophysiology of neurologic injury. The experimental work in this thesis utilizes magnetic
resonance imaging (MRI) in order to assess the cerebral hemodynamic environment in children
with SCD therefore this chapter will serve to introduce basic concepts in MRI and how MRI can
be used to obtain measurements such as CBF and cerebrovascular reserve.
1.5.1 Basic MRI Principles and Contrast Mechanisms
1.5.1.1 MRI Architecture
The work in this thesis was performed on a clinical 3T Siemens MRI scanner, however
there are other vendors such as Philips and General Electric, Hitachi, Toshiba, etc. Nevertheless,
36
MRI scanners are fundamentally comprised of three types of coils, which generate 3 distinct types
of magnetic fields; the superconducting coil creates the main magnetic field, the transmission coil
and the gradient fields. There are four major types of magnets used to create the main magnetic
field (Bo); air-cored resistive magnets, iron-cored electromagnets, permanent magnets and
superconducting magnets. For the purpose of this thesis, only the superconducting magnet will be
referred to as it is the one utilized in most clinical MRI scanners. The Bo is produced by a current
running through a coil, which is cooled by liquid helium to approximately 2o Kelvin (-271.16oC)
in order to allow for super-conductance. The main benefit of achieving super conductance is to
achieve a state of approximately 0 electrical resistance, therefore the current will continue to
circulate through the wire indefinitely. The superconducting magnet is permanently on as it is
incredibly costly to turn the magnet off and on again. The main field strength is expressed in units
of Tesla (T), which is the unit of magnetic flux density or induction. One T is equivalent to 10,000
Gauss. The transmission coil transmits radiofrequency (RF) pulses perpendicular to Bo, which
oscillates at a set frequency. The magnetic field from the transmission coil is extremely small (1/10,
000T). The transmission coil surrounds the body part destined to be imaged (brain, neck, body
coils, etc.) and is used to 'excite' the hydrogen atoms through the phenomenon of nuclear
resonance, which will be described below. The gradient fields (Gx, Gy, Gz) are produced by three
independent coils whose general function is to localize the MR signal in 3-D space.
The target and source of signal in MRI can include isotopes such as; H-1, C-13, Xe-129,
etc. The critical component is that the nucleus must have a net integral nuclear spin, which
produces a magnetic moment, therefore nuclei such as C-12, O-16, S-32, which have 0 spin, cannot
undergo magnetic resonance. The most common is H1-MRI, due to the naturally high abundance
of hydrogen nuclei within the body (75-80%).
37
1.5.1.2 Magnetic Resonance
For simplistic purposes, in a constant external magnetic field, the spin of the hydrogen
nuclei align parallel to the external magnetic field. When aligned with Bo, nuclei precess at a
particular frequency as demonstrated by the following equation;
𝒘𝟎 = 𝒚𝑩𝟎 Equation 10
Where 𝒘𝟎 is the Larmor frequency of the precessing spin in radians per second, 𝐵0 is the strength
of the external magnetic field in Tesla, and 𝑦 is the gyromagnetic ratio of a proton, which is 2.67
X 108 radians s-1 T-1 (or 42.58 MHz/T). The net magnetization (M) of a proton in an external
magnetic field can be classically viewed as longitudinal (Mz) and transverse (Mxy) vector
components as seen in Figure 1.7 below. When aligned with Bo, the entire magnetization is in Mz
axis. When a radiofrequency (RF) pulse is applied at a frequency equal to that of the hydrogen
nuclei's Larmor frequency, the hydrogen nucleus absorbs the RF pulse and the Mz is tipped by 90o
into the Mxy plane. This process is called nuclear magnetic resonance.
38
When the precessing hydrogen nuclei absorbs the RF pulse, it is placed in a high energy
state ('excited') and will eventually return back down to its ground state magnetization, Mz, after
the RF field is turned off. In the transition from the high energy to low energy state, the hydrogen
nuclei releases energy in the form of RF radiation. This process is called relaxation. The RF energy
released is read by the receiver coil and is the raw signal that the MRI uses. The relaxation
processes can be described as the recovery of Mz and decay of Mxy, which have exponential
profiles and will be detailed separately.
If an RF pulse was applied, the information retrieved would have very poor spatial
specificity because all the hydrogen nuclei would be affected by a uniform magnetic field and
therefore would be precessing at the same Larmor frequency according to Equation 5.1. Therefore,
Figure 1-7. Vector representation of hydrogen nuclei magnetization.
Obtained with permission from Dr. Kassner's Lab.
39
when an RF pulse is applied at the Larmor frequency, most of the hydrogen would undergo
resonance, undergo relaxation and provide signal. Generally speaking, spatial specificity is
accomplished through the use of gradient coils. The use of gradient coils distorts Bo so that it is no
longer uniform and changes in field strength along a predefined gradient. Therefore, the hydrogen
nuclei along the gradient magnetic field no longer precess at the same Larmor frequency but at a
different frequencies depending on the field strength at that position in space. Now, when a RF
pulse is applied at a particular frequency, only the hydrogen nuclei with the matching Larmor
frequency will undergo nuclear resonance, whereas the other nuclei along the gradient would not.
Only the hydrogen nuclei that underwent resonance are able to undergo relaxation and provide
signal. Therefore, by precisely adjusting the gradients, one is able to obtain signal from certain
imaging slices, which is the general basis under slice-selection.
1.5.1.3 T1 – Recovery
The recovery of Mz following the excitation by the RF pulse is called the spin-lattice
relaxation or T1 relaxation and can be described by the following equation;
𝑴𝒁 = 𝑴𝟎 × (𝟏 − 𝒆 𝑻𝟏
−𝒕) Equation 11
Where 𝑀𝑍 is the longitudinal magnetization of the nuclei at a time = 𝑡, 𝑀0 is the
magnetization vector , 𝑡 is the time, and 𝑇1 is the time it takes for 63% of the equilibrium
magnetization (𝑀0
) to recover. This equation is depicted in Figure 1.8 below.
40
The mechanism at which T1 occurs is as follows. Hydrogen nuclei are in constant motion,
which includes both vibrational and translational processes. The frequency at which these motions
occur is called the spectral density function (SDF). Considering each nuclei has specific motion
frequencies, then the magnetic moment of each nuclei will also have a frequency distribution. In
order for an excited atom to undergo T1-relaxation (or spin-lattice relaxation), its neighbouring
proton's magnetic moment fluctuation frequency have mirror the excited atom's Larmor frequency.
Once these frequencies match, the excited atom can release the energy and transition back to the
ground-state, which can be described as the recovery of the Mz. The critical feature is that hydrogen
in different environments have varying SDFs and therefore have different T1s. In other words, the
Figure 1-8 Graph depicting T1 recovery following a 90o excitation pulse. Adapted
from http://www.revisemri.com/tools/timeconst/images/90recovery.gif
41
rate of Mo recovery is dependent on the environment of the hydrogen nuclei. Fluids such as blood
or cerebral spinal fluid (CSF) have long T1s (1500-2000ms), fatty tissues such as adipose or
cerebral white-matter have short T1s (100-150ms), and water-based tissues such as muscle or grey-
matter have intermediate T1s (200-1200ms). Therefore, according to Equation 2, after a time t,
hydrogen nuclei in different environments will have different Mz and it is this difference that
produces contrast between different tissue types.
1.5.1.4 T2 – Decay
The decay of Mxy following the excitation by the RF pulse is due to the collective dephasing
of nuclear spins and is called the spin-spin relaxation or T2 relaxation and can be described by the
following equation;
𝑴𝑿𝒀 ≅ 𝑴𝟎 × 𝒆𝑻𝟐
−𝒕 Equation 12
Where Mxy is the transverse component of the magnetization, Mo is the magnetization
vector, t is the time, and T2 is the time it takes for the magnetization to reduce to 37% (1/e) of its
original value. This equation is graphically depicted in Figure 1.9 below.
42
T2 decay (spin-spin relaxation) occurs due to the fact that there are several hydrogen nuclei
within proximity of one another. A lone hydrogen nucleus precesses at a frequency set by the Bo,
however a spinning nucleus also produces its own magnetic field, which can interact with
neighboring hydrogen nuclei causing an increase or decrease in their precession frequencies.
Because the nuclei are in constant motion, as the nuclei move further apart, they return to the
original Larmor frequency precession. However, although they once again are precessing at the
Larmor frequency, they do so in different phases. This spin-spin interactions ultimately results in
a loss of phase across all the excited hydrogen nuclei, which results in a decay in signal (T2). The
Figure 1-9. Graph depicting T2 decay following a 90o excitation pulse. Adapted from
http://www.revisemri.com/tools/timeconst/images/90decay.gif.
43
critical feature is that hydrogen atoms in different environments have different SDF where the fast
moving spins (ex CSF) experiences the neighbouring hydrogen's magnetic fields as fluctuating
rapidly, which essentially averages out over a few milliseconds (ms). Therefore hydrogen in a fast-
moving, less constrained environment see a relatively homogeneous local field and produce little
dephasing (long T2) than the protons in slower-moving, constrained environment, which
experience the local field in-homogeneities (due to the other protons magnetic fields) to a greater
extent and therefore dephase quicker (short T2s). The T2 of fluids such as CSF or blood is the
longest (700-1200ms), fatty tissues such as adipose or white-matter are the shortest (10-100ms),
while fluid-tissues such as muscle or grey-matter are intermediate (40-200ms). Therefore
according to equation 3, after a time t, hydrogen nuclei in CSF would experience less Mxy decay
than white matter, therefore show contrast once the image is generated.
1.5.1.5 T2* - Decay
In the perfect world, Bo is homogeneous and T2 decay occurs only due to the random
interaction of different nuclei's generated magnetic fields (spin-spin interaction). However, due to
imperfections in the super conducting magnet, there are inhomogeneities in the external magnetic
field. Furthermore, every tissue has different magnetic susceptibilities, which causes main field
distortion at tissue boundaries, in particular air/tissue interfaces. In addition, patients may have
metal implants, which can also cause field distortions. The local-field distortions in the external
field due to the magnetic fields of precessing spins are random in nature and in the perfect world
would account for the T2 decay. However, the distortions from imperfections in the magnet, tissue
magnetic susceptibilities, and possible metal implants are fixed. Therefore, in reality, the Mxy
44
decays faster than T2 would predict. The rate at which this decay occurs is called T2* and is the
sum of the fixed and random T2 effects as shown by the equation
𝑴𝑿𝒀 ≅ 𝑴𝟎 × 𝒆𝑻𝟐∗
−𝒕 Equation 13
𝟏
𝑻𝟐∗ =
𝟏
𝑻𝟐 𝑹𝒂𝒏𝒅𝒐𝒎+
𝟏
𝑻𝟐 𝑭𝒊𝒙𝒆𝒅 Equation 14
1.5.1.6 Contrasts in MRI
The time between the application of an excitation pulse to the application of the subsequent
excitation pulse is known as the repetition time (TR). Particular TR and TE can be used to produce
images with certain weightings because hydrogen nuclei in different environments have different
relaxation rates as seen in below in Figure 1.10. An image with T1-weighting is one that maximizes
T1 signal contributions and tissue contrast while simultaneously reducing T2 contributions.
Therefore to create a T1-weighted image, TR is kept short in order to maximize contrast between
tissues of different T1s, and TE is kept short in order to reduce T2 contrast. To create a T2-weighted
image, TR is kept long in order to allow for full T1 relaxation thus minimizing signal contribution
from T1 processes, TE is kept long in order to maximize T2 signal contribution. There are two other
combinations for TR and TE, having short TR and long TE does not produce a useful image as
having a short TR provides T1 weighting and a long TE provides T2 weighting therefore the
contrast is not with meaning. However, using a short TR and TE removes weighting from both T1
and T2 contributions and produces an image called proton density, which is useful in certain
45
clinical scenarios such as in knee imaging to distinguish articular cartilage from the cortical bone
and menisci.
Figure 1-10 Generating T1 contrast with changing Repetition and Echo Time in
Gray Matter, White Matter, and Cerebrospinal Fluid. Obtained with permission
from Dr. Kassner's lecture slides.
46
1.5.2 Cerebrovascular Imaging
1.5.2.1 Time of Flight Angiography
Simplistically speaking, motion during an MRI scan results in an inaccurate storing of raw
MR signal. When the image is reconstructed using the data from k-space (a matrix that temporary
stores raw MR data), the motion will produce image blurring/repetition artifacts. Motion can
include gross patient motion, swallowing, but can also be physiological in nature such as a heart
beating or the flow of blood through vessels, which can all cause artifact. However, MRI Time of
flight (TOF) angiography (MRA) takes advantage of this phenomenon in order to visualize the
arterial system. When exciting an imaging slice that contains a major vessel, during the TR of a
pulse sequence, the bolus of blood within the imaging slice moves and is replaced by fresh bolus
of blood. In spin-echo sequences, if the blood speed is such that the magnetized blood water spins
leave the imaging slice in the time between the 90o pulse and 180o inversion pulse, then no spin
echo is generated and therefore there is no signal. The relatively stationary spins, such as those
within tissue, remain in the imaging slice and therefore generate their spin echoes and produce
signal.
GE sequences do not use the slice selective 180o inversion pulses, therefore do not experience
these signal washouts but instead only flow enhancement effects. Therefore when the blood flow
is such that a fresh bolus of blood replaces the RF tipped bolus, then the next time an RF pulse is
applied, the fresh blood will be able to produce more signal as seen below in Figure 1.11. In
addition to other things, MRA images are useful as it allows for the radiologist to look for vessel
narrowing (stenosis).
47
1.5.2.2 Assessing Microcirculatory Flow
The microcirculation includes the flow within capillaries and capillary beds.
Microcirculatory flow, which is typically referred to as perfusion in units of mL/min/100g, is the
flow of blood to an amount of tissue and will be referred to as cerebral blood flow (CBF) from
now on. CBF data can be acquired using various imaging techniques such as; nuclear imaging, and
computed tomography (CT), and MRI. Nuclear imaging primarily includes O-15 H2O positron-
emission tomography (PET). PET allows for quantification of CBF in physiological units and is
the current gold standard for measuring CBF (Chen, Wieckowska, Meyer, & Pike, 2008).
However, PET requires the injection of radioactive tracers, which limits studies where repeat scans
are required and is unappealing in pediatric populations. In addition, PET has low temporal and
spatial resolution, low signal-to-noise ratio (SNR), and requires cyclotrons, which are not readily
available everywhere (Chen et al., 2008). CT can be used to measure CBF by using xenon as a
contrast agent. The benefits of xenon CT are its ability to measure absolute CBF in physiological
units. In addition, xenon CT is particularly advantageous in low flow conditions, such as brain
ischemia. The drawbacks of xenon CT includes; exposure to x-ray radiation, low SNR, and xenon's
Figure 1-11 Time of Flight Concept. Obtained with permission from Dr. Kassner's
Lecture Slides.
48
anesthetic properties. The anaesthetic properties can alter baseline physiology and x-ray irradiation
is not favourable in a pediatric population (Duncan, 1997). MRI techniques for measuring CBF
include dynamic susceptibility contrast (DSC) MRI or arterial spin labeling (ASL). Dynamic
Susceptibility Contrast (DSC) DSC uses a contrast agent (typically gadolinium gadopentetate) and
tracer kinetics in order to quantify CBF, however, the gadolinium gadopentetate has recently been
shown to remain in the tissues for extended periods of time, which limits its use in pediatric
populations (FDA Safety Announcement, 2015). The main common drawback with PET, CT, and
DSC is its unfavorable implementation in pediatric populations. The alternative means to acquire
CBF data, non-invasively, without the use of ionizing radiation, is through the use of ASL MRI.
ASL was used to quantify baseline CBF in both experimental studies and will be introduced in
detail next.
1.5.2.2.1 ASL MRI Basic Concepts and Analysis for Baseline CBF Measurements
ASL measurements of CBF are based on the principle that hydrogen nuclei can be
magnetically labeled/tagged’ by a RF pulse therefore allowing the use of endogenous blood water
as the tracer (Silva, Stefanovic, & Paiva, 2004). ASL magnetically tags a bolus of blood, typically
at the level of common carotids, and collects the raw MRI signal at the level of the brain tissue
after a pre-set time delay, known as the post label delay (PLD) (Silva et al., 2004). Following the
time delay, the magnetically labeled spins flow from the inversion plane at the level of the common
carotids and into the brain where the image is acquired as seen in Figure 1.12 below. As the
magnetized bolus circulates into the brain, it begins mixing and exchanging with unmagnetized
blood water and extravascular water, which causes a net decrease in magnetization. This decrease
49
in net magnetization is what is used in order to calculate CBF. Thus far, the raw image obtained is
called the tagged image. Subsequently, a control image is acquired without any magnetic tagging.
Subtraction of the signal difference from the tag and control images allows for the measurement
of CBF. An example ASL CBF map is seen below in Figure 1.13 below.
Figure 1-12. Basic representation of labeling and Imaging planes in ASL.
Obtained with permission from Dr. Kassner's slides.
Figure 1-13 Representative arterial spin labeling image of cerebral blood flow.
Obtained with permission from Dr. Kassner's lab.
50
One of the fundamental drawbacks of ASL is that the magnetically labeled blood
comprises only 1% of the voxel volume and that ASL sequences tag only a small portion of that
volume at the level of the carotids, which limits the available SNR. In addition, the subtraction
process also reduces SNR (Golay X, Hendrikse J, Lim TCC (2004) Therefore, several averages
are required in order to have sufficient SNR, which makes ASL susceptible to motion artifacts.
Another issue with ASL is that the PLD is set prior to the scan and is dependent on mean transit
time. In low flow environments where the transit time is increased such as during cerebral
ischemia, the PLD may be too short. In a low flow environment, a too short of a PLD would result
in insufficient time for any magnetically tagged spins to enter the imaging plane therefore
underestimating CBF. In this case, if the PLD was increased the magnetization may be lost due to
T1 by the time the spins arrive in the tissue of the imaging plane. Alternatively, the same PLD
would be too long in a high flow environment, which would result in the tagged blood to partially
or entirely leave the imaging plane prior to image acquisition thereby underestimating CBF (Silva
et al., 2004).
ASL techniques differ based on how the blood is tagged. Continuous ASL (CASL)
provides a continuous RF pulse, pulsed ASL (pASL) tags a slab of blood water, and
pseudocontinuous ASL (pCASL) uses several short trains of inversion pulses. In the experimental
sections of this thesis, pASL was used to measure CBF. In pASL, a large imaging slab is inverted
at the level of the feeding arteries. The benefits is that the labeling efficiency is very high and is a
function of slab size not labeling duration. Also, the energy deposition is lower than the other
tagging pulse sequences. (Buxton et al., 1998; Grade et al., 2015). =
With pASL, regional CBF can be quantified from the mean signal difference between ASL
tag and control images using a single compartment kinetic model with the following equation;
51
𝑪𝑩𝑭 =𝟔𝟎𝟎𝟎𝝀(𝑺𝑰𝑪𝒐𝒏𝒕𝒓𝒐𝒍− 𝑺𝑰𝑳𝒂𝒃𝒆𝒍)𝒆
𝑻𝑰𝟐𝑻𝟏𝒂
𝟐𝜶𝑻𝑰𝟏𝑺𝑰𝑷𝑫 Equation 15
where CBF is the regional cerebral blood flow in mL/min/100g, λ is the blood/tissue water partition
coefficient (0.9mL/g), SIControl and SILabel are the time-averaged signal intensities in the control and
label images, α is the inversion efficiency (98%), SIPD is signal intensity of a proton density-
weighted image, TI1 is the time between inversion tagging and bolus termination, TI2 is the
duration between the inversion tagging pulse and the excitation pulse, and T1α is the longitudinal
relaxation time of blood at 3T (1650ms) (Aslop, et al 2015).
1.5.2.3 Cerebrovascular Reactivity for Measuring Cerebrovascular Reserve
1.5.2.3.1 Introduction
As has been recently detailed in Chapter 1.3, the control of vascular radius has profound
effects on CBF and is mediated by several physiological mechanisms. However, there is a finite
limit to which a vessel can dilate, which is referred to as vasodilatory capacity or cerebrovascular
reserve. Sufficient vascular reserve is critical in maintaining CBF across a range of cerebral
perfusion pressures, increasing CBF during times of increased O2 consumption or reduced oxygen
content. Therefore it is beneficial to measure vascular reserve in order to characterize the
hemodynamic status of the cerebral flow environment in both healthy and disease processes.
Cerebrovascular reactivity (CVR) reflects the vasculature's ability to accommodate an
increase in the demand for CBF (Mark, Mazerolle, & Chen, 2015). CVR is determined by
measuring the change in cerebral blood flow (CBF) following the administration of a vasoactive
52
stimulus, such as CO2, acetazolamide injection, etc. In the healthy brain, vasoactive stimuli such
as CO2 results in dilation of resistance-arterioles, which reduces the vascular resistance thereby
increasing blood flow. A vessel has a finite degree to dilate therefore CVR can reflect
cerebrovascular reserve (Gupta et al., 2012). CVR has provided insights into various disease
processes that affect cerebral vessels such as arterial venous malformations, cerebral gliomas,
Alzheimer's disease, etc. (Fierstra et al., 2011; Glodzik, Randall, Rusinek, & de Leon, 2013; Hsu
et al., 2004). In particular, CVR has shown significant utility in diabetes and carotid artery disease,
where inadequate CVR was shown to contribute to a higher risk of stroke (Fülesdi et al., 1999;
Yonas, Smith, Durham, Pentheny, & Johnson, 1993).
Under conditions when resting blood flow is normal, a low CVR indicates that the
vasculature is unable to provide the same increase in CBF compared to healthy vasculature. Low
CVR can be attributed to endothelial injury/dysfunction, which impairs the vasculature's response
to dilatory stimuli. In addition, low CVR can also mean the vessel response is delayed or that the
brain area in question has increased arterial transit time, such as in the white matter. Apart from
being reduced, CVR can also be negative, which means that the vasodilatory stimuli produces a
paradoxical decrease in CBF to the brain region. Negative CVR has been demonstrated in carotid
occlusive diseases or moya moya vasculopathy, where the vessels downstream to the stenosis are
maximally dilated in order to maintain CBF (Mikulis et al., 2005). In the event that a patient with
stenotic large feeding vessels is given a vasodilatory stimulus, then the maximally dilated vessels
are unable to further dilate , whereas the less affected vasculature surrounding the area still have
reserve intact and their dilation causes a redistribution of blood flow from the affected area to the
healthy area . To that end, CVR has been well used in patients with carotid artery disease in order
53
to identify and stratify the disease based on severity and to assess post surgical outcome (Gupta et
al., 2012).
1.5.2.3.2 Vasoactive Stimuli and Delivery
Measuring CVR requires the use of a vasodilatory stimulus in order to increase CBF. There
are various methods to cause dilation of cerebral resistance-vessels, however the most common
include some form of CO2 inhalation, breath-hold, or acetazolamide injection. The method used
in the experimental work of this thesis was CO2 inhalation, which increases the CO2 partial
pressures in the blood stream (hypercapnia). CO2 inhalation has the benefit of being inexpensive,
non-invasive, and well tolerated. Hypercapnia can be achieved through CO2 gas inhalation or
accumulation via breath hold. Breath hold techniques rely on innate metabolism of cells to produce
CO2 and decrease pH thereby causing SMC relaxation and consequent vasodilation. The benefit
of using a breath hold technique is that it can be done at the bedside and does not require expensive
equipment. However the drawbacks are several; firstly under a breath-hold there is the
confounding vasodilatory effect of hypoxia that needs to be accounted for, which may change CBF
and the metabolic rate of oxygen. Furthermore, the sensitivity of this technique is poor as the O2
and CO2 blood gases progressively decrease and increase respectively. The reproducibility is also
an issue whereas each individual has a different metabolic rate and therefore produce CO2 at
different levels, in addition, the cooperation of the subject varies and can be limited in disease
states. Lastly, breath-holding can result in compliancy issues especially in younger children
thereby limiting its applicability to compliant adults (Saito et al., 2011; Sasse, Berry, Nguyen,
Light, & Mahutte, 1996; Totaro, Marini, Baldassarre, & Carolei, n.d.). As opposed to relying on
metabolic production of CO2, CO2 gas can be administered exogenously either through fix-
inhalation or through a computer controlled gas blender. The computer controlled gas blender,
54
which administers patient specific concentrations of both O2 and CO2 simultaneously in order to
prospectively target particular end-tidal O2 (PETO2) and CO2 (PETCO2) levels thus providing the
operator with appreciable control over the gas stimuli (Spano et al., 2013). A schematic of the
computer-controlled gas delivery system used in our experiment is shown below in Figure 1.14.
Figure 1-14. Computer-controlled gas blender and delivery apparatus. Obtained with
permission from Dr. Kassner's Lab.
55
1.5.2.3.3 Measuring CVR
For any CVR experiment, a change in CBF or velocity must be calculated. Measuring a
change in CBF using ASL MRI sequences is difficult as ASL has limited temporal resolution due
to the time required for the labeled spins to traverse across from the tagging plane to the imaging
plane. Limited temporal resolution restricts the number of possible tag and control images
obtained, which impacts the potential SNR. Furthermore, ASL sequences suffer from poor SNR
due to the fact that blood comprises only 4% of an imaging voxel which limits the maximum
amount of signal available, as well as the nature of the subtraction method. Finally, the change in
CBF typically requires a vasodilatory stimuli in order to increase the CBF therefore the post
labeling delay assumed for ASL may not be suitable for the increased CBF. The benefit of ASL
CVR is that the parameter is calculated using physiological relevant units of mL/min/100g.
Furthermore, the ASL signal comes from the arterial side thus providing ASL CVR values with
high specificity. While using ASL allows for the direct measurement of CBF changes, it is not yet
standardized and the technical hurdles mentioned above and faces challenges under high flow
environments of hypercapnia (Tancredi et al., 2012). Alternatively, Blood Oxygen Level
Dependent (BOLD) measures of CVR is an attractive option due to its high temporal and spatial
resolution and high SNR. BOLD imaging was used in the experimental work of this thesis for
CVR measurements and will be detailed below. In addition, a recent paper has demonstrated that
CVR mapping using a prospective CO2 stimuli and BOLD imaging is well tolerated, safe, and
feasible in a clinical patient population with a wide age range (Spano et al., 2013). Also, the use
of ASL-CVR has never been validated in advanced cerebrovascular disease.
56
1.5.2.3.4 Blood Oxygen Level Dependent
BOLD imaging is based on the magnetic properties of Hb where oxygenated hemoglobin
is diamagnetic and deoxygenated hemoglobin is paramagnetic (Kim & Ogawa, 2012). The
paramagnetic property of deoxygenated Hb creates a strong local magnetic field that can increase
or decrease the precessing speed of nearby water nuclei. After interacting with the magnetic field
of the deoxyhaemoglobin, hydrogen nuclei of water molecules diffuse across the red blood cell
membrane and return to their normal precessing frequency but are now out of phase. The bulk
dephasing of water nuclei due to deoxyhaemoglobin shortens the T2* and hence reduces the MR
signal. Therefore venous blood, which is approximately 60% oxygenated, has a shorter T2* than
arterial blood, which is approximately 100% oxygenationed. The BOLD effect occurs when the
proportion of oxygenated/deoxygenated blood changes during a task/stimuli, which results in a
change in T2* and therefore MR signal as seen below in Figure 1.15 and 1.16 (Bold schematic and
image).
Figure 1-15 Blood-Oxygen Level-Dependent Schematic adapted with
permission from Dr. Kassner Neuroimaging Course Slides.
57
Assuming the metabolic rate of oxygen (CMRO2) remains unchanged, an increase in CBF
following CO2 inhalation will decrease oxygen extraction fraction (OEF), which leads to an
increase in venous blood oxygenation and therefore increase the MRI signal(Kim & Ogawa, 2012).
Therefore the amplitude of the BOLD effect following CO2 administration is a function of the
magnitude of CBF increase. In addition, the increase in CBF is mirrored by an increase in cerebral
blood volume (CBV) due to the dilation of the vessels, which increases CBV in the arterial and
venous circulation (Zhou, Rodgers, & Kuo, 2015). An increase in CBV increases the absolute
number of dHb within the voxel, which decreases T2*. Therefore the BOLD effect following the
administration of CO2 is mainly due to flow driven-change in dHb proportions, as well as an
increase in CBV. An example image of how the BOLD signal changes with CO2 is seen below in
Figure 1.17
Figure 1-17 Relationship Between Blood-Oxygen Level-Dependent Signal and Carbon
Dioxide. Obtained with permission from Dr. Kassner's Lab.
Figure 1-16 Blood-Oxygen Level-Dependent Image
obtained with permission from Dr. Kassner's Lab.
58
2 Study 1: The impact of anemia on cerebrovascular reserve and cerebral blood flow in children with sickle cell disease: a quantitative MRI Study
2.1 Introduction
Sickle cell disease is a hemoglobinopathy characterized by chronic hemolytic anemia,
painful vaso-occlusive crises, endothelial dysfunction, and chronic organ damage. SCD children
with abnormally elevated transcranial Doppler ultrasound (TCD) velocities in the large intracranial
arteries have an associated 40% risk of overt stroke within 3 years (Weiner et al., 1997). Elevated
TCD velocities are due to two synergistic mechanisms; large-vessel vasculopathy and increased
cerebral blood flow (CBF) due to anemia (Doepp et al., 2012). However, approximately 1 in 3
children who suffer an overt stroke do not present with visible vasculopathy, thereby placing
emphasis on the role of anemia (Helton et al., 2014).
Anemia is defined as the reduction of red blood cell mass, which accounts for 98% of total
available oxygen content whereas the remaining 2% of available oxygen is dissolved in blood
plasma (Finch & Lenfant, 1972). Red cell mass is tightly associated with hematocrit (Hct), which
is the proportion of red blood cells in a centrifuged volume of whole blood and is approximately
0.36-0.50 in healthy adults (Lewis et al., 2001). SCD patients are anemic due to chronic hemolysis
of red blood cells, which ranges in severity of approximately 0.15-0.30 Hct (Steinberg, 1999). To
compensate for a state of chronically reduced oxygen content, CBF is increased through cerebral
vessel dilation in order to increase the rate of oxygen delivery (I. Prohovnik et al., 1989). The goal
of this compensatory mechanism is to attempt to maintain a sufficient oxygen supply to the highly
metabolically active cerebral tissue in an anemic environment. However, there is limit to which a
vessel can dilate, which is collectively referred to as cerebrovascular reserve. Insufficient
59
cerebrovascular reserve has been shown to increase the risk for stroke in patients with carotid
artery disease (Gupta et al., 2012). In addition to anemia, children with SCD have endothelial
dysfunction resulting from endothelial injury from ongoing ischemia-reperfusion injury and
impaired NO bioavailability secondary to hemolysis, which may also affect the total
cerebrovascular reserve. Therefore, investigating cerebrovascular reserve in SCD children may
provide information on the effectiveness of their compensatory capacities.
In-vivo assessment of cerebrovascular reserve can be obtained by measuring cerebral
vascular reactivity (CVR), which reflects the cerebral vasculature’s ability to accommodate an
increased demand for CBF (Goode, Krishan, Alexakis, Mahajan, & Auer, 2009). CVR can be
determined by inducing arterial vasodilation with a vasoactive stimulus, such as CO2 gas, and
measuring the resulting change in CBF using various imaging modalities such as; TCD, 133-
Xenon enhanced computer tomography (Xe-CT), near-infrared spectroscopy, or magnetic
resonance imaging (MRI). CVR is a dynamic measure of vessel responsiveness and acts as a
marker for endothelial dysfunction and reserve and therefore may able to better describe the
hemodynamic environment in SCD children than CBF measures alone (Manwani & Frenette,
2013; Nur et al., 2009a; Wood et al., 2008). MRI is a particularly advantageous imaging modality
for measuring CVR in the pediatric population as it does not require the use of ionizing radiation
or the injection of radio-labeled tracers. Furthermore, MRI benefits from high spatial-temporal
resolution and the ability to obtain both functional and anatomical data thus allowing for the
acquisition of several parameters within a single scan session.
60
Previous imaging studies using TCD and Xe-129 computer tomography have shown that
cerebrovascular reserve, as indicated by CVR, is impaired in adults with SCD when compared to
healthy controls (Nur et al., 2009a; Isak Prohovnik, Hurlet-Jensen, Adams, De Vivo, & Pavlakis,
2009). Neither study was able to demonstrate a relationship between CVR and the degree of
anemia, however the previous studies assessed CVR and CBF in adults with SCD as opposed to
children. This is important as it has been well documented that adults and children have different
cerebral hemodynamic environment as children exhibit higher metabolic demands and CBF, lower
blood pressure, and higher cardiac output than adults (Biagi et al., 2007; Chiron et al., 1992;
Takahashi et al., 1999). In addition, the risk for ischemic stroke is highest in children whereas
adults face a greater risk for hemorrhagic stroke (Ohene-frempong et al., 1998). Therefore, the
differences in hemodynamic environments and stroke risks warrants investigation into CVR and
CBF in children with SCD and to determine how the severity of anemia affects these measures.
2.2 Purpose and Hypothesis
The aim of this study was to determine if cerebrovascular reserve is impaired in SCD
children compared to healthy pediatric controls and if this impairment is associated with the
severity of anemia, as indicated by Hct, and CBF in non-stenotic children with SCD using
quantitative MRI. We hypothesized that CVR is decreased and CBF is increased in children with
SCD compared to healthy pediatric controls. Furthermore, we hypothesized that CVR decreases
and CBF increases with increasing severity of anemia in children with SCD. In addition, we
hypothesized that CVR decreases with increasing CBF in both SCD children and healthy pediatric
controls.
61
2.3 Materials & Methods
2.3.1 Subject recruitment
Twenty seven pediatric patients with documented sickle cell disease (HbSS = 22, HbS βO/+-
thalassemia = 4, HbSC = 1) between the ages of 9 and 18 were recruited from the hematology
clinic at our institution between 2011 and 2014 for our study. Exclusion criteria included; a history
of overt stroke, chronic transfusion program or simple transfusion within the last 6 months,
abnormal TCD velocity, hydroxyurea treatment, hospitalization due to vaso-occlusive crises
within 3 months prior to the scan, and visible major stenosis on magnetic resonance angiography
scans. In addition, 22 healthy controls were also recruited as controls from members of the
community, which included family members and student volunteers. The study was approved by
the Research and Ethics Board at our institution (REB: 1000013967). Patients and volunteers were
asked to abstain from consuming caffeinated and alcoholic beverages within 48 hours of the MR
scan. Informed written consent was obtained from each patient and volunteer or parent/guardian
prior to the study.
2.3.2 CO2 Challenge
A computer-controlled CO2 stimulus was used to induce global-vasodilation of cerebral vessels.
The CO2 stimulus was administered using a model-driven prospective end-tidal (MPET) system
(RespirActTM; Thornhill Research Inc.; Toronto, Canada), which regulates the flow and
composition of CO2, O2, and N2 gases based on each subject’s individual physiological parameters
(age, gender, height, and weight) and delivers the gas mixture via a re-breathing mask and circuit.
The delivery system of the gas mixture allows for fast and accurate simultaneous targeting of end-
62
tidal CO2 and O2 partial pressures, (PETCO2 and PET O2, respectively). Additional information on
the MPET system is provided by Slessarev et al (Slessarev et al., 2007). For the CO2 breathing
challenge, we utilized a block design consisting of 60 second periods of normocapnia (PETCO2 =
40 mmHg) alternating with a 45 second iso-oxic step increase to targeted hypercapnia (PETCO2 =
45 mmHg). The PETO2 was maintained at 100 mmHg. The total duration of the breathing challenge
was 8 minutes.
2.3.3 Magnetic Resonance Imaging
Imaging data were obtained on a clinical 3T MRI (MAGNETOM Tim Trio; Siemens Medical
Solutions; Erlangen, Germany) equipped with a 32-channel head coil. The CVR protocol consisted
of an 8-minute blood-oxygen-level dependent (BOLD) acquisition using a single-shot T2*-
weighted echo-planar (EP) imaging sequence (TE/TR=30/2000 ms, FOV = 220, voxel
size=3.4x3.4x4.5mm, FA = 70o) that ran in parallel with the above CO2 breathing challenge. High
resolution 3D T1-weighted anatomical images (MPRAGE TE/TR = 2.96/2300ms, FOV = 256,
voxel size = 1.0 X 1.0 X 1.0mm, FA = 9o, PAT = 2) were collected under free-breathing for co-
registration and segmentation purposes. Arterial spin labeling (ASL) data were obtained using a
EP-2D Pulsed Arterial Spin Labeling (PASL) images with PICORE Q2T TE/TR=13/2500ms,
FOV=220, voxel size = 3.4 X 3.4 X 4.5, FA=90o. 3D Time-of-Flight (TOF) Magnetic Resonance
Angiography (MRA) images were obtained using TE/TR = 3.59/20ms, FOV = 200mm, voxel size
= 0.5 X 0.5 X 0.5mm to assess possible stenosis within intracranial vessels. Axial T2-weighted
fluid-attenuated inversion-recovery (FLAIR) images were obtained with TE/TR= 85/9000ms,
FOV=220mm, voxel size= 0.8x0.7x4.5mm, FA=120o to assess silent infarcts and other white
matter hyper-intensities.
63
2.3.4 MRI Review – Anatomical
Patient and volunteer T1-weighted anatomical, FLAIR, and MRA images were assessed by
an experienced neuroradiologist (M.S.) blinded to the nature of the study in order to screen for the
presence for white matter abnormalities on FLAIR and possible stenosis on MRA.
2.3.5 Data processing (CVR and CBF)
Patient and healthy volunteer MRI data were transferred to an independent workstation for
post-processing and further analysis. The dynamic BOLD measures were first corrected for
motion, spatially smoothed to reduce noise, and temporally filtered to remove low frequency
artifacts. The PETCO2 waveform then had to be temporally aligned to its respective corrected
BOLD signal. A linear regression of each voxel’s corrected BOLD signal was performed against
the aligned PETCO2 waveform (General Linear Model), which yielded the raw CVR map. The raw
CVR data were then normalized to the temporal mean BOLD signal map to represent CVR in
terms of % ΔMR signal / mmHg (CO2). The CVR map was then co-registered to the high resolution
T1-weighted anatomical images. Grey matter (GM) and white matter (WM) masks were generated
from the T1-weighted images by first using a brain extraction algorithm (FSL-BET) to remove
non-brain areas, followed by automated tissue segmentation (FSL-FAST). The masks were used
in order to retrieve CVR measurements in the gray and white matter. White matter lesions masks
were created on FLAIR images, which were then co-registered and applies to the CVR map. CBF
was quantified from the mean signal difference between ASL tag and control images using a one
compartment kinetic model as described by Buxton, et al. (Buxton, 1998).
64
2.3.6 Hematological and Clinical Measures
Patient Hct values were recorded from complete blood count (CBC) reports, obtained from
scheduled phlebotomy at the sickle cell clinic. CBCs were obtained within 30 days of each patient's
MRI scan. In addition, we obtained standard clinical measures of BMI and age from the online
clinical reports. For the healthy volunteers, a Hct of 0.45 was assumed for males and 0.41 was
assumed for females based on literature values (Dacie and Lewis, 2001).
2.3.7 Statistical analysis
Statistical analysis were performed using SPSSv22. A two-tailed Student's t-test was used to test
whether the mean CVR in SCD children was significantly different than in healthy controls.
Pearson Correlation Coefficients were calculated for associations between CVR and CBF in both
the gray and white matter of patients and healthy volunteers. Pearson Correlation Coefficients were
also calculated for associations between CVR and Hct, CBF and Hct, in the grey and white matter
of SCD children. All statistical analyses used p<0.05 for the statistical significance threshold.
65
2.4 Results
Imaging data were obtained from 27 SCD patients. One patient had bilateral narrowing of
the MCA and distal basal artery and was therefore excluded from analysis. The remaining imaging
data from 26 SCD patients underwent the full analysis listed in the protocol. 26 SCD children (7
male/ 19 female) were scanned with an average age of 14.2 ± 2.43 years and an average Hct of
0.26 ± 0.0427. 22 healthy controls (12 male/ 10 female) were also scanned with an average age of
14.7 ± 2.82 years and a Hct of 0.45 and 0.41 for males and females respectively. Review of healthy
control data was unremarkable. See Table 6.1, for a complete listing of patient and healthy control
demographics.
Figure 2-1. Patient Demographics and Hematological Parameters
Parameter SCD (±SD) Controls (±SD) P
Sample Size (N) 26 22
Gender (M/F) 7/19 12/10 NS
Age (years) 14.2 ± 2.43 14.9 ± 2.76 NS
BMI (Kg/m2) 18.5 ± 2.16 20.6 ± 3.68 p < 0.05
Hematocrit 0.264 ± 0.042 0.36-0.50 (Literature) -
CVR Gray Matter (%ΔBOLD/mmHg)
0.141 ± 0.049 0.256 ± 0.056 p < 0.001
CVR White Matter (%ΔBOLD/mmHg)
0.092 ± 0.030 0.150 ± 0.035 p < 0.001
CBF Gray Matter (ml/100g/min)
65.5 ± 12.0 36.5 ± 8.78 p < 0.001
CBF White Matter (ml/100g/min)
44.2 ± 9.19 25.2 ± 7.09 p < 0.001
66
All SCD patients and healthy controls successfully completed the breathing challenge in
the MRI. The average normocapnic and hypercapnic PETCO2 for SCD patients was 39.8 ± 1.21
and 43.5 ± 1.49, respectively. In the healthy controls, the average normpcanic and hypercapnic
PETCO2 was 39.8 ± 1.34mmHg and 43.8 ± 1.41 mmHg, respectively. Normocapnic and
Hypercapnic PETO2 was 104 ± 4.22 mmHg and 105 ± 3.17 mmHg for SCD patients. Normocapnic
and Hypercapnic PETO2 was 105 ± 3.76 mmHg and 106 ± 4.33 mmHg for healthy controls. See
Table 6.2 for the results of the CO2 challenge.
Both SCD patients and healthy control data analysis resulted in high quality CVR, CBF,
and anatomical images. Representative CVR and CBF maps in a SCD patient and healthy control
is shown in Figure 6.1. Review of SCD patient's FLAIR images by an experienced neuroradiologist
(M.S.) demonstrated that 10/26 (38%) patients had at least one silent infarct. Review of MRA
images by an experienced neuroradiologist showed that 5/26 (19.2%) patients had evidence of
mild tortuosity.
Figure 2-2 CO2 Challenge Results
Parameter SCD (±SD) Controls (±SD) P
PETCO2(mmHg), normocapnic 39.8 (1.21) 39.8 (1.34) NS
PETCO2(mmHg), hypercapnic 43.5 (1.49) 43.8 (1.41) NS
PETO2(mmHg) , normocapnic 104 (4.22) 105 (3.76) NS
PETO2(mmHg), hypercapnic 105 (3.17) 106 (4.33) NS
67
CVR of SCD children was significantly decreased compared to CVR in healthy controls in
both the gray matter (GM = 0.141 ± 0.049 SD vs. 0.256 ± 0.056 SD %ΔBOLD/mmHg CO2,
p<0.001) and white matter (WM= 0.092 ± 0.006 SD vs. 0.150 ± 0.035 SD %ΔBOLD/mmHg CO2,
p<0.001) as seen in Figure 6.2 (A,B). CBF of SCD children was significantly increased when
compared to the CBF of healthy controls in both the gray matter (GM 65.5 ± 12.0 SD vs. 36.5 ±
8.78 SD mL/100g/min, p<0.001) and white matter (WM = 44.2 ± 9.19 vs. 25.2 ± 7.09 SD
mL/100g/min, p<0.001) as seen in Figure 6.3 (A,B).
Figure 2-3. Representative Cerebrovascular Reactivity (CVR)(A) and Cerebral Blood Flow (CBF)(B)
maps in both SCD patients (Bottom) and Healthy Controls (Top).
68
Figure 2-4 Cerebrovascular reactivity (CVR) in patients with sickle cell disease
(SCD) and healthy controls. CVR, expressed as a relative change in blood-oxygen
level-dependent magnetic resonance signal per millimeter of mercury change in end-
tidal partial pressure of carbon dioxide was significantly decreased in children with
SCD (red, n=26) when compared to healthy controls (green, n=22) in the gray matter
(A) and white matter (B). Bars represent mean ± standard error of the mean.
0
0.05
0.1
0.15
0.2
0.25
0.3
GM
CV
R (
%Δ
MR
/mm
Hg) Sickle Cell Disease
Healthy
p < 0.001
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
WM
CV
R (%
ΔM
R/m
mH
g)
Sickle Cell Disease
Healthy
p < 0.001
A
B
69
Figure 2-5. Cerebral blood flow in patients with sickle cell disease (SCD) and healthy
controls. Cerebral perfusion, expressed as the volume of blood signal in milliliters per minute
in 100g of tissue, was significantly increased in children with SCD (red, n=26) when
compared to healthy controls (green, n=22) in the gray matter (A) and white matter (B). Bars
represent mean ± standard error of the mean.
A
B
70
Correlation analysis revealed a statistically significant relationship between CVR and Hct
in the gray matter (r= 0.81, p<0.001) and white matter (r= 0.74, p<0.001) of children with SCD as
seen in Figure 6.4 (A,B). In addition, CBF and Hct and was significantly correlated in the gray
matter (r=-0.56, p<0.05) and white matter (r= -0.46, p<0.05) in children with SCD as seen in Figure
6.5 (A,B). Furthermore, CVR was significantly associated with CBF in the gray matter (r=-0.72,
p<0.001) and in the white matter (r=-0.64, p < 0.001) in both SCD children and healthy controls
as seen in Figure 6.6 (A,B). In patients with SCD, CVR was significantly associated with CBF in
the gray matter (r=-0.5, p<0.05) but not in the white matter (r=-0.35, p = 0.08). Furthermore, in
healthy controls, CVR and CBF were not significantly associated in neither the gray matter (r=-
0.24, p>0.05) or white matter (r=-0.1, p>0.05).
71
Figure 2-6. Relationship between cerebrovascular reactivity (CVR) and the degree of anemia
(Hct) in patients with sickle cell disease (SCD). Cerebrovascular reactivity expressed as a
relative change in blood-oxygen level-dependent magnetic resonance signal per millimeter of
mercury change in end-tidal partial pressure of carbon dioxide (PETCO2) was significantly
associated with the degree of anemia in children with SCD (n=26) in the gray matter (A) and
white matter (B).
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.15 0.17 0.19 0.21 0.23 0.25 0.27 0.29 0.31 0.33 0.35
WM
CV
R (%
ΔM
R/m
mH
g)
Hematocrit
r = 0.74p < 0.001
A
B
72
Figure 2-7. Relationship between cerebral blood flow and the degree of anemia (Hct) in
patients with sickle cell disease (SCD). Cerebral blood flow, expressed as the volume of blood
signal in milliliters per minute in 100g of tissue, was significantly associated with Hct in
children with SCD (n=26) in the gray matter (A) and white matter (B).
A
B
73
Figure 2-8. Relationship between cerebrovascular reactivity (CVR) and cerebral blood flow in
patients with sickle cell disease (SCD) and healthy controls. CVR expressed as a relative change
in blood-oxygen level-dependent magnetic resonance signal per millimeter of mercury change in
end-tidal partial pressure of carbon dioxide was significantly associated with cerebral blood flow,
expressed as the volume of blood signal in milliliters per minute in 100g of tissue in children with
SCD (n=26) and healthy controls (n=22) in the gray matter (A) and white matter (B).
A
B
74
2.5 Discussion
Our study has demonstrated that cerebrovascular reserve, as assessed by MR-based CVR,
is significantly reduced in children with SCD children compared to age-matched controls in both
gray and white matter, reflecting an impaired ability of cerebral vessels to accommodate to an
increased demand for CBF. In addition, the data shows that cerebrovascular reserve and CBF are
associated with the severity of anemia. This finding suggests that the cerebral dilatory vessels in
severely anemic SCD children are less responsive to a dilatory stimulus compared to SCD patients
with a lower degree of anemia. The decreased vasodilatory response to a dilatory stimulus in
severely anemic children implies that the cerebral vessels are approaching their cerebrovascular
reserve and/or have reduced dilating capabilities due to endothelial dysfunction.
Although CVR has been shown to be reduced in the adult SCD population, CVR has not
been formally studied in the pediatric SCD population. Our results demonstrate that CVR in
children with SCD in the GM (0.141 %ΔBOLD/mmHg CO2 +0.049D) and WM (0.092
%ΔBOLD/mmHg CO2 + 0.006SD) is significantly reduced compared to healthy pediatric controls
in the gray matter (0.256 %ΔBOLD/mmHg CO2 ± 0.056 SD) and white matter (0.150
%ΔBOLD/mmHg CO2 ± 0.035 SD). This suggests that relative to healthy controls, SCD children
have a decreased capacity for further vasodilation. This is important as vasodilation is crucial in
maintaining sufficient CBF in the face of decreasing perfusion pressure as well as acute anemic or
hypoxemic events (Tsivgoulis & Alexandrov, 2008). Reduced CVR may aid in explaining why
the cerebral parenchyma in SCD is at a constant threat of ischemia (Quinn et al., 2013).
Children with SCD present with chronic anemia due to ongoing hemolysis of sickled
erythrocytes, however the rate at which these cells hemolyze is variable therefore the degree of
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anemia is different for each individual with SCD manifesting in a range of Hct (0.197-0.334 in our
demographic) (Bensinger & Gillette, 1974). In order to attempt to maintain a sufficient supply of
oxygen to neural tissue in a global state of reduced oxygen carrying capacity, the body increases
blood flow by increasing cardiac output and decreasing vascular resistance of downstream
arterioles through vasodilation. However, there is a limit to which a vessel can dilate and when the
vasculature reaches its capacity to vasodilate then it relies on suboptimal mechanisms of increasing
oxygen extraction fraction in order to maintain the cerebral metabolic rate of oxygen (CMRO2)
(Tsivgoulis & Alexandrov, 2008). Ultimately, the body maintains a compromised ability to
circumvent hypoxia during events of acute anemia or hypoxemia such as in acute chest syndrome
(ACS), parvovirus B19 infection, and splenic sequestration, which are associated with ischemic
stroke and silent infarction(Ohene-frempong et al., 1998). Furthermore, CVR has been described
as an independent risk factor for stroke in patients with carotid disease(Gupta et al., 2012; Markus
& Cullinane, 2001). Therefore our data suggests that children with precariously low hematocrit
may be exhausting their ability to respond to an increased demand for CBF, such as during an
acute anemic or hypoxemic event, therefore may be at risk for tissue hypoxia and subsequent
infarction, particularly in the more sensitive watershed regions of the deep white matter. The
importance of baseline anemia to the susceptibility of neurologic injury is supported by several
studies showing that lower hemoglobin is an independent risk factor for both SCI and overt strokes.
In order to effectively assess the effect of anemia on cerebrovascular reserve we obtained
MRA images in order to exclude potential patients with significant stenosis on MRA. Previous
studies have demonstrated that stenosis of the major intracranial cerebral vessels result in
downstream arteriole vasodilation as reflected by regionally reduced CVR values (Markus &
Cullinane, 2001; Silvestrini et al., 2000a). This mechanism by which stenosis affects CVR is
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independent of the degree of anemia therefore would act to confound our results. This study does
not argue against the link between stenosis and stroke in children with SCD, as there is convincing
evidence for its involvement in ischemic stroke. However, imaging studies of the SCD children in
the Stroke Prevention Trial in Sickle Cell Anemia and the Stroke Witch Transfusion Changing to
Hydroxyurea trials provided compelling evidence that stenosis is not present in a significant
proportion of children with abnormal TCDv and with histories of stroke therefore stenosis cannot
be the sole or main contributor to abnormal TCDv and risk of stroke(Abboud et al., 2004; R. J.
Adams, 1998; R E Ware, Zimmerman, & Schultz, 1999). The data in this study suggests that the
risk of neurologic injury in children with SCD is associated with the degree of anemia which
reintroduces the plausible role of anemia and hemodynamic insufficiency with stroke risk in SCD
children.
We have demonstrated that children with SCD have reduced CVR compared to healthy
controls, reflecting a compromised ability to accommodate a demand for increased CBF of
cerebral tissue. Furthermore, we have shown for the first time that the CVR is strongly associated
with the degree of anemia, suggesting precariously anemic children may have exhausted
vasodilatory capacity and therefore may be unable to increase CBF during an acute anemic or
hypoxemic event. This finding places emphasis on the importance of hct levels in a clinical setting
and suggests the link between anemic and neurologic injury may perhaps be hemodynamic in
nature.
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3 Study 2: Transfusion and Hydroxyurea Increases Cerebrovascular Reserve in Children with Sickle Cell Disease: A Quantitative MRI Study
3.1 Introduction
Sickle Cell Anemia (SCD) is a lifelong hemoglobinopathy, which is characterized by
chronic hemolytic anemia, painful vaso-occlusive crises, and progressive organ damage. Overt
ischemic stroke is the most devastating complication in children with sickle cell disease (SCD),
affecting roughly 1 in 10 children before the age of 20 (R. Adams et al., 1992). The
pathophysiology of stroke in children with SCA is still unclear. Currently, stroke is understood to
be an acute complication arising from intracranial vasculopathy, a progressive narrowing of the
major cerebral arteries that reduces perfusion pressure to the downstream tissue to the point of
infarction (Fasano, Meier, & Hulbert, 2014). In the face of decreasing perfusion pressure,
cerebral blood flow (CBF) is maintained by cerebral vasodilation until the cerebrovascular
reserve is exhausted, whereupon the body relies on suboptimal mechanisms to maintain tissue
viability (Powers, 1991). In addition, children with SCD are chronically anemic, which requires
a compensatory increase in CBF, which may further limit to degree to which the cerebral vessels
can dilate (I. Prohovnik et al., 1989; Isak Prohovnik et al., 2009). Previous work has
demonstrated that reduced cerebrovascular reserve is strongly associated with an increased risk
of ischemic events in patients with stenoocclusive carotid disease. (Gupta et al., 2012; Markus &
Cullinane, 2001; Silvestrini et al., 2000b; Szabo, Sheth, Novak, Rozsa, & Ficzere, 1997).
Therefore cerebrovascular reserve may be a useful hemodynamic measure in stroke
susceptibility in SCD and would warrant improvement through therapeutic intervention.
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Currently, prophylactic chronic transfusion regimens (cTx) are the gold standard for
primary and secondary stroke prevention in children with abnormal transcranial Doppler (TCD)
ultrasounds (R. J. Adams, 1998). The target of cTx is to reduce and maintain the hemoglobin S%
(HbS) to below 30%, indefinitely, as the Stroke Prevention Study in Sickle Cell Anemia 2
(STOP2) demonstrated that discontinuation of cTx results in an increased risk of stroke (R. J.
Adams & Brambilla, 2005). The mechanism in which cTx reduces the risk of stroke is not well
understood, however cTx decreases HbS concentration, increasing oxygen carrying capacity,
reducing intravascular hemolysis and increasing deformability is believed to play a role
(Detterich et al., 2013). Although cTx reduces the risk of stroke from 10% to 1%, it is associated
with a host of potentially fatal adverse side effects including; iron overload, allo- and
autoimmunization, contamination of blood products with infective agents, hyperviscosity, and
delayed hyperhaemolysis reactions(Chou, 2013). In addition, cTx has a direct impact on hospital
resources and requires specialized equipment and personnel, which may not be readily accessible
in developing countries where SCD is most prevalent. Therefore there is a need to locate a
relatively safer and less costly alternative to cTx for stroke prevention in children with SCD.
Hydroxyurea (HU) is a myelosuppressive agent used for, amongst other things, to reduce
the number of vaso-occlusive crises, ameliorate disease severity, and is currently being
investigated as an alternative to cTx for primary stroke prevention in the TWITCH trial
(Charache et al., 1995; Platt et al., 1994). The therapeutic effect of HU acts by increasing
expression of fetal hemoglobin (HbF), which does not copolyermerize with sickle hemoglobin
(HbS), thereby reducing the rate of sickling (Charache et al., 1992). Furthermore, HU has been
shown to improve RBC deformability and hydration, decrease the number of circulating
leukocytes and reticulocytes, increase arterial O2 saturations, and reduce the rate of hemolysis
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(Ballas, Marcolina, Dover, & Barton, 1999b; Pashankar, Manwani, Lee, & Green, 2015; Russell
E Ware, 2010). However, it is currently unknown has HU affects cerebrovascular reserve and
CBF in SCD children.
Cerebrovascular reserve can be inferred through measures of cerebrovascular reactivity
(CVR), which is defined as the change in blood flow following the administration of a
vasodilatory stimulus. CVR can be obtained using MRI by administering CO2 gas and measuring
the resulting change in relative blood flow using a blood-oxygen-level-dependent (BOLD) MR
sequence. In addition, MRI has the unique advantage of being non-invasive, without harmful
radiation, while offering high spatial and temporal resolution, which is particularly advantageous
in the pediatric population.
3.2 Purpose and Hypothesis
The purpose of this study was to assess how cTx and HU affects cerebrovascular reserve,
as indicated by CVR, and CBF in children with SCD. By understanding the therapeutic effect of
transfusion on CVR and CBF, it will produce a bench mark of expected results for future studies
that are testing alternative treatments for stroke prevention. We hypothesized that children
treated with cTx or HU will have increased CVR and decreased CBF compared to non-treated
SCD children.
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3.3 Methods
3.3.1 Subject Recruitment
Children with documented sickle cell anemia (HbSS) on HU, cTx, and age-matched
untreated SCA children were recruited for this study from the Department of Haematology at The
Hospital for Sick Children. In addition, healthy volunteers were recruited from the community.
Exclusion criteria for SCA patients on hydroxyurea were; treatment length less than a year in order
to account for escalation of dose and response time. Patients on cTx were scanned up to 14 days
following their scheduled transfusion. General exclusion criteria included; previous stroke,
emergency room visits in the preceding 3 months, acute vaso-occlusive events (crises, acute chest
syndrome, splenic sequestration, or priapism) in the preceding 3 months, hematological evidence
of non-compliance to treatment (HbF, MCV, neutrophil assessment). Hematological parameters
were obtained from complete blood counts during each patient’s scheduled clinic visits. Patients
and healthy controls were asked to abstain from consuming caffeinated and alcoholic beverages
prior to the day of scanning. Written informed consent was obtained from each patient, healthy
control or parent/guardian prior to the study.
3.3.2 MRI
Imaging data were obtained on a clinical 3T MRI (MAGNETOM Tim Trio; Siemens Medical
Solutions; Erlangen, Germany) equipped with a 32-channel head coil. The CVR protocol
comprised of an 8-minute blood-oxygen-level dependent (BOLD) acquisition using a single-shot
T2*-weighted echo-planar (EP) imaging sequence (TE/TR=30/2000 ms, FOV = 220, voxel
size=3.4x3.4x4.5mm, FA = 70o) that ran in parallel with the CO2 breathing challenge outlined
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below. Arterial spin labeling (ASL) data were obtained using a EP-2D Pulsed Arterial Spin
Labeling (PASL) images with PICORE Q2T TE/TR=13/2500ms, FOV=220, voxel size = 3.4 X
3.4 X 4.5, FA=90o. 3D T1-weighted anatomical images (MPRAGE TE/TR = 2.96/2300ms, FOV
= 256, voxel size = 1.0 X 1.0 X 1.0mm, FA = 9o, PAT = 2) were acquired under free-breathing for
co-registration and segmentation purposes. 3D Time-of-Flight (TOF) Magnetic Resonance
Angiography (MRA) images were obtained using TE/TR = 3.59/20ms, FOV = 200mm, voxel size
= 0.5 X 0.5 X 0.5mm to assess stenosis within intracranial vessels. Axial T2-weighted fluid-
attenuated inversion-recovery (FLAIR) images were obtained with TE/TR= 85/9000ms,
FOV=220mm, voxel size= 0.8x0.7x4.5mm, FA=120o to assess silent infarcts and other white
matter hyper-intensities.
3.3.3 CO2 challenge
The CO2 gas was administered using a model-driven prospective end-tidal (MPET) system
(RespirActTM; Thornhill Research Inc.; Toronto, Canada), which regulates the flow and
composition of CO2, O2, and N2 gases based on each subject’s individual physiological parameters
(age, gender, height, and weight) independent of minute ventilation and delivers the gas mixture
via a re-breathing mask and circuit. The delivery of the gas mixture allows for fast and accurate
simultaneous targeting of end-tidal CO2 and O2 partial pressures, (PETCO2 and PET O2,
respectively). Additional information on the MPET system is provided by Sleesarev et al. For the
CO2 breathing challenge, we implemented a block design consisting of 60 second periods of
normocapnia (PETCO2 = 40 mmHg) alternating with a 45 second iso-oxic step increase to targeted
hypercapnia (PETCO2 = 45 mmHg). The PETO2 was maintained at 100 mmHg throughout the entire
breathing challenge. The total duration of the breathing challenge was 8 minutes.
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3.3.4 Data Processing
MRI data were transferred to an independent workstation for post-processing and further analysis.
The dynamic BOLD maps were first corrected for motion, spatially smoothed to reduce noise, and
temporally filtered to remove low frequency artifacts. The mean BOLD signal was then temporally
aligned to its respective PETCO2 waveform. The BOLD signal was regressed against the aligned
PETCO2 waveform on a voxel-wise basis (General Linear Model), which yielded the raw CVR
map. The raw CVR data were then normalized to the temporal mean BOLD signal map to represent
CVR in terms of % ΔMR signal / mmHg (CO2). The CVR map was then co-registered to the high
resolution T1-weighted anatomical images. Grey matter (GM) and white matter (WM) masks were
generated from the T1-weighted images by first using a brain extraction algorithm (FSL-BET) to
remove non-brain areas, followed by automated tissue segmentation (FSL-FAST). The CVR
measurements were then averaged over the GM and WM. CBF was quantified from the mean
signal difference between ASL tag and control images using a one compartment kinetic model.
3.3.5 Statistical analysis
All statistical analysis was performed using SPSS v22. An independent samples, two-tailed,
Student's t-test was used to test for significant differences in CVR, CBF between the HU-treated
SCA group, untreated SCA group, and healthy controls. After correcting for family-wise error rate
due to multiple comparisons using the Bonferonni correction, statistical analyses used p<0.025 for
the statistical significance threshold.
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3.4 Results
Imaging data were obtained from 17 HU-treated and 5 ctX-treated children with SCA, as well as
21 age-matched untreated SCA children. One HU-treated patient was excluded due to excessive
motion. The remaining image data underwent full analysis of the protocol listed in the methods.
16 HU-treated SCA children (10 male /6 female) with an average age of 14 ± 2.57 years, 5 cTx-
treated SCA children (2 male/ 3 female), and 21 SCA children not on treatment (5 male / 16
female) were scanned The hematological parameters are outlined in Figure 3.1. Hct (as seen in
Figure7.1), Hb, and MCV were all significantly higher in the HU-treated group compared to
untreated SCA children as can be seen in Figure 3.1. The absolute reticulocyte count was lower in
the HU-treated group, however this did not reach significance (p=0.12). The Hct was significantly
increased in the cTx group and HU group compared to the no treatment group as can be seen in
Figure 3.2.
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Figure 3-1 Patient Demographics
Parameter No Treatment SCA Hydroxyurea SCA Transfusion SCA
Sample Size (N) 21 16 5
Gender (M/F) 5/16 10/6 2/3
Age (years) 14.1 ± 2.42 14.0 ± 2.57 12.8 ± 2.16
Hematocrit 0.253 ± 0.037 0.292 ± 0.035* 0.328 ± 0.013γ
Hemoglobin F% - 15.8 ± 8.07 -
Absolute Reticulocyte (K/uL) 270 ± 140 212 ± 79 -
Mean Corpuscular Volume (fL)
80.3 ± 10.8 97.0 ± 10.6* -
SaO2 0.97 ± 0.03 0.99 ± 0.01 -
Neutrophil (KuL) 6.1 ± 3.1 3.9 ± 1.6* -
• * Denotes p<0.025 between no treatment and HU SCA
• γ Denotes p<0.025 between no treatment and Transfusion SCA
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Apart from the single HU-treated patient, the data analysis resulted in high quality CVR,
CBF, and anatomical images. The HU-treated group demonstrated significantly increased CVR
compared to the CVR of healthy controls in the gray matter (0.171 ± 0.009 vs. 0.132 ± 0.010
%ΔMR/mmHg PETCO2; p=0.008) but not in the white matter (0.107 ± 0.006 vs. 0.087 ± 0.007
%ΔMR/mmHg PETCO2; p=0.034). The cTx-treated group demonstrated significantly increased
CVR compared to the CVR of healthy controls in the gray matter (0.168 ± 0.009 vs. 0.132 ± 0.010
%ΔMR/mmHg PETCO2; p=0.015) but not in the white matter (0.092 ± 0.006 vs. 0.087 ± 0.007
%ΔMR/mmHg PETCO2; p=0.638). As seen in Figure 3.3.
Figure 3-2. Hematocrit is significantly increased in Hydroxyurea-treated and Transfusion-treated
children with sickle cell anemia compared to no treatment children with SCA. Error bars depict
standard error of the mean.
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Figure 3-3 Cerebrovascular Reactivity is significantly increased in the gray matter (A) but not
the white matter (B) of hydroxyurea-treated and transfusion-treated children with sickle cell
anemia compared to non treated children with sickle cell anemia. Error bars denote standard error
of the mean.
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There was no significant difference in CBF between the HU-treated group and no treatment
group in the gray matter (70.9 ± 2.89 vs. 68.1 ± 2.42 ml/100g/min; p=0.472) or in the white matter
(43.2 ± 1.43 vs. 44.6 ± 1.52 ml/100g/min; p=0.500). The cTx-treated group demonstrated
significantly decreased CBF compared to the no treatment group in the gray matter (54.3 ± 1.25
vs. 68.1 ± 2.42 ml/100g/min; p<0.001) and in the white matter (33.3 ± 0.618 vs. 44.6 ± 1.52
ml/100g/min; p<0.001). As seen in Figure 3.4.
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Figure 3-4 Cerebral blood flow (CBF) was signficantly decreased in the gray matter (A) and
white matter (B) of transfusion-treated children with sickle cell anemia (SCA) compared to non
treated SCA children. HU-treated SCA children showed no signficantly different CBF changes
in either gray matter or white matter compared to non treated SCA children. Error bars
represent standard error of the mean.
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3.5 Discussion
This study is the first to demonstrate the effect of HU and cTx on MR-based measures of
cerebrovascular reserve and CBF in children with sickle cell anemia. The foremost finding is that
SCA children treated with HU or cTx had significantly increased cerebrovascular reserve as
indicated by CVR, suggesting that the vasculature can better accommodate an increased demand
for CBF compared to the no-treatment SCA group. In addition, ASL measures of CBF were
significantly decreased in cTx compared to the no treatment SCA group whereas CBF in the HU-
treated group was not significantly different than the SCA group. Overall, these findings suggest
that the cerebrovasculature in HU treated or cTx treated children with SCA can better cope with
events requiring changes in vessel tone such as large-vessel vasculopathy or acute reductions in
oxygen carrying capacity such as during splenic sequestration (SS), acute chest syndrome (ACS)
and parvovirus B19 infection.
Hemolysis of red blood cells causes anemia, endothelial injury, and reduced nitric oxide
bioavailability, which can call affect the degree to which cerebral vessels can dilate. With respect
to anemia, the severity ranges from 015-0.30 Hct (Steinberg, 1999). The body responds to the
individual severity of anemic through vasodilation of arterioles. In addition, due to the nature of
the disease, children with SCA are at risk for acute drops in oxygen carrying capacity (hematocrit).
These acute anemic events include SS, ACS, and parvovirus B19 Infection and are independent
risk factors for silent infarcts (Bernaudin et al., 2011; Debaun et al., 2012; Kwiatkowski et al.,
2010) and are temporally linked to a risk of overt stroke (Ohene-frempong et al., 1998; Wierenga,
Serjeant, & Serjeant, 2001). Several authors have proposed a non-vasculopathic mechanism of
stroke whereby the continuous drop in oxygen carrying capacity is first met with vasodilation
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similar to the response to decreases in perfusion pressure, however, instead of being held constant,
CBF is increased in order to maintain oxygen supply (DeBaun et al., 2012; Hulbert & Ford, 2015;
Isak Prohovnik et al., 2009). In stage two, oxygen delivery is maintained by increasing oxygen
extraction fraction (OEF). After OEF is exhausted, the body cannot maintain sufficient oxygen
delivery to meet the cerebral metabolic rate of oxygen (CMRO2), thereby resulting in tissue
hypoxia and infarction. Furthermore, patients with SCD have impairments in CVR (Nur et al.,
2009a; Isak Prohovnik et al., 2009) therefore placing them in a precarious situation during these
acute anemic events. However, not all children with SCA may suffer from tissue hypoxia
following such events, either these children have sufficient hemodynamic capacity to circumvent
hypoxia or the AEE can also range in severity whereby the less severe AEE may be insufficient in
stressing the hemodynamic system to cause hypoxia. Our results show that children on HU and
cTx can better respond to vasodilatory stimuli therefore perhaps affording these children with
greater hemodynamic capabilities to deal with such events.
Endothelial injury is a complex result of chronic occlusion and reperfusion, as well as
oxidative stress from iron in heam, which is released from hemolysis. In addition, hemolysis also
releases arginase and hemoglobin into the circulation, which can impact NO bioavailability (Reiter
et al., 2002). NO is one of the most potent vasodilator of the cerebral vasculature (Morris et al.,
2000). NO is produced by nitric oxide synthases from L-arginine by one of three isoforms of nitric
oxide synthases (NOSs). Therefore, by releasing arginase into the circulature, hemolysis reduces
the bioavailability of NO (Morris et al., 2005). The release of soluble hemoglobin results in the
reaction of heme-iron with NO producing nitrate, an inert molecule at speeds nearing the rate of
diffusion thus further reducing NO bioavailability (Reiter et al., 2002). This reduction in NO
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bioavailability may impair the vasculature's ability to respond to a dilatory stimuli thus resulting
in a low CVR.
HU increases hemoglobin F, which does not copolyermize with hemoglobin S thereby
reducing the rate of hemolysis(Charache et al., 1992, 1995). By reducing hemolysis, HU is
clinically proven to increase Hct and hypothesized to improve NO bioavailability for reasons
outlined above. In our study we saw that the HU cohort has significantly greater oxygen carrying
capacity (Hct), in addition to reduced reticulocyte count and increased mean corpuscular volume,
which are all clinical effects of HU. Our results demonstrate that HU-treated children with SCA
show greater increases in CBF following a standard administration of CO2 when compared to SCA
children not on treatment. This can be due to main two processes; (I) the chronically higher levels
of hct in the HU group affords a lower degree of baseline vasodilation required to maintain oxygen
delivery therefore the HU group may have greater remaining vasodilatory reserve than the more
anemic untreated SCA group. (II) the reduction in hemolysis improved NO bioavailability for the
reasons outlined above, which afforded the HU group with improved vasodilatory abilities.
Furthermore, HU contains an NO moiety and has been shown to release NO through currently
unknown pathways, which may further help restore the dilatory ability of the vasculature (Gladwin
et al., 2002; Russell E. Ware, 2010). Or, it may be the case that both mechanisms are working in
concert, in any case, improvements in vasodilatory capabilities affords children with HU group
with greater hemodynamic capacity to deal with drops in perfusion pressure or oxygen carrying
capacity.
The goal of cTx therapy is to dilate the circulating sickled cells and to improve oxygenation
in order to prevent sickling (Serjeant, 1992). Children on cTx have their hemoglobin S fraction
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maintained less than 30%, therefore the majority of the blood in cTx treated SCA children is
comprised of healthy red blood cells that do not have the propensity to sickle. By introducing
healthy red blood cells into the circulation, not only does the severity of anemia decrease, but so
does the endothelial injury from the aforementioned downstream effects of intravascular
hemolysis. This may explain why SCA children treated with cTx have a greater cerebrovascular
response to CO2 compared to non-treated SCA children. In addition, SCA children on cTx
comprise the most severe clinical phenotype of the disease whereas the no-treatment SCA group
comprises more of the clinical norm, therefore the fact that both CVR and CBF were improved on
cTx therapy speaks to the potency of the treatment.
Prohovnik et al. showed that SCD patients with greater oxygen carrying capacity (Hct)
have lower blood flow than their more anemic counterparts using Xenon-CT (I. Prohovnik et al.,
1989). Furthermore, Zimmerman demonstrated that HU was able to decrease cerebral blood flow
velocities (CBFv) in the major cerebral arteries using Transcranial Doppler (TCD) ultrasound
(Zimmerman, Schultz, Burgett, Mortier, & Ware, 2007). Therefore, we hypothesized that CBF
would be lower in the less anemic HU group than in the controls, however, no significant CBF
differences were observed. A reason we did not find differences in CBF between groups whereas
Zimmerman et al. found reductions in CBFv may be due to the measure itself, where pASL
measures blood flow per 100g of tissue (perfusion) whereas TCD measures bulk velocity at the
major feeding vessels far removed from the tissue. Fluid dynamic principles state that the flow of
a uniformly moving fluid through a rigid vessel is equal to the product of the fluid’s velocity and
the cross sectional area of the tube. Although Zimmerman found that velocity decreases with HU
treatment, a previous study using MRA images demonstrated that SCD patients treated with either
transfusion or HU had an 8% increase in the diameter in 42 intracranial vessels compared to
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untreated SCD patients. Therefore, the increase in vessel diameter following HU treatment may
serve to balance the decreased velocity expected from HU treatment thereby resulting in a
relatively equal CBF. Alternatively, Zimmerman et al. found that the decrease in CBFv following
HU treatment was associated with maximal baseline CBFv meaning that the children with higher
CBFv prior to treatment tended to show the greatest reduction. The average CBFv prior to HU
treatment was 173 ± 22 cm/s, which means that more than half of their patients were in the
conditional/abnormal range, whereas all our patients had normal TCDv therefore the expected
change in blood flow may be blunted because of their lowered baseline velocities. However, there
are currently no studies that looked at the long-term effect of HU in children with SCD. A
limitation of our study is the sample size where we have 16 HU treated patients and 21 non HU-
treated patients, which is not powered to detect the difference in CBF. Prohovnik et al.
demonstrated that CBF is negatively associated with Hct in patients with SCD. In our study, the
HU group has significantly greater Hct (0.292 ± 0.035) compared to the untreated group (0.253 ±
0.037) owing to HU's ability to reduce hemolysis and increase Hct therefore the HU group would
be expected to have lower blood flow requirements than the untreated group. However, HU also
introduces fetal hemoglobin into the red blood cell which has a higher oxygen affinity than both
adult or sickle hemoglobin, therefore the oxyhaemoglobin dissociation curve may be left-shifted
in patients on HU, which has been corroborated by HU's ability to increase oxyhaemoglobin
saturation (Narang et al., n.d.; Pashankar et al., 2015). Fetal hemoglobin's greater affinity for
oxygen may result in a reduced oxygen extraction fraction, which is met by an increase in blood
flow in order to maintain the cerebral metabolic rate of oxygen CMRO2. Therefore we have two
opposing forces on cerebral blood flow at the tissue level, where the higher hematocrit would
reduce CBF but the greater O2 affinity would lead to an increase in CBF, therefore blunting the
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net change of CBF of patients on HU treatment. Future studies looking at measures such as tissue
CBF, CBFv, etc. would greatly benefit from obtaining oxygenation measures and CMRO2
measures in order to determine what compensatory pathway the body is utilizing.
In summary, we have shown for the first time that SCA children treated with either HU or
cTx have a normalized ability to deal with demands for blood flow than no treatment SCA children.
The improvement in vasodilatory capabilities affords the HU and cTx group with greater abilities
to deal with decreases in perfusion pressure or decreases in oxygen carrying capacity.
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4 Overall Discussion and Future Directions
4.1 Overall Discussion
In this thesis, cerebrovascular dilatory reserve was assessed in children with SCD using
BOLD MRI in combination with a CO2 breathing challenge. We assessed how the severity of
anemia affects cerebrovascular dilatory reserve and if hydroxyurea or chronic transfusion can be
used to improve it. The experimental work in this thesis demonstrates that cerebrovascular reserve,
as indicated by CVR, is decreased in children with SCD compared to healthy pediatric controls.
Furthermore, we have shown that CVR is associated with Hct in children with SCD, which
suggests that SCD children with greater severity of anemia utilize more dilatory reserve than less
anemic SCD children. The second experimental study demonstrated that SCD patients on HU or
cTx therapy have improved cerebrovascular reserve compared to SCD children not treated with
HU or cTx. The HU and cTx treated SCD children had significantly increased Hct relative to
untreated SCD children, which according to the results in the first study may be one of the reasons
why treated children have greater cerebrovascular reserve. In addition, experimental work from
our lab has shown that compared to healthy adults, healthy children have lower cerebrovascular
and higher CBF that is thought to provide sufficient oxygen and nutrients to fuel the increased
demands of brain development (Leung, 2015). Considering that SCD children face complications
that further reduce their Hct and SaO2 then it is suggestive that the children with greater baseline
anemia may perhaps lack the necessary reserve to circumvent failure of tissue oxygenation. Failure
of tissue oxygenation is defined as hypoxia and when hypoxia is a result of anemia, it is referred
to as anemic hypoxia (Samuel & Franklin, 2008). Therefore, patients treated with HU and cTx
should be have a lower risk of neurologic injury from failure of tissue oxygenation due to a greater
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capacity to deal with reductions in Hct or SaO2 such as during splenic sequestration, acute chest
syndrome, or parvovirus B19 infection, which are associated with both overt stroke and SCIs.
Corroborating this assertion is the evidence that cTx reduces the risk of overt and secondary stroke,
as well as the incidence of silent infarcts(R. J. Adams, 1998; DeBaun et al., 2014; Hulbert,
McKinstry, Lacey, Moran, Panepinto, Thompson, Sarnaik, Woods, Casella, Inusa, Howard,
Kirkham, Anie, Mullin, Ichord, Noetzel, Yan, Rodeghier, & DeBaun, 2011; Scothorn et al., 2002)
HU is currently being tested as an alternative to cTx for primary stroke prevention in the TCD with
Transfusions Changing to Hydroxyurea (TWiTCH) Phase III, multicenter trial. Subsequently, the
next hurdle is to assess whether HU can reduce the incidence of SCIs.
The link between severe anemia and neurologic injury in SCD children is becoming a well
supported observation. A prospective study demonstrated that approximately 20% of children with
SCA that had a hemoglobin less than 5.5g/dl (~0.165 Hct) from an acute anemic event, developed
an acute silent cerebral infarct during their hospitalization (Quinn et al., 2013). Within our study,
the most anemic SCA patient in our study had a Hct of 0.197 and a CVR of 0.053
%ΔBOLD/ΔPETCO2, whereas the least anemic SCA patient had a Hct of 0.334 and a CVR of
0.2265 %ΔBOLD/ΔPETCO2, for the same increase in PETCO2. These are steady-state Hct and CVR
values, in that the children weren't hospitalized within 3 months of the scan date. This comparison
goes to show that if an acute anemic event occurred, the more anemic child would theoretically
have less cerebrovascular reserve to deal such reductions in oxygen content. The results therefore
bring the Hct/Hg blood parameters to a forefront, where even though two children may not be
symptomatic, this doesn't mean that both children are equally at risk for neurologic injury during
such events. Quinn's recent commentary on SCI places particular emphasis on the anemia and
poses the question "how low can you safely go" and suggests that waiting for 'symptomatic anemia'
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may be too late (Quinn et al., 2013). In addition, this study confirms several findings in the
pediatric SCD population that is observed in the adult SCD population; the first that CVR is
significantly reduced compared to healthy controls and the second being that CVR is associated
with normocapnic CBF (Nur et al., 2009b; Isak Prohovnik et al., 2009). In other words, SCD
children have less dilatory reserve than healthy children and that the reduction in dilatory reserve
is used to increase CBF to the parenchymal tissue. This is an important finding as low CVR can
be a result from endothelial injury and dysfunction alone (and not anemia) as has been found in
multiple sclerosis and diabetes (Marshall et al., 2014). Alternatively, patients with end stage renal
disease have low Hct originating from low/impaired erythropoietin and have the same relationship
with vascular reactivity and CBF and had a low CMRO2 compared to healthy controls.
Furthermore, once erythropoietin was used to increase the Hct, the CBF reduced, the CVR
increased, and importantly the CMRO2 was returned to healthy levels(Hirakata et al., 1992). The
low CVR found in SCD children may be a result of endothelial injury from the oxidative stress
originating from cell free haem or reperfusion injury or a natural response to low Hct levels. It is
currently not known what CMRO2 the SCD children have and whether these parameters can be
returned to normal after correcting for the anemia. This is where the second study comes into
perspective. The results showed that HU or cTx patients had higher Hct than non-treated SCD
patients, which may account for the higher CVR seen in the treated group. It would be interesting
if increasing Hct to healthy levels would result in a normalization of CVR or whether there is
irreversible endothelial damage that would prevent from CVR return to healthy levels. However,
the current cTx guidelines recommend not to transfuse over 10g/dl (30% Hct) in order to prevent
viscosity complications, which may reduce oxygen delivery despite increasing Hct (Chou, 2013;
Wc & Dwan, 2013). The second study also noted that HU treated children had increased GM CBF
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compared to untreated SCD children potentially due to the increased affinity of HbF, however it
was only approaching significance, whereas cTx treated SCD children had reduced GM CBF
compared to untreated SCD children. Both HU treatment and Tx treatment have been shown to
reduce TCD velocities therefore the discrepancy may result in the relationship between
macrocirculation flow as assessed by TCD and microcirculatory flow as assessed by pASL
(Zimmerman et al., 2007).
A recent commentary has proposed a non vasculopathic mechanism of stroke, which took
into account the current knowledge of risk factors and imaging findings (Hulbert & Ford, 2015).
A host of risk factors for overt stroke have been elucidated, which include; large-vessel stenosis,
low Hct, young age, high blood flow, high blood pressure, and acute anemic events (Kugler et al.,
1993; Miller et al., 2001; Ohene-frempong et al., 1998). Although stenosis is not present in all
SCD patients that suffer a stroke, large-vessel stenosis reduces perfusion pressure and is matched
with autoregulatory dilation to maintain CBF. Therefore having a stenosis already utilizes the
limited vascular reserve of vessel, which in addition to baseline compensation for anemia, will
possibly drastically reduce vascular reserve and perhaps even increase OEF. In this vulnerable
state, if an acute event occurs such as a febrile illness which increases oxygen demand, or, instead
an acute anemic event which reduces oxygen content then the brain relies on the remainder of
vascular reserve and oxygen extraction reserve which may be insufficient to maintain oxygen
supply thereby resulting in tissue hypoxia and infarction (Hulbert & Ford, 2015). High blood
pressure can result from stiffening of the arteries, which may result in SCD due to the
vasoconstrictive phenotype elicited by nitric oxide signaling disruption (Potoka & Gladwin, 2015).
Perhaps the vasoconstrictive/stiff arteries may be less responsive to dilatory stimuli which
theoretically would further limit the dilatory capabilities of the vasculature. The fact that these
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strokes occur in young children, where the incidence peakS at the age of 4, is an important piece
of information. Previous literature has shown significant effects of development on cerebral
hemodynamics. CBF is highest in children and work from our lab has shown that the increased
CBF is accomplished through using cerebrovascular reserve (Leung J, 2016(Biagi et al., 2007).
Therefore in terms of ability to deal with acute anemic events, it seems that the pediatric population
is particularly susceptible as it is using a greater proportion of its compensatory mechanisms for
what seems to be development.
4.2 Limitations
A limitation in our study is the generalizability of our results to the general clinical
population of children with SCD. First, SCD is an umbrella term for various inherited
hermoglobinopathies and includes HbSS, HbSC, HbSβo/+, etc. In the first study, we attempted to
address the genetic spread by including other variants of SCD in addition to HbSS, which included
4 patients with HbSβo/+ and 1 patient with HbSC. One of the differences amongst the SCD variants
is the disease severity, where HbSS is the most common and most severe variant, followed by
HbSβo, HbSβ+, and HbSC. Including these variants allowed for increasing the range in the degree
of anemia and CBF, which was beneficial in demonstrating that the physiological relationship
interactions with anemia, CVR, and CBF. However, the main limitation is within our HbSS (SCA)
population. Although HbSS patients all have the same genotypic mutation, the clinical phenotype
of the disease is variable due to coinheritance of other common mutations such as glucose-6-
phosphate dehydrogenase (G6PD) deficiency, variable deletions of the alpha globin (α-
thalassemia), and perhaps epigenetics and socioeconomic status. G6PD deficiency is thought to
increase the hemolytic severity of patients with SCA, whereas the α-thalassemia actually
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ameliorates disease severity and is a protective factor against overt stroke. Socioeconomic status
differences may also play a significant role not only with respect to selection bias in our study but
also access to healthcare, education levels, and perhaps nutrition, which may all play a complex
role in affecting the phenotypic expression of the disease. The range in clinical severity amongst
patients with SCA can also be seen in the risk for overt stroke based on TCD velocities. The fact
that all children with confirmed TCD velocities over 200cm/s in the MCA are placed on cTx and
that cTx is an exclusion criteria for the first study produces a selection bias for SCA patients that
are less severe. In other words, the SCA patients that have an abnormally high risk for an overt
stroke could not be included in the patient population, which must be taken into consideration
when interpreting the data. HU reduces the risk of painful vaso-occlusive crises and is
recommended to patients who have a history of these events, which warrant management.
Therefore by excluding HU patients in our first study, we introduce another selection bias for SCA
patients that for whatever reason do not have a high reoccurrence of painful crises or opted not to
intitate HU therapy. These exclusion criteria were necessary in the first study to determine the
effect of anemia on the cerebrovasculature of children with SCA because both cTx and HU actively
change the disease. For example, cTx introduces healthy adult red blood cells into the circulation
and the HbA levels are maintained above 70%, where adult red blood cells do not undergo sickling
and have a different hemoglobin structure, which significantly alters the pathophysiology of SCA
and therefore must be grouped separately as a treatment cohort as we did in the second study. In
our second study, clinical severity plays a significant role in our patient demographics therefore
when comparing the non-treated SCA group to the cTx SCA group it must be taken into account
that the cTx group constitutes the sickest of all children with HbSS. A different approach would
be to scan children when their abnormal TCDv are confirmed in the clinic prior to initiating the
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cTx program and perform the second scan immediately after, however such a study design would
be difficult to execute owing to the limited availability of subjects. Another limitation in terms of
patient demographics is that a blood draw could not be performed in our healthy control
population, which was not approved by the research ethics board at our institution and we therefore
used literature values of healthy Hct in healthy children. Information on healthy Hct would have
allowed us to determine if the relationship between anemia and CVR or CBF extends into the
healthy pediatric population. Although CVR was strongly associated with Hct in children with
SCD, increasing the Hct of patients to healthy levels may not necessarily normalize CVR due to
the hyperviscous blood of patients with SCD and the potential for irreversible endothelial damage
to have occured. In addition, it has been observed that healthy males have higher CVR than healthy
females, perhaps owing to greater Hct as well as the influence of female sex hormones on CBF
(Kassner A, 2010). However, we saw no sex differences of CVR in patients with SCD, which may
be due to the fact that a large proportion of our patients are assumed to not have gone through
puberty. Nevertheless, the effect of sex on Hct on patient cerebral hemodynamics needs to be
assessed in future studies of SCD. As well, due to patient availability, there was a delay between
the scheduled clinic visits where the hematological reports are generated and the MRI scanning,
which is approximately 33 days on average.
With respect to the CVR measurements, the BOLD response following CO2 administration
is assumed to be primarily weighted by CBF, thereby allowing BOLD changes to infer CBF
changes (Kim & Ogawa, 2012; Spano et al., 2013). However, the BOLD signal relies on T2*
effects, which is intrinsically dependent on the amount of hemoglobin molecules per voxel.
Therefore, all things considered equal, reducing Hct would serve to reduce the BOLD response
through physical rather than physiological mechanisms. This is an important argument to consider
102
as it applies to the observations in both the first and second studies. To address this matter, a recent
fMRI study in SCD contained a demographic with a greater Hct range than the one within this
study and reported no significant effect of Hct on the BOLD response (Zou, Helton, & Smeltzer,
2011). The authors argued that although a low Hct indicates less hemoglobin in an imaging voxel,
which would reducing the BOLD signal, there is a simultaneous increase in cerebral blood volume
(CBV) that increases the amount of hemoglobin molecules present in that voxel. The effect being
that the drop in BOLD signal due to anemia is buffered by the increase in CBV (Zou et al., 2011).
In addition, in our study we are not measuring the BOLD signal per se, instead we measure the
change in the BOLD signal therefore may be circumventing any potential effect of Hct on BOLD
signal. In addition, Kuwabara et al. assessed the vasodilatory response in anemic patients
secondary to chronic renal failure and using O-15 H2O PET following CO2 inhalation and showed
a similar relationship between CVR and Hct (r=0.79) (Hirakata et al., 1992). Therefore, the
relationship between CVR and Hct is believed to represent physiological relationships and not
primarily the Hct dependency of the BOLD signal.
The pASL-based CBF measurements in our study are quite a bit lower compared to the
literature values using PET, which is the current gold-standard for absolute CBF quantification
(Ibaraki et al., 2008). This may be a result of the pASL sequence, which suffers from relatively
low signal to noise ratio (SNR) and is fundamentally dependent on PLD between the tag and
control images. The conventional PLD assumes normal arterial transit times, whereas children and
patients with SCA have elevated CBF and therefore reduced arterial transit times. Therefore,
following the PLD the tagged bolus has traveled further into the imaging plane, which may lead
to slight reductions in CBF measurements as the tail-end of the bolus is being caught. In addition,
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ASL is dependent on the T1 of blood, which may perhaps be different in sickle cell disease and
thus be contributing to the underestimation.
4.3 Future Directions
Using MRI to obtain physiologically relevant measurements such as CVR or CBF relies
on knowing the T1, T2, and T2* of tissue and especially blood. Although these parameters are
known in healthy populations, currently, it is not well established how the blood T1,T2,and T2* of
oxygenated and especially deoxygenated blood of patients with sickle cell disease. Considering
that dephasing of hydrogen nuclei spins involves the diffusion of water in and out of the red blood
cell where it interacts with the magnetic field of deoxyhemoglobin, it would be important to
determine how the diffusion changes with a dehydrated, sickled cell. In addition, typically
hemoglobin is sequestered within the red blood cell, however due to the chronic hemolysis in SCD,
there is significant cell-free haem in the vasculature. The cell-free haem would then interact with
water molecules without the need of diffusion across the red blood cell membrane, thus affecting
the T2 and T2*. In chronically transfused patients, the increased iron storage in ferritin due to the
iron overload may also pose an additional mechanisms in which T2 and T2* can be altered.
Considering that T2* is important when using BOLD sequences and T2 is important for things such
as determining oxygenation using methodologies such as the T2 Under Spin Tagging method,
therefore there is a great need to quantify SCD blood T2 and T2* under varying levels of
oxygenation as assuming healthy blood values may not be suitable. In addition, Blood T1 can also
theoretically be different in SCD, which would affect CBF quantification through ASL methods.
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In addition to CBF and CVR, future studies should perhaps include the measurements of
OEF as it further characterizes the oxygen availability of the cerebral tissue than CBF or CVR
alone. As previously mentioned, oxygen availability to the tissue can be altered by managing CBF
or changing OEF. In the first study, we have seen that cerebrovascular reserve depletes with
increasing severity of anemia, however, it would be important to determine if OEF is also changing
or whether it remains unchanged until cerebrovascular reserve is completely exhausted. Measuring
OEF in this circumstance would allow for further stratification of patients based on who is utilizing
their compensatory mechanisms to a greater degree, which may provide a novel method of stroke
risk or silent infarct detection. A prospective trial quantifying OEF and cerebrovascular reserve
with a neurologic injury as an endpoint may be able to set a threshold value of OEF or CVR below
which an increased risk of injury would exist. In the second study, the results showed that CBF in
the HU-treated SCD children is not significantly different than untreated SCD children, despite
having greater Hct, which may be due to the increased affinity of HbF to O2. However, it is difficult
to speculate whether this is a result of treatment, where HU increases red cell deformability and
therefore may improve microcirculatory flow or if it could be explained by differences in OEF.
For example, If the HU-treated group had significantly lower OEF as a result of higher oxygen
affinity of HbF then it may very well be the case that CBF remains elevated despite a greater Hct
in order to maintain CMRO2. In other words, the fetal Hb is holding onto the oxygen molecule and
therefore the delivery of flow must be increased to compensate. In this case, measuring OEF would
be useful in determining treatment effects of HU and future therapeutics aimed at playing a role in
the prevention of neurologic injury. To that end, when comparing patients and controls we assume
that the CMRO2 is equal, which may not be the case in a hypoxic environment. Therefore,
105
measurements of CVR, CBF, OEF, and CMRO2, should be acquired in order to better characterize
the cerebral hemodynamic environment and to determine the mechanism of treatments.
In the experimental work of this thesis we primarily focused on the degree of anemia and
its relationship with CVR and CBF in children with sickle cell disease. However, SCD is a state
of reduced NO bioavailability, which is a potent vasodilator and anti-inflammatory agent and has
been noted to be improved following HU-treatment. Therefore future studies would benefit from
obtaining measures of NO, as well as its precursor arginine and metabolized products such as
nitrate in order to investigate the role of this key players with respect to the dilatory ability of
cerebral endothelia and smooth muscle. Importantly, future studies should assess both oxygen
dependent and oxygen independent roles of NO physiology. In addition, although we assessed the
cerebral hemodynamic environment, the human physiology is tightly coordinated with several
organ systems. Of particular importance in SCD is lung function as children with SCD have a
significantly greater proportion of asthma and a major reason for mortality in females with SCD
is due to lung disease. Considering that the lungs are the primary source of oxygen saturation and
that they are drastically affected in patients with SCD, future studies should assess the lung
physiology and perform pulmonary function tests in order to determine its effect on oxygen
delivery and compensation in the brain. In addition, it would be significant to determine if the flow
of blood to the lungs of patients with SCD is significantly different than healthy controls,
essentially a pulmonary vascular reactivity test.
CVR is showing promise in predicting the risk of stroke in carotid artery disease.
Therefore, stroke research in children with SCD would benefit from a longitudinal study using
CVR and OEF quantification as possible stroke risk factors. SCD patients would have their CVR
106
quantified and followed-up longitudinally to determine if CVR and OEF can predict the event of
an outcome such as silent infarction. Another direction may be to measure CVR and OEF in SCD
children following an acute complication requiring hospitalization such as ACS, B19 infection,
SS, or febrile illness and determine if CVR or OEF can partly predict the occurrence of acute silent
cerebral infarctions. Currently, CVR is measured globally or stratified by gray and white matter
when MRI is present, which may be sufficient for large scale complications such as overt-stroke,
however may not be sensitive enough for smaller lesions such as silent infarctions. Therefore
future research should be aimed at overcoming the technical hurdle of obtaining accurate CVR, as
well as OEF measurements on a regional level. Obtaining CVR and OEF on a regional level may
provide information on the compensatory strain in different areas of the brain, which is thought to
occur before the injury and therefore may be reversible to a degree. Particular areas such as the
internal watershed zone may see great benefit in having its hemodynamic environment
characterized by these parameters as this area is thought to be susceptible to injury from
hemodynamic failure and is an area commonly affected in children with SCD. As mentioned
previously, silent cerebral infarctions occur without overt neurologic symptoms and occur in
approximately 40% of children by the age of 18 with significant impact on cognitive abilities.
Although chronic transfusion therapy is known to reduce the occurrence of silent infarcts, we have
no means at predicting which children are at risk and may benefit from therapeutic intervention.
Therefore one of the next big hurdles in SCD research is to investigate a suitable method of
detecting silent infarcts.
4.4 Conclusion
In this thesis, we investigated the cerebral hemodynamics and the effect of transfusion
and hydroxyurea treatment in children with SCD using advanced MRI protocols. Understanding
107
the cerebral hemodynamics is important in understanding why the brain in children with SCD is
at an increased risk for injury in the form of overt stroke and silent infarcts. In addition, the
understanding of treatment effects on the cerebral vessels not only serves to better understand the
mechanism of how these therapies may work but may also provide a basis to which future
therapies aimed at stroke prevention can be compared to. In the first study, we investigated the
effect of anemia on cerebrovascular reserve as reflected by CVR measurements, as well as the
effect that anemia has on CBF, which are both important hemodynamic parameters. The first
study demonstrated that children with SCD that have greater degrees of anemia have less
cerebrovascular reserve and higher CBF than less anemic SCD children. Considering
cerebrovascular reserve is utilized to increase oxygen delivery, therefore children with SCD that
have critically low Hct may have insufficient reserve to compensate for acute anemic events or
other stressors that impact oxygen. This finding may help explain why lower hematocrit is a risk
factor for overt stroke and silent infarcts in children with SCD and may potentially be used in
future work to stratify patients based on stroke risk. The second study investigated how children
with SCD on chronic transfusion, hydroxyurea, and non-treated compare in their CVR and CBF.
The results have shown that children with SCD that are treated with either HU or cTx have
greater cerebrovascular reserve than non-treated children. In addition, the study showed that
children with SCD on cTx have lower CBF than non-treated children, whereas HU-treated
children have no significantly different CBF than non-treated children. These results suggest that
these therapeutics improve cerebrovascular reserve, which may recover the cerebrovasculature's
ability to deal with changes in oxygen demands to some extent. The fact that the CBF of HU-
treated children with SCD does not significantly differ from non-treated children is interesting
and warrants further investigation into the mechanisms of HU. Further studies in SCD should
108
characterize the cerebral hemodynamic environment by obtaining OEF and CMRO2 in addition
to CBF and CVR measurements, longitudinally, in order to assess the risk of neurologic injury.
109
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