3T BOLD MRI MEASURED CEREBROVASCULAR RESPONSE TO

HYPERCAPNIA AND HYPOCAPNIA: A MEASURE OF CEREBRAL

VASODILATORY AND VASOCONSTRICTIVE RESERVE

Jay S. Han

A thesis submitted in conformity with the requirements for the degree of

Master of Science,

Graduate Department of Physiology,

Faculty of Medicine

University of Toronto

© Copyright by Jay Shou Han (2010)

ii

Jay Shou Han

Masters of Science (2010)

Graduate Department of Physiology

Faculty of Medicine

University of Toronto

Toronto, Ontario, Canada

Abstract

Cerebrovascular reactivity (CVR) - defined as a change in cerebral blood flow (CBF) in

response to a given vasodilatory stimulus - is a measure of the ability of the cerebral

vasculature to maintain a constant CBF despite reductions in perfusion pressure. A decrease

in CVR (which is interpreted as a reduction in the vasodilatory reserve capacity) in the

vascular territory downstream of a stenosed supply artery correlates strongly with the risk of

a hemodynamic stroke. As a result, the use of CVR studies to evaluate cerebral

hemodynamics has clinical utility. Application of CVR studies clinically, depends on a

thorough understanding of the normal response. The goal of this thesis therefore was to map

CVR throughout the brain in normal healthy individuals using Blood Oxygen Level

Dependant functional Magnetic Resonance Imaging as an index of CBF and precisely

controlled changes in end-tidal partial pressure of carbon dioxide as a vasodilatory stimulus.

iii

Acknowledgements

First and foremost I would like to thank both my supervisor Dr. Joseph A. Fisher, and Dr.

David J. Mikulis for introducing to me the world of clinical research and medicine. Both

have provided me with invaluable opportunities and guidance. Under their mentorship I have

matured both as a student and as an individual and I am forever indebted.

I would also like to thank Dr. Daniel M. Mandell, Dr. Adrian P. Crawley, Julien Poublanc

and Dr. James Duffin for their expert personal and professional advice and time spent

reviewing the data and the thesis.

I would also like to collectively thank Alexandra Mardimae, Dr. Marat Slessarev and Dr.

David Preiss, for their unconditional support at the beginning as colleagues, and now as close

friends. I would also like to express my sincere gratitude to Cliff Ansel, Stephanie Dorner,

Keith Ta, Eugen Hlasny, David Johnstone and Jerry Plastino for their assistance as I

conducted my research.

Lastly, I want to thank my friends and family for their patience, understanding and

unconditional support.

iv

Table of Contents

Abstract ................................................................................................................................ ii

Acknowledgements…………………………………………………………………………..iii

List of Figures ................................................................................................................... viii

List of Tables ........................................................................................................................x

List of Graphs .......................................................................................................................x

Abbreviations…………………………………………………………………………………xi

1. Chapter 1 .......................................................................................................................1

Background Review of Literature ..........................................................................................1

1.1 Neuro-anatomy .......................................................................................................1

1.2 Cerebral Arterial Circulatory Anatomy ....................................................................1

1.2.1 Anterior Cerebral Artery .................................................................................. 4

1.2.2 Middle Cerebral Artery .................................................................................... 4

1.2.3 Posterior Cerebral Artery ................................................................................. 4

1.2.4 Collateral Circulation ....................................................................................... 5

1.3 Cerebral Blood Flow, Metabolism, and Auto-regulation ..........................................7

1.3.1 Cerebral Blood Flow and Metabolism .............................................................. 7

1.3.2 Cerebral Autoregulation ................................................................................... 7

1.3.3 Cerebral Vasodilatory Reserve ......................................................................... 9

1.4 Cerebrovascular Reactivity (CVR) ........................................................................ 11

1.4.1 Quantification Testing of CVR ....................................................................... 12

1.4.2 CVR and carbon dioxide (CO2) ...................................................................... 13

1.4.3 Mechanism of carbon dioxide mediated vascular response ............................. 13

1.5 Administration and Manipulation of CO2 Stimuli .................................................. 15

1.5.1 Acetozolamide (Diamox) ............................................................................... 15

1.5.2 Breath Holding ............................................................................................... 16

1.5.3 Varying Minute Ventilation ............................................................................ 17

1.5.4 Inspired concentrations of O2 and CO2 - Non-rebreathing .............................. 17

1.5.5 Inspired concentrations of O2 and CO2 - Rebreathing .................................... 18

v

1.5.6 Inspired concentrations of O2 and CO2 – Modified Prospective End Tidal

Targeting (MPET) Breathing circuit ............................................................................ 19

1.6 Imaging of Cerebrovascular Reactivity .................................................................. 20

1.6.1 Trans – Cranial Doppler (TCD) ...................................................................... 20

1.6.2 Xenon – 133 Wash Out .................................................................................. 21

1.6.3 Positron Emission Tomography (PET) ........................................................... 22

1.6.4 Single Photon Emission Computed Tomography (SPECT) ............................. 23

1.6.5 Near Infrared Spectroscopy ............................................................................ 23

1.6.6 Magnetic Resonance Imaging (MRI) – Blood Oxygen Level Dependant MRI 24

2. Chapter 2 ..................................................................................................................... 25

Rationale and Objectives ..................................................................................................... 25

2.1 Rationale and Objectives ....................................................................................... 25

2.2 Optimizing the vasoactive Stimuli ......................................................................... 29

2.2.1 Illustration of CBF response changes in PCO2 ................................................ 29

2.3 Optimizing BOLD MR Imaging ............................................................................ 30

2.3.1 Static magnetic field strength ......................................................................... 31

2.3.2 Repetition Time (TR) ..................................................................................... 31

2.3.3 Echo Time (TE) ............................................................................................. 31

2.3.4 Voxel Size and Slice thickness ....................................................................... 32

3. Chapter 3 .................................................................................................................... 33

Quantification of Brain CVR to CO2 ................................................................................... 33

3.1 Introduction ........................................................................................................... 33

3.2 Materials and Methods .......................................................................................... 35

3.2.1 Ethics and Consent ......................................................................................... 35

3.2.2 Magnetic Resonance Imaging ......................................................................... 35

3.2.3 Control of PETO2 and PETCO2 ...................................................................... 35

3.2.4 Determination of Cerebrovascular Reactivity ................................................. 36

3.2.5 Grey and WM Segmentation .......................................................................... 37

3.2.6 Statistical Analysis ......................................................................................... 38

3.3 Results .................................................................................................................. 40

vi

Control of PETCO2 and PETO2 ................................................................................... 40

3.3.1 Global BOLD CVR ........................................................................................ 43

3.3.2 Grey and White Matter BOLD CVR .............................................................. 44

3.3.3 Grey Matter BOLD CVR ............................................................................... 44

3.3.4 WM CVR ....................................................................................................... 45

3.4 Discussion ............................................................................................................. 47

3.5 Conclusion ............................................................................................................ 50

4. Chapter 4 .................................................................................................................... 52

Quantification of Regional Brain BOLD CVR to CO2 ......................................................... 52

4.1 Introduction ........................................................................................................... 52

4.2 Materials and Methods .......................................................................................... 53

4.2.1 Ethics and Consent ......................................................................................... 53

4.2.3 Magnetic Resonance Imaging ......................................................................... 53

4.2.4 Control of PETO2 and PETCO2 ...................................................................... 53

4.2.5 PETCO2 and PETO2 Data Processing ........................................................... 56

4.2.6 BOLD MRI Data Analysis and Determination of CO2 Cerebrovascular

Reactivity .................................................................................................................... 56

4.2.7 Cortical Vascular Territory Segmentation and Determination of CVR ............ 56

4.2.8 Periventricular and subcortical WM Segmentation and Determination of CVR

…………………………………………………………………………………57

4.2.1 Statistical Analysis ......................................................................................... 57

4.3 Results .................................................................................................................. 58

4.3.1 End Tidal Gas Values: Control of PETCO2 and PETO2 .................................. 58

4.3.2 GM vascular territory CVR ............................................................................ 60

4.3.3 WM territory vascular response to CVR ......................................................... 64

4.4 Discussion ............................................................................................................. 67

4.5 Conclusion ............................................................................................................ 70

5. Chapter 5 ..................................................................................................................... 71

Extended Discussion, Conclusions, and Future Directions ................................................... 71

vii

5.1 Cerebral blood flow vs changes in CO2: Sustained Hypercapnia and hypocapnia at

Rest ……………………………………………………………………………………...72

5.2 The Effect of aging on CVR to CO2 ...................................................................... 77

5.3 The effect of sex on CVR to CO2 .......................................................................... 81

5.4 The repeatability of CVR to CO2 ........................................................................... 82

5.5 Influence of CO2 on Mean Arterial Pressure and CVR to CO2 ............................... 82

5.6 Vasocontrictive Reserve ........................................................................................ 83

5.7 Future Studies ....................................................................................................... 85

viii

List of Figures

Figure 1-1. 3 Tesla Time of Flight MR Angiography of the author‟s Circle of Willis

demonstrating vessels of interest. ..........................................................................................3

Figure 1-2. 3 Tesla Time of Flight MR Angiography of the Circle of Willis. Multiplanar

reconstruction (MPR) Posterior – Anterior view of the Circle of Willis demonstrating the

vessels of interest. .................................................................................................................5

Figure 1-3. Cerebral Blood Flow vs. Cerebral Perfusion Pressure. Between cerebral perfusion

pressures of 50mmHg and 150mmHg cerebral blood flow is constant. When cerebral

perfusion pressure is below 50mmHg or above 150mmHg cerebral blood flow becomes

directly proportional to changes in cerebral perfusion pressure. .............................................9

Figure 1-4. Progression of encroachment on Vasodilatory Reserve. A) Normal CPP and

Vascular tone. B) Reduction in CPP and encroachment on vasodilatory reserve. C) Severe

reduction in CPP and exhausted vasodilatory reserve (maximal vasodilatation). .................. 11

Figure 2-1. MCAV vs PETCO2. Red points demonstrate the change in MCAV in the .......... 29

Figure 3-1. Anatomical Segmentation Maps. A) Representative segmented GM (red)

anatomical map (B) Representative segmented WM (red) anatomical map. ......................... 37

Figure 3-2. Formula for Calculating Proportional Change in BOLD CVR ........................... 39

Figure 3-3. Removing the effect of blood volume on measured BOLD CVR to allow GM and

WM comparison of non-linearity. A) Raw WM BOLD CVR . B) Raw GM BOLD CVR .

Notice BOLD CVR scale is different in A) and B). C) BOLD signal normalized for blood

volume for both GM and WM, allowing the use of the same ordinate scale. ........................ 40

Figure 3-4. Raw PETO2 tracing (Green), Raw PETCO2 tracing (red) and corresponding

averaged Raw whole brain BOLD MRI signal (blue) for (a) hypercapnic and (b) hypocapnic

studies. ................................................................................................................................ 42

Figure 3-5. BOLD MRI CVR Maps. A) Representative hypercapnia BOLD MRI CVR

response. B) Representative hypocapnia BOLD MRI CVR response. .................................. 43

Figure 3-6. Grey Matter BOLD MRI CVR at hypercapnia and hypocapnia. ......................... 46

Figure 3-7. White matter BOLD MRI CVR at hypercapnia and hypocapnia ........................ 46

Figure 4-1. Raw PETO2 tracing (Green), Raw PETCO2 tracing (red) and corresponding

averaged Raw whole brain BOLD MRI signal (blue) for (a) hypercapnic and (b) hypocapnic

studies. ................................................................................................................................ 55

Figure 4-2. A) Subcortical WM Mask. B) Periventricular WM Mask................................... 58

ix

Figure 4-3. Right Cortical ACA, MCA and PCA vascular territory CVR to Hypercapnia .... 62

Figure 4-4. Right Cortical ACA, MCA and PCA vascular territory CVR to Hypocapnia ..... 62

Figure 4-5. Left Cortical ACA, MCA and PCA vascular territory CVR to hypercapnia. ...... 63

Figure 4-6. Left Cortical ACA, MCA and PCA territory CVR to hypocapnia. ..................... 64

Figure 4-7. Periventricular WM CVR to hypercapnia and hypocapnia. ................................ 66

Figure 4-8. Subcortical WM CVR to hypercapnia and hypocapnia. ..................................... 66

Figure 4-9. Representative vascular anatomy in different vascular territories with theoretical

degree of accompanied compensatory vasodilatation. Modified from Marinknovic et al.

Anatomic and Clinical Correlations of the Lenticulostriate Arteries. Clinical Anatomy

14:190–195 (2001). ............................................................................................................. 68

Figure 5-1. Normal Vascular Response Curve to Changes in PaCO2 .................................... 72

Figure 5-2 Cerebral Vascular response curve shift downward due to sustained hypercapnic

levels of PaCO2 at rest. A) Initial vascular response to hypercapnia; Increase in CBF. B)

Normalization of CBF to sustained hypercapnia with attenuated vascular response to further

hypercapnia: a downward shift of the physiological response curve. ................................... 74

Figure 5-3. Cerebral Vascular response curve shift upward due to sustained hypocapnic

levels of PaCO2 at rest. A) Initial vascular response to hypocapnia; decrease in CBF due to

vasoconstriction. B) Normalization of CBF to sustained hypocapnia with enhanced vascular

response to hypercapnia....................................................................................................... 76

Figure 5-4. Step wise reduction in perfusion pressure accompanied by loss of

vasoconstrictive reserve. A) Normal CPP and maintenance of vasoconstricitive ability. B)

Progressive reduction of CPP loss of vasodilatory ability (due to compensatory dilatation)

and preservation of vasoconstrictive ability. C) Severe Reduction of CPP both vasodilatory

and vasoconstrictive ability are abolished (a presumed magnitude of vasoconstriction at this

point might lower CBF to ischemic thresholds). .................................................................. 84

x

List of Tables

Table 3-1. Hypercapnic PETCO2 and PETO2 values at each stage of protocol (Mean ±

Standard Error). ................................................................................................................... 41

Table 3-2. Hypocapnic PETCO2 and PETO2 values at each stage of protocol (Mean ±

Standard Error). ................................................................................................................... 41

Table 3-4. Grey Matter BOLD CVR values at hypercapnia and hypocapnia. ....................... 44

Table 3-5. White Matter BOLD CVR values at hypercapnia and hypocapnia. ...................... 45

Table 4-1. Hypercapnic PETCO2 and PETO2 values at each stage of protocol (Mean ±

Standard Error). ................................................................................................................... 59

Table 4-2. Hypocapnic PETCO2 and PETO2 values at each stage of protocol (Mean ±

Standard Error). ................................................................................................................... 59

Table 4-3 Mean cortical vascular territory BOLD MRI CVR to hypercapnia (expressed as

Mean ± Standard Error). ...................................................................................................... 60

Table 4-4. Mean cortical vascular territory BOLD MRI CVR to hypocapnia (expressed as

Mean ± Standard Error). ...................................................................................................... 60

Table 4-5. Mean subcotical WM BOLD MRI CVR to hypercapnia and hypocapnia.

(expressed as Mean ± Standard Deviation) .......................................................................... 64

Table 4-6. Mean Periventricular WM CVR to hypercapnia and hypocapnia (expressed as

Mean ± Standard Deviation). ............................................................................................... 64

List of Graphs

Graph 5-1. Global Brain BOLD measured CVR to hypercapnia Vs. Age. ............................ 78

Graph 5-2. Global Brain BOLD measured CVR to hypocapnia Vs. Age. ............................. 78

Graph 5-3. Grey Matter BOLD measured CVR to hypercapnia Vs. age. .............................. 79

Graph 5-4. Grey Matter BOLD measured CVR to hypocapnia Vs. age ................................ 79

Graph 5-5. White Matter BOLD measured CVR to hypercapnia Vs. age. ............................ 80

Graph 5-6. White Matter BOLD measured CVR to hypocapnia Vs. age .............................. 80

xi

Abbreviations

ACA – Anterior Cerebral Artery

ACZ- Acetozolamide

ANOVA - Analysis of Variance

BOLD MRI – Blood Oxygen Level Dependant Magnetic Resonance Imaging

CBF – Cerebral Blood Flow

CBV - Cerebral Blood Volume

CCA – Common Carotid Artery

cGMP - Cyclic Guanosine Monophosphate

CO2 - Carbon Dioxide

CPP – Cerebral Perfusion Pressure

CSF - Cerebral Spinal Fluid

CVR – Cerebral Vascular Reactivity

ECA – External Carotid Artery

FICO2 - Inspired Fractional Concentration of Carbon Dioxide

GM – Grey Matter

ICA – Internal Carotid Artery

ICP – Intracranial Pressure

MAP – Mean Arterial Pressure

MCA – Middle Cerebral Artery

MPET - Modified Prospective End-Tidal Targeting

MRI - Magnetic Resonance Imaging

N2 - Nitrogen

NIRS - Near Infrared Spectroscopy

NO - Nitric Oxide

O2 - Oxygen

OEF - Oxygen Extraction Fraction

PaCO2 - Arterial Parital Pressure of Carbon Dioxide

PaO2 - Arterial Parital Pressure of Oxygen

PCA – Posterior Cerebral Artery

PCO2 - Partial Pressure of Carbon Dioxide

xii

PET - Positron Emission Tomography

PETCO2 – End –Tidal Partial Pressure of Carbon Dioxide

PETO2 - End-Tidal Parital Pressure of Oxygen

PICO2 - Inspired Parital Pressure of Carbon Dioxide

PIO2 - Inspired Parital Pressure of Oxygen

PO2 - Partial Pressure of Oxygen

PVWM - Periventricular White Matter

rCBF - Regional Cerebral Blood Flow

rCPP – Regional Cerebral Perfusion Pressure

ROI - Region of Interest

SCWM - Subcortical White Matter

SNR - Signal to Noise Ratio

SPECT - Single Photon Emission Tomography

TCD - Transcranial Doppler

TE - Echo Time

TR - Reptition Time

WM – White Matter

1

1. CHAPTER 1

BACKGROUND REVIEW OF LITERATURE

1.1 NEURO-ANATOMY

The brain consists of paired frontal, parietal, temporal, and occipital lobes, which collectively

form the cerebrum and the cerebellum. Contained within each lobe and cerebellum is tissue

that is composed predominantly of cell bodies (Grey Matter) and of interconnecting neurons

(White Matter).

Approximately 40% of brain tissue is composed of Grey Matter (GM). GM is mainly

composed of neuronal cell bodies and accompanying dendrites and axons which terminate in

synapses between neighboring neurons. The human brain contains an estimated 50-100

billion neurons and about 100-500 trillion synapses. The thickness of the GM ranges from

1.5 mm to 4 mm depending on the location, and is organized into 4 to 6 distinct layers.

The other 60% of the brain tissue is composed of White Matter (WM). WM consists mainly

of myelinated axons; the myelin, which contributes to its white appearance, is produced by

oligodendrocytes. The individual axons are the conduits for which information is transferred

throughout the brain. Myelination of the individual axons serves to increase the speed of

action potentials that propagate down an axon. Also located within the WM are a number of

cells that support the WM, such as glial cells, astrocytes and a smaller number of microglia.

1.2 CEREBRAL ARTERIAL CIRCULATORY ANATOMY

Blood flow throughout the brain is directed by a complex cerebral arterial circulatory system

that can be studied as two smaller arterial circulatory pathways; an anterior and a posterior

arterial circulatory system.

The anterior arterial cerebral circulatory system begins as the level of the two common

carotid arteries (CCA); the left CCA usually arises from the aortic arch and the right CCA

2

branches off from the innominate artery. The two common carotid arteries course cephalad

from behind the sterno-calvicular joint to the upper border of the thyroid cartilage. At that

level each CCA bifurcates into an external carotid artery (ECA) - supplying the superficial

muscles and skin- and an internal carotid artery (ICA) - that becomes the primary source of

blood flow for each cerebral hemisphere. The ECA courses behind the neck of the mandible

and after passing the parotid glands, bifurcates into 7 major arteries including the superficial

temporal and maxillary arteries.

Each ICA continues intra-cranially to the supra-clinoid region from which it divides into the

primary segments of the anterior (ACA) and middle cerebral (MCA) arteries. Other branches

that arise from the ICA include the ophthalmic, posterior communicating, and anterior

choroidal arteries.

The posterior arterial circulatory system consists of the left and right vertebral arteries which

originate as a branch of the respective subclavian artery. The two vertebral arteries then

traverse superiorly along the vertebral bodies before coming together to form a single vessel,

the basilar artery, at the junction between the medulla oblongata and the pons. Prior to

joining as the basilar artery, each vertebral artery gives rise to an anterior spinal artery, -

which supplies the spinal cord - and the posterior inferior cerebellar arteries - which supply

the inferior cerebellum and lower brainstem. The basilar artery bifurcates intra-cranially into

the right and left posterior cerebral (PCA) arteries respectively (Figure 1-1).

3

Figure 1-1. 3 Tesla Time of Flight MR Angiography of the author‟s Circle of Willis

demonstrating vessels of interest.

The two circulatory systems are connected through three smaller vessels, the anterior

communicating artery and the left and right posterior communicating arteries (PCA) at the

base of the brain – this forms the collateral network named the Circle of Willis which will be

discussed in further detail in section 1.2.4. From the Circle of Willis arise the six principle

cerebral blood vessels that supply the brain, the Left Anterior Cerebral Artery (LACA), the

Left Middle Cerebral Artery (LMCA), the Left Posterior Cerebral Artery (LPCA), the Right

Anterior Cerebral Artery (RACA), the Right Middle Cerebral Artery (RMCA) and the Right

Posterior Cerebral Artery (RPCA).

4

1.2.1 ANTERIOR CEREBRAL ARTERY

Each ACA branches directly from the ICA (Figure 1-2). The ACA courses below the anterior

cerebral hemispheres on each side and ramifies over the cortical surface at the front of the

brain. The ACA also extends along the longitudinal sulcus between the two hemispheres on

each side and continues up the medial aspect of the hemisphere giving off penetrating

branches supplying the interior of the respective hemisphere. Connecting the LACA and

RACA is the anterior communicating artery which appears just as the ACA are entering the

interhemispheric sulcus.

Each ACA principally supplies the following ipsilateral hemispheric regions:

frontal pole of the hemisphere

the whole medial surface of the frontal and parietal lobes to the parieto-

occipital suclus, where it then anastamoses with the posterior cerebral artery

1.2.2 MIDDLE CEREBRAL ARTERY

Each MCA is a direct continuation of the main branch of the ICA coursing in a horizontal

plane, laterally and slightly anteriorly (Figure 1-2); they are the predominant arteryies in the

brain. Major proximal vessels that branch of the MCA are the lenticulo- striate arteries.

The MCA principally supplies

the insula,

the inferior and middle frontal gyri

two thirds of the precentral and postcentral gyri,

the superior and inferior parietal lobules

the superior and middle temporal gyri

1.2.3 POSTERIOR CEREBRAL ARTERY

The PCA is a direct continuation of the basilar artery (Figure 1-2).

PCA is primarily responsible for supplying blood flow to the medial and

inferior surfaces of the occipital lobe

the inferior surface of the gyrus of the temporal lobe,

5

part of the superior parietal lobule and all of the calcarine cortex

Figure 1-2. 3 Tesla Time of Flight MR Angiography of the Circle of Willis. Multiplanar

reconstruction (MPR) Posterior – Anterior view of the Circle of Willis demonstrating the

vessels of interest.

1.2.4 COLLATERAL CIRCULATION

In addition to the principle cerebral supply vessels there is a subsidiary network of vascular

channels that serves to stabilize blood flow if there is a failure of one of the principle vessels.

These collateral vessels serve to redirect cerebral blood flow from patent primary vessels into

the vascular beds of failed primary vessels for which there is a cerebral insufficiency.

While some of these vessels are “anatomically patent” others represent “potential

anastomotic connections” and are recruited only under ischemic conditions (Liebeskind

6

2003b). This section will briefly review these collateral networks which is divided into

primary and secondary collateral networks.

1.2.4.1 PRIMARY COLLATERAL PATHWAY

The Circle of Willis is considered the primary collateral pathway. Anatomically, three major

vessels, a single anterior communicating artery and two PCA form the Circle of Willis. These

vessels together link the left and right ACAs, MCAs and PCAs forming a circular network,

which allows inter hemispheric blood flow.

This “ideal” configuration of the Circle of Willis, shows many variants. The anterior

communicating artery is absent in 1.8 % of subjects, and either posterior communicating

arteries may be absent (1%) or hypoplastic in 13.2% of individuals (Kapoor et al. 2008).

1.2.4.2 SECONDARY COLLATERAL NETWORKS

Secondary collateral networks serve to augment the flow provided by the Circle of Willis.

Secondary collateral networks include the leptomenningeal vessels, which consists of pial

arteries that connect the arterial trees of two major cerebral arteries, serving two different

cortical territories and the ophthalmic artery, which forms a potential conduit between the

ICA and ECA.

Additionally, there are other collateral networks that are not commonly encountered but may

be recruited or developed over time in the presence of occlusive pathology (Liebeskind

2003a).

The following is a list along with their intended collateral circulatory pathways:

1) Tectal Plexus – Joins the supratentorial branches of the PCA with the infratentorial

branches of the superior cerebellar artery.

2) Orbital plexus – which joins the ophthalmic artery with the facial, middle

menningeal, maxillary, and ethmoidal arteries

7

3) Rete Mirabile caroticum – which connects the internal carotid artery with the external

carotid artery

1.3 CEREBRAL BLOOD FLOW, METABOLISM, AND AUTO-REGULATION

1.3.1 CEREBRAL BLOOD FLOW AND METABOLISM

Cerebral blood flow (CBF) is both a vital and tightly regulated process. Though the human

brain only weighs approximately 1,300 – 1,400 g, it receives a disproportional 15% of the

total cardiac output. The normal average CBF throughout the entire brain is approximately

50 mL/100g/min (Lassen 1985). However, considered separately, blood flow to the GM is

higher at 80mL/100g/min compared to the WM which is 20 mL/100g/min. (Vavilala et al

2002) due to the difference in metabolic demand.

Globally, the effect of lower cerebral blood flows become evident as cerebral metabolism is

disrupted. At a CBF of less than 35 ml/100g/min, protein synthesis is reduced (Hossmann

1994) despite normal neurological function (Marshall et al. 2001b). Neurological deficits

become evident when CBF less than 27 ml/100g/min (Marshall et al. 2001a) and cortical

EEG activitiy is abolished at CBF values of 18 ml/100g/min or less (Trojaborg and Boysen

1973). If the brain is subjected to blood flows of less than 15ml/100g/min for an hour or less,

then permanent infarction is thought to ensue (Pulsinelli 1992).

1.3.2 CEREBRAL AUTOREGULATION

When CBF is below normal values, there is potential for ischemic damage to occur. CBF is

therefore precisely maintained at 45 – 50 ml/100g/min.

However, changes in physiological factors such as mean arterial blood pressure (MAP),

metabolism, chemical factors and neuronal input can all influence CBF values (Vavilala et al.

2002b). The ability to maintain a constant CBF despite these dynamic changes is due to the

presence of cerebral autoregulation (Vavilala et al. 2002a).

8

In this thesis, the most pertinent aspect of cerebral autoregulation is that which controls the

regional cerebral perfusion pressure (rCPP).

The global CPP is the net pressure gradient that drives blood flow to the brain. Under normal

conditions where intracranial pressure is low, CPP is entirely defined by changes in MAP.

CPP = Mean Arterial Pressure (MAP) – Intracranial Pressure (ICP)

Cerebral autoregulation compensates for fluctuations in CPP through reflexive

vasoconstriction and vasodilatation which alters the resistance in downstream vascular beds,

thereby maintaining CBF.

The cerebral autoregulatory response is effective over the MAP range of approximately 50 to

150 mmHg (Lassen 1959). Beyond these limits the cerebral autoregulatory response is

exhausted and blood flow becomes proportional to the MAP (Figure 1-3).

9

Figure 1-3. Cerebral Blood Flow vs. Cerebral Perfusion Pressure. Between cerebral

perfusion pressures of 50mmHg and 150mmHg cerebral blood flow is constant. When

cerebral perfusion pressure is below 50mmHg or above 150mmHg cerebral blood flow

becomes directly proportional to changes in cerebral perfusion pressure.

1.3.3 CEREBRAL VASODILATORY RESERVE

Cerebrovascular occlusive disease may result in stenotic lesions partially or totally occluding

a feeding vessel to the brain. This results in a reduction of the downstream rCPP.

Autoregulation then results in downstream dilation of the resistance vessels in an attempt to

restore/maintain sufficient blood flow to sustain neuronal cellular metabolic function. As the

narrowing of the lumen of the feeding vessel progresses, so does the magnitude of

compensatory vasodilation by downstream resistance vessels (Yonas and Pindzola 1994).

Once the resistance vessels have reached maximal dilation, this compensatory response is

said to be “exhausted” and any further reductions in rCPP results in proportional reduction in

blood flow (Figure 1-4).

10

A)

B)

11

C)

Figure 1-4. Progression of encroachment on Vasodilatory Reserve. A) Normal CPP and

Vascular tone. B) Reduction in CPP and encroachment on vasodilatory reserve. C) Severe

reduction in CPP and exhausted vasodilatory reserve (maximal vasodilatation).

However, the availability of collateral circulatory conduits will mitigate the degree of

compensatory dilation of the brain blood vessels. This is illustrated in a study in which

patients with a complete carotid artery occlusion where found to have no evidence of

intracranial hemodynamic compromise due the existence of collateral circulation which

compensated for reductions in rCBF (Vernieri et al. 2001a).

A measure of the cerebral vascular vasodilatory reserve capacity therefore represents not

only a functional assessment of the degree of lumen narrowing of a major feeding vessel, but

also of the extent of collateral vascular supply and overall, the ability to augment CBF.

1.4 CEREBROVASCULAR REACTIVITY (CVR)

While structural angiography imaging methods may show anatomical continuity of vessels,

and the presence of recruited collateral conduits, they do not provide a measure of the actual

perfusion contribution of such vessels (van Everdingen et al. 1998) to preserve rCPP and

maintain CBF (Hofmeijer et al. 2002). Cerebro-vascular reactivity (CVR) - broadly defined

12

as the change in cerebral blood flow (CBF) per unit change in vasoactive stimulus - on the

other hand, is a method that it is capable of measuring the physiological impact of occlusive

lesions on reducing rCPP indicating the net effect of both the reserve of vascular dilatation

and recruitment of collateral blood flow (Allcock 1967) (Matteis et al. 1999;Silvestrini et al.

2000;Cupini et al. 2001).

CVR has been shown to be a prognostic indicator of future ischemic events. Vernieri et al

(Vernieri et al. 2001b) determined that in the presence of severe carotid artery disease, an

impaired CVR was associated with an increased probability of stroke of 32.7%/yr compared

to 8%/yr if CVR was normal. Similarly, Kleiser et al. (Kleiser and Widder 1992) measured

the CVR in 85 patients with internal carotid artery occlusions using TCD as an indicator of

CBF. In follow-up studies over 38 ± 15 months they found that in the group with greater

CVR, none developed a stroke, whereas in the group with diminished CVR, 32% suffered

ipsilateral events consisting of TIA‟s and strokes.

These observations therefore underscore the significance of studying and quantifying the

vasodilatory capacity through CVR measures as a means to evaluate the state of the cerebral

vasculature and/or vascular disease progression.

1.4.1 QUANTIFICATION TESTING OF CVR

With the application of a vasodilatory stimulus, CVR can be used to assess the capacity to

cerebral autoregulate or, alternatively, the cerebral vascular vasodilatory reserve capacity.

The quantification normative absolute CVR values in healthy population therefore represent

a means to determine the deviations from normal values in the presence of pathology.

Though many methods have been devised to assess CVR, the most widely used method is the

application of Carbon dioxide (CO2) as dilator stimulus, and an imaging method to measure

the resultant changes in CBF.

13

1.4.2 CVR AND CARBON DIOXIDE (CO2)

While the cerebral vasoautoregulation responds to changes in rCPP, the cerebral vasculature

is also very sensitive to changes in its chemo-environment, specifically, to changes in the

arterial partial pressure of CO2 (PaCO2). The time course of response is on the order of

seconds. Increases in PaCO2 elicits a vasodilatory response and decreases in PaCO2 elicits a

vasoconstrictive response.

With its potent effect on vascular system and the combined safety and ease of use, CO2 is the

most commonly used vasodilatory stimulus in the study of CVR.

In this thesis I will use PaCO2 when referring to the direct stimulus of vascular change (i.e.,

the direct independent variable), and PETCO2 when referring to the measured parameter that

reflects PaCO2 (Robbins et al. 1990d).

1.4.3 MECHANISM OF CARBON DIOXIDE MEDIATED VASCULAR RESPONSE

The physiological mechanism by which dissolved arterial CO2 elicits vasoreactive effects is

not fully understood. It has been hypothesized that CO2 or a CO2–mediated change in the

extracellular pH, or both, induces the change in the cerebral vascular tone, with the site of

action appearing to be directly on the vessel wall. This has been demonstrated by the

application of either an acidic or alkalotic solution onto the brain which has been shown to

dilate or constrict cortical surface cerebral arteries in vivo (Wahl et al. 1970). Once the pH is

altered, a series of second messengers systems (prostinoids, nitric oxide, cyclic nucleotides,

potassium channels, and intracellular calcium) are recruited to exert its effects on the smooth

muscle in the cerebral vessels.

The three principally vasoactive prostinoids in the brain are 1) Prostaglandin E2, 2)

Prostacyclin (PGI2) – which are both vasodilator prostinoids and 3) Prostaglandin F2 alpha –

which is a vasoconstrictor prostinoid (Hsu et al. 1993). Nitric Oxide (NO) is also an

important regulator of cerebral vascular tone and consequently CBF. In the brain NO is

produced by NO-synthase enzymes in the cerebral vascular endothelial cells, some

perivascular nerves, parenchymal neurons and glia (Faraci and Brian, Jr. 1994b).

14

NO exerts its effects by activating guanylate cyclase in the vascular smooth muscle resulting

in an increase in the intracellular concentration of cyclic guanosine monophosphate (cGMP)

causing vasodilatation (Faraci and Brian, Jr. 1994a). There is however conflicting data with

regards to the overall effect of NO as a mediator on CO2-induced cerebral vasodilatation.

Studies have shown that the inhibition of NO-synthase reduces the magnitude of cerebral

vasodilation due to hypercapnia (Wang et al. 1992b;Iadecola and Zhang 1994a) however the

response is not completely abolished as between 10 - 70% of vasodilatory response

remains(Wang et al. 1992a;Iadecola and Zhang 1994b). Moreover, NO does not appear to

have an effect on mediating hypocapnia cerebral vasoconstriction as the inhibition of NO-

synthase does not alter cerebral vasoconstriction (Wang et al. 1992c). Thus, while NO may

have a role in CO2-mediated vasodilatation, the role is not exclusive.

Cyclic nucleotides are important secondary messengers in the CO2 mediated changes in

vascular tone. NO activates guanylate cyclase in the vascular smooth muscle, resulting in an

increase in cyclic GMP concentration and prostanoids activate adenylate cyclase and increase

the cyclic AMP concentration(Parfenova et al. 1994). Both cyclic GMP and cyclic AMP

then activate their complimentary protein kinases which phosphorylates calcium channels,

reducing the entry of calcium into vascular smooth muscle (Sperelakis et al. 1994).

The cyclic nucleotides also known to activate a subset of potassium channels which result in

membrane hyperpolarization and inactivation of voltage gated calcium channels which also

reduces the intracellular calcium concentration (Kitazono et al. 1995). The resultant decrease

intracellular calcium is the mechanism by which arterial tone is decreased.

Another subset of Potassium channels also appear to be acted on directly by decreasing

extracellular pH. The decrease in pH is thought to increase the open-state probability of

KATP channels which would hyperpolarize cells and cause vascular smooth muscle

hyperpolarization and cerebral vasodilation (Davies 1990).

15

This is supported by in vitro evidence which demonstrates that vasodilatation caused by

hypercapnia (PaCO2 ~ 55mmHg) – which induces extracellular acidosis - can be attenuated

by the blockade of KATP channels (Kontos and Wei 1996).

In summary, the exact mechanism by which CO2 affects vascular tone is still not well

understood. The mechanism appears not be mediated by one pathway but rather through the

interaction of multiple pathways. Moreover the mechanism that appears to function in adults

also appears to be different in neonates (Rosenberg et al. 1982) thus further research is

required to elucidate the exact underlying mechanisms of CO2 mediated changes in vascular

tone.

1.5 ADMINISTRATION AND MANIPULATION OF CO2 STIMULI

Changes in PaCO2 are potent vasoactive stimuli, and given the ease and safety of use, are

commonly employed in CVR studies. Increases in PaCO2 are easily reversed with

hyperventilation and changes in CBF are predictable at about 3% per mmHg change in

PaCO2 (Ringelstein et al. 1988). As the measure of PaCO2 is invasive, requiring an arterial

blood sample, the partial pressure of end-tidal CO2 (PETCO2) is most frequently used as a

suitable surrogate (Robbins et al. 1990c). In this section the various methods of manipulating

PaCO2 are described along with the advantages and disadvantages of each method.

1.5.1 ACETOZOLAMIDE (DIAMOX)

Acetozolamide (ACZ) has been administered to induce increases in PaCO2 in CVR studies.

ACZ is a competitive inhibitor of the carbonic anhydrase. Carbonic anhydrase is a zinc-

containing enzyme that catalyzes the following reversible reaction.

CO2 + H2O ↔ HCO3-

+ H+

In the presence of ACZ the carbonic anhydrase is inhibited from catalyzing the

aforementioned reaction resulting in an increase in PaCO2 (Leaf and Goldfarb 2007).

16

Advantages

administration does not alter the systemic blood pressure making it a good surrogate

for measuring CVR in the presence of hypotension

Safe

Disadvantages

must be injected intravenously rendering its use somewhat invasive.

The time course of response to oral administration is highly variable

PaCO2 changes in response to changes in ventilation are superimposed on those of

ACZ

The mechanism of affect of ACZ does not allow a quantifiable measure of change in

CO2 to be made thereby making each application an independent stimulus that is non

standardized.

1.5.2 BREATH HOLDING

Breath holding is another method of inducing changes in PaCO2. This method works by

eliminating the flux of both O2 and CO2 at the lung allowing for the alveolar PO2 and PCO2

to equilibrate with those in the mixed venous blood. Once the alveolar and mixed venous

PO2 and PCO2 are equilibrated then any changes that occur will do so only according the

metabolic production of CO2 (Parkes 2006a).

Advantages

No external gas sources are needed

Relatively safe

Disadvantage

Cannot measure PETCO2 and PETO2

The rates of change in PaO2 and PaCO2 are relatively slow and vary from

subject to subject based on and as well as the circulation time required for

blood to travel from the lungs to the tissues and then back to the lungs.

The CO2 capacitances of the body are very large relative to metabolic

changes, resulting in a buffering of end tidal partial pressure changes relative

17

to content changes and thereby limiting the change in PaCO2 from those at

steady state.

The PaCO2 and PaO2 change continuously in opposite direction.

The changes in PaCO2 and PO2 are not linear therefore very sensitive to time

of breath-hold

The length of the stimulus is limited by the subject‟s ability to hold his breath

1.5.3 VARYING MINUTE VENTILATION

Variations in minute ventilation will result in corresponding changes in alveolar ventilation,

which leads to changes in the alveolar (and arterial) PO2 and PCO2. Hyperventilation results

in increase in PaO2 and decrease in PaCO2, whereas hypoventilation has the opposite effect.

Advantages

No external gas sources required

The rates of change in PaO2 and PaCO2 with hyperventilation are more rapid

than with breath holding (or hypoventilation)

Relatively safe and simple to perform

Disadvantages

Changes in PaO2 and PaCO2 are inversely linked

The magnitude of change in PaO2 and PaCO2 is limited by maximal voluntary

increase and decrease in ventilation

Voluntary efforts are resisted by ventilation control mechanisms

1.5.4 INSPIRED CONCENTRATIONS OF O2 AND CO2 - NON-REBREATHING

In this method the subject, breathes via a non-rebreathing valve, inhales from a reservoir

containing a gas composed of a mixture of O2, CO2 and N2. The composition of the mixture

can be adjusted prior to the start of experiment or corrected after every breath (as in dynamic

end-tidal forcing)(Robbins et al. 1982).

18

Advantages

The precise concentration of the inspired gas is known

Inspired PO2 (PIO2) and PCO2 (PICO2) can be varied independently

Inspiring gas of known composition will result in a particular PaO2 and PaCO2

based on the metabolic parameters and alveolar ventilation in a given subject.

The composition of the inspired gas can therefore be varied to yield required

arterial concentrations of O2 and CO2.

Disadvantages

PaCO2 and PaO2 are not a direct function of the PICO2 and PIO2 abut also of

the minute ventilation. As the minute ventilation in response to a given PICO2

can vary from person to person, so will the PaCO2 and PaO2 .

Changes in arterial gases (low PaO2 and/or high PaCO2) stimulate peripheral

and central chemo-receptors, altering ventilation

The requirement of mixing of pure O2, CO2 and N2 exposes the subject to the

risk of inhaling a hypoxic mixture resulting from the inadvertent mixing of

excess/or pure CO2 or N2.

Complex calculations are required to account for individual, resting PETCO2

and PETO2

Breath-by-breath variability in tidal volume, and hence alveolar ventilation,

results in variation in PETCO2 and PETO2

1.5.5 INSPIRED CONCENTRATIONS OF O2 AND CO2 - REBREATHING

In this method the subject re-breathes from a bag primed with a concentration of CO2 and O2

forming a semi-closed system where the PaCO2 rises progressively as a result of the addition

of metabolically produced CO2 into the system. The PaO2 is kept constant by an infusion of

O2 from an external source equal to the O2 consumption.

Advantages

This method allows studies of physiological responses to a steadily increasing PaCO2

with simultaneous control of PaO2 levels.

19

Breath-by-breath changes in tidal volume have little effect on the observed PETCO2,

Disadvantage

This method results only in a slow steady increase in PETCO2 with or without a

steady level of PETO2

1.5.6 INSPIRED CONCENTRATIONS OF O2 AND CO2 – MODIFIED PROSPECTIVE END

TIDAL TARGETING (MPET) BREATHING CIRCUIT

This novel method of manipulating PaCO2 and PaO2 independently and with fine control is

the only method to have PETCO2 values correlated with PaCO2 (Ito et al. 2008). Based on the

use of sequential re-breathing circuits, the exact targeting and control of PaCO2 and PaO2 is

achieved by delivering a volume of fresh gas into alveoli on each breath(Slessarev et al.

2007d). The fresh gas is a composition of the following three gases 1) 100% oxygen, 2) 10%

Oxygen, balance Nitrogen (90%) and 3) 10% Oxygen, 20% Carbon Dioxide, balance

Nitrogen (70%) – the mixture of which is determined by taking into account the subjects VO2

and VCO2 and the target of PaCO2 and PaO2 that is to be achieved (Slessarev et al. 2007e).

All this performed through an automated gas delivery system.

Advantages

Only method whereby PETCO2 has been shown to be equal to the independent

variable, PaCO2

Independent control of PETCO2 and PETO2 and thereby PaCO2 and PaO2 .

Control of PETCO2 and PETO2 is independent of subject‟s respiratory rate or

breathing pattern

Delivery of a standardized and repeatable stimulus

Requires minimal cooperation

Disadvantages

The use of a face mask which may prohibit use in subjects with claustrophobia or a

beard

Requires sufficient cooperation to provide a minimal minute ventilation

20

Requires custom equipment consisting of an automated gas blender, sources of O2,

air, and two specialty mixed gases, computer control, special program for blender

control and gas analysis, gas analyzers, along with all magnetic limitations and

precautions, in order to administer method.

1.6 IMAGING OF CEREBROVASCULAR REACTIVITY

Previous studies of CVR have incorporated various methods to image changes in blood flow.

However each method differs in a number of ways such as duration of quantitative accuracy,

data acquisition, brain coverage and spatial resolution, all of which can confer both

advantages and disadvantages in measuring CVR. This section will provide a brief overview

of each commonly used imaging method.

1.6.1 TRANS – CRANIAL DOPPLER (TCD)

Trans – Cranial Doppler ultrasonography allows the measure of CVR through the measure of

blood flow velocities (Aaslid et al. 1982). The method is based on the Doppler affect which

detects the change in frequency of sound waves reflected from moving objects. In this

particular instance the moving objects are the red blood cells which are flowing in the blood

vessels being insonated. Its main use is in measuring flow velocities of various large basal

vessels (depending on the placement of the probe) which are considered not to change

diameter with changes in CO2. Thus TCD has poor resolution with respect to regional

impairments in the smaller cerebral arteriole beds.

Advantages

The Gold standard for measuring CVR

Non – invasive

Ease of use – bedside

High temporal resolution

Repeatable

21

Disadvantages

Limited in spatial resolution provides one value for each hemisphere; focal

impairments secondary to downstream branch vessel pathology may be

undetecable

Accuracy issues inherent to errors and dependence on angle, flow velocity,

pulsatility on vessel diameter operator dependant

Subjects must have a “temporal bone window” (5- 10% of population do not)

Velocity changes in large arteries are but surrogates of changes in blood flow

in the downstream cerebral arterioles.

1.6.2 XENON – 133 WASH OUT

First described by Glass and Harper in 1963, this method derives CBF measurements from

inert gas clearance methods based on the FICK principle using a depth focusing collimator

(HARPER et al. 1964).

In this method xenon133

is injected into the carotid artery until a sufficient time has elapsed so

that the brain tissue and venous blood concentration of Xe133

is in equilibrium. Once

equilibrium is reached the infusion stops, and the arterial blood which is now free of

radioactive Xe133

, will begin to wash out the Xe133

in brain. The rate at which the washout

occurs depends on the rate of CBF. Using the collimator, an area of the cerebral cortex is

focused on the level of radioactivity (reduction in) is detected and CBF is determined.

This method is therefore purely dependant on the physical properties of diffusion and

solubility.

Advantages

Body eliminates XE 133 rapidly ~ 20 – 30 mins this allows repeated studies

Disadvantages

Highly invasive

Lacks anatomical correlation – negates comparison studies

22

Xe-133 produces a weak signal, it therefore provides little information on blood flow

in deep regions of the brain

Xe-133 has a long half life and is a radioactive hazard for both subjects and

laboratory personnel

Brain tissue is not entirely homogeneous thus the injection time must be long enough

for the brain to ensure that the Xe133 concentration is equilibrated throughout

1.6.3 POSITRON EMISSION TOMOGRAPHY (PET)

Positron Emission Tomography (PET) is scanning method that relies on the use of unstable

positron emitting isotopes such as 15O, 18F, and 11C that are synthesized by a cyclotron

(Ter-Pogossian et al. 1975;Phelps et al. 1978). The isotopes are created by bombarding the

oxygen, fluorine or carbon with protons. These isotopes once created can then be

incorporated into many reagents compounds such as water and glucose. The radio-labeled

compounds can then be injected in the circulatory system where they are then distributed and

taken up into areas that are physiologically more active. The unstable isotope within the

compound then underogoes decay as the extra proton is broken down into a neutron and a

positron. The positron then proceeds to collide with an electron (usually within a few

millimeters) resulting in a release of gamma rays in opposite directions (180 degrees). The

gamma rays are then collected by detectors positioned around the head. By reconstructing

the site of collision with the election active regions or uptake are reconstructed providing a

three dimensional map.

Advantages

Whole brain coverage

Non invasive tomographic images with quantitative parameters regional CBF,

regional CBV, region OEF

Disadvantages

Intravenous injection of Tracers combined with arterial blood sampling

Need a cyclotron PET for CBF measurements

Not available at the bedside

23

Spatial resolution of PET studies ranges from ~ 4 to 6 mm

1.6.4 SINGLE PHOTON EMISSION COMPUTED TOMOGRAPHY (SPECT)

Single Photon Emission Computed Tomography (SPECT) is a non invasive method of

measuring regional CBF, derived from older techniques of imaging CBF (Budinger et al.

1977). This technique is based on the use a radio-labeled compound with a short half life

such XE133

that is either inhaled or injected into the circulatory system. Once in the

circulatory system the radio-labeled compound binds to red blood cells which are carried

throughout the body while undergoing radioactive decay emitting protons. The emitted

photons are then collected by a gamma camera that is rotated around head ultimately

generating a localized three dimensional image of CBF.

Advantages

Lower start up costs

More widely available compared to PET and MRI

Simpler to use; Radiolabeled probes are commonly available and do not

require an onsite cyclotron such as required by PET

Disadvantages

Not as flexible as PET or as accurate

Limitation in spatial resolution ~ 4 - 6 mm

1.6.5 NEAR INFRARED SPECTROSCOPY

Near Infrared Spectroscopy (NIRS) is a non-invasive method for studying changes in CBF.

The method is dependent upon the near infrared light absorption properties of both

oxyhemoglobin and deoxyhemoglobin (Pellicer and Bravo 2010). By transmitting light into

the brain and analyzing what is reflected by sensors, changes in both deoxyhemoglobin and

oxyhemoglobin as well as the change in total hemoglobin can be measured providing both

hemodynamic and metabolic CBF measures.

Advantages

High temporal resolution up to hundreds of hertz

24

Disadvantages

Low spatial resolution

Depth - measurement in deep structures of the brain cannot be obtained due

to limitations in optical penetrance

1.6.6 MAGNETIC RESONANCE IMAGING (MRI) – BLOOD OXYGEN LEVEL DEPENDANT

MRI

Blood Oxygen Level Dependant Magnetic Resonance Imaging (BOLD MRI) is a method of

indirectly measuring changes in CBF in the microvasculature, using deoxy-hemoglobin as an

endogenous contrast agent (Ogawa et al. 1990b). Changes in CBF are inferred from flow

induced changes in the absolute paramagnetic deoxy-hemoglobin concentration, which alters

the local magnetic field in giving rise to the BOLD MRI signal. An increased concentration

of paramagnetic deoxy-hemoglobin, such as that arising from a reduction in CBF in the

setting of stable neuronal activity, increases the magnetic field inhomogeneity which distorts

the magnetic field resulting in decreased T2* relaxation decreasing the measured BOLD

signal in an imaging voxel. Conversely an increase in CBF in the setting of stable neuronal

activity, reduces the concentration of deoxy-hemoglobin, resulting in less local magnetic

field inhomgeneity and an increase in the BOLD signal.

However, other factors such as the intra-voxel cerebral blood volume (CBV), cerebral

metabolic rate of oxygen consumption, arterial partial pressure of oxygen (PaO2), and

hematocrit can also influence the magnitude change in BOLD signal(Ogawa et al. 1993).

That said, empirical evidence still suggests that, within healthy subjects, changes in BOLD

signal to changes in PaCO2 are dominated by CBF effects (Shiino et al. 2003;Ziyeh et al.

2005)

Advantages

Good spatial resolution – down to 3 mm

Whole brain coverage

Non – invasive – no radiation

Repeatable

25

Lower cost than PET

Disadvantages

Availability

Claustrophobia, noise

Intolerant of subject movement

The BOLD signal drifts with time

2. CHAPTER 2

RATIONALE AND OBJECTIVES

2.1 RATIONALE AND OBJECTIVES

The varying methodology in measuring CVR – both in the manipulation of PaCO2 and CBF

imaging - has made CVR an unreliable, unrepeatable, and un-standardized measure – all of

26

which need to be addressed for any type of diagnostic technique to be considered and

adopted for use in a clinical setting.

To date, the normal physiological response of the cerebral vasculature response to changes in

physiological range of PaCO2 has yet to be characterized in humans with a standardized

method – due to limitations in PaCO2 manipulation methods - which would allow the

accurate representation of the relationship between changes in PaCO2 and CBF.

The most common error is assuming that the inspired fractional concentration of CO2

(FICO2) is the independent stimulus. As discussed above, the independent stimulus is the

PaCO2. The PaCO2 is a function of the minute ventilation response to the FICO2. So, for

example, when a 5% CO2 –air mixture is administered to two theoretical subjects the

resulting ventilatory response may differ. One may have a brisk ventilatory response which

increases PETCO2 by 8 mmHg and the other may have a lesser ventilatory response with an

increase in PETCO2 of 3 mmHg. If the denominator in the calculation of both is the same

(i.e., when the stimulus is taken as the independent variable), the CVR calculation would be

quite different which is the approach of most published CVR studies.

Additionally, CVR is often calculated as a „slope‟ in the sense of „rise over run‟, which

falsely assumes a linear relationship over a measured range of PaCO2 which has been shown

to be curvilinear (Ide et al. 2003e).

Thus a change in CBF in response to a change in PaCO2 would depend on both the

magnitude of the change PaCO2 as well as the starting point. At extreme levels of

hypocapnia (~ less than 20 mmHg) and hypercapnia (~ greater 100 mmHg) the vascular

response appears to plateau and level off (REIVICH 1964c). If the PaCO2 falls outside of this

range during measurement, there would be an underestimation of the slope, or CVR.

Between these two levels the reported relationship of CBF to PaCO2 has varied. Some have

reported a linear relationship (REIVICH 1964b), whereas others have not (Clark et al.

1996b;Ide et al. 2003g). However, the majority of studies of CVR to PaCO2 – both in

27

healthy and diseased states - have confined themselves to study CVR in the hypercapnic

range (Clark et al. 1996a;Yezhuvath et al. 2009c) or have disregarded the effect of range of

PaCO2 on the CVR (Mikulis et al. 2005).

Many studies have also ignored the independent effect of PaO2. An increasing PaO2 has

been determined to have an independent vasoconstrictive affect on the cerebral vasculature

(Floyd et al. 2003b) and on BOLD signal (Prisman et al. 2008c). Methods commonly used to

manipulate PaCO2 in the study of CVR – with the exception of dynamic end tidal forcing

(Wise et al. 2007b) - do not allow the independent control of O2, which otherwise varies.

Thus the true independent affect of PaCO2 or PaO2 on CVR still remains to be resolved.

The use of CVR imaging methods has also varied, resulting in measures of CVR without a

spatially structured approach.

The reported spatial distribution of CVR has differed according to the imaging modality:

transcranial ultrasonography (TCD) (Aaslid et al. 1989), Positron emission tomography

(PET) (Ito et al. 2000d), Perfusion Computed Tomography (Liu et al. 2008) and blood

oxygen level dependant BOLD MRI (Ogawa et al. 1990a).

The majority of studies have reported global CVR using TCD. However, the resolution of

TCD is low, as it only provides response information from large intracranial vessels. The

vascular response to PaCO2 is not limited to large intracranial vessels and changes do occur

at the mircrocirculatory level. The use of TCD may therefore mask regional level changes.

Alternatively, imaging methods with higher spatial resolutions have been used to measure

vascular responses to PaCO2. Grey and WM vascular responses have been reported

independently of each other using SPECT (Shirahata et al. 1985b), Xenon133

(Reich and

Rusinek 1989a) and PET (Ito et al. 2000c) with GM vascular reactivity demonstrated to be

higher in some studies (Shirahata et al. 1985a;Reich and Rusinek 1989b). However, the

spatial resolutions of these methods are also limited in terms of brain volume coverage,

consisting of a few transverse imaging planes of limited thickness.

28

Within the GM and WM, it is not known if the vascular response to changes in PaCO2 varies

from region to region in the brain, or is uniform according to arterial anatomy. However

differences in CVR between specific neural structures have been reported (Ito et al. 2000b)

(Bright et al. 2009d).

The use of BOLD MR imaging to indirectly image changes in CBF potentially provides good

spatial resolution (voxels 2 x 2 x 3 mm at 3 Tesla) with full brain coverage. However BOLD

CVR studies to date been limited to reports of CVR in brain regions associated with specific

visual or motor functions (Rostrup et al. 2000c;Bright et al. 2009c) without regard to the

cerebral arterial anatomy.

Previously, it has been suggested that variations in cerebral arterial anatomy may influence

the vascular response to changes in PaCO2 (Mandell et al. 2008a). With variations in cerebral

vascular anatomy known to be present within regions of grey and WM (Reina-De La et al.

1998b;Nonaka et al. 2003a;Nonaka et al. 2003d) the vascular response to PaCO2 may thus be

heterogeneous throughout the brain.

Collectively, various questions therefore still remain, in the study of CVR to changes in

PaCO2. The objective of this thesis was to therefore to develop a standardized test of CVR

that will address the following 2 key questions:

1) Is there a difference in the magnitude of CVR to a standardized normoxic 10 mmHg

change in PaCO2 if tested above, (hypercapnic), or below (hypocapnia) resting

PaCO2?

2) Is there a difference in CVR regionally according to vascular territory throughout the

brain?

29

2.2 OPTIMIZING THE VASOACTIVE STIMULI

MPET was used to administer two repeatable standardized stimuli – the first, an iso-oxic

(PETO2 150 mmHg) hypercapnic stimulus in which PaCO2 was cycled in the hypercapnic

range (from 40 mmHg to 50 mmHg) and the second an iso-oxic hypocapnic stimulus

consisting of cycling PaCO2 in the hypocapnic range from 30 mmHg to 40 mmHg. As there

is hysteresis with respect to the direction of change (Ide et al. 2003f), both changes were

those of increasing PaCO2.

2.2.1 ILLUSTRATION OF CBF RESPONSE CHANGES IN PCO2

For the purpose of illustration, we reproduced a CBF-PaCO2 curve as reported by Ide et al.

(Ide et al. 2003). We monitored the change in MCAV in response to 5 mmHg incremental

step changes in PETCO2 from 25 mmHg to 55 mmHg while maintaining a PETO2 of 150

mmHg. The graph below demonstrates a curvilinear relationship resulting in two distinct

CVR slopes if two point changes are made in the hypo- and hypercapnic regions (25 mmHg

to 40 mmHg and 40 mmHg to 50 mmHg) (Figure 2-1).

Figure 2-1. MCAV vs PETCO2. Red points demonstrate the change in MCAV in the

hypocapnia range of PETCO2 (from 25 mmHg to 40 mmHg). Blue points demonstrate the

change in MCAV in the hypercapnic range of PETCO2 (from 40 mmHg to 50 mmHg). A

constant PETO2 (150 mmHg) was maintained throughout the entire study.

30

2.3 OPTIMIZING BOLD MR IMAGING

BOLD MR imaging is an indirect method of detecting changes in CBF. Echo planar imaging

is one of the fastest methods of measuring changes in T2*, the relaxation time constant that is

responsible for the BOLD contrast. T2* along with T2 and T1 represent time constants of the

spin relaxation which are properties of the tissue being imaged. When tissue is placed in a

static magnetic field, protons in the tissue will align parallel to the magnetic field resulting in

a net longitudinal magnetization. The application of a perpendicular (90°) radiofrequency

(RF) pulse through an imaging slice of the tissue will tilt the protons away from the parallel

alignment into a transverse plane, while precessesing around the direction of the net

magnetization. As the protons slowly realign parallel to the magnetic field, energy is released

and detected by a receiving coil which provides the spatially encoded information. The

imaging contrast seen between different tissues is generated by the different rates in which

protons in different tissues return to their equilibrium state. T2* reflects the tissue

microenvironment or the relative inhomogeniety of the local magnetic field (susceptibility).

T2* is highly influenced by the interface of tissues of different magnetic characteristics (such

as at the interface of bone and soft tissue) or in the case of the BOLD contrast, the

31

concentration of paramagnetic deoxyhemoglobin which disrupts the local magnetic field in

an imaging voxel and results in a decrease in BOLD signal.

Specific MR imaging parameters can be optimized to obtain the best signal to noise ratio

(SNR) in generating BOLD signals. While there is probably no single combination of

acquisition parameters that will be optimal for every BOLD MR imaging study, the

understanding of how each of following parameters can affect the BOLD signal will allow

the optimization of data acquisition.

2.3.1 STATIC MAGNETIC FIELD STRENGTH

As the static magnetic field strength is doubled the measurable signal increases 4 fold.

However, the noise also increases 2 fold resulting in a 2 fold increase in SNR(Okada et al.

2005).

2.3.2 REPETITION TIME (TR)

The TR is the time in milliseconds between successive RF pulses applied to the same

imaging slice. Longer TR theoretically would allow for a proton to fully recover to the

longitudinal plane of magnetization after an RF pulse allowing for a higher SNR. However,

in a given fixed imaging time, a longer TR limits the number of images that are acquired for

a particular imaging volume. (Constable and Spencer 2001)

2.3.3 ECHO TIME (TE)

TE represents the time in milliseconds between the application of the 90° pulse and the

peak signal detected. Shorter TE reduces the influence of susceptibility artifacts (field

inhomogenieties) as well as increase the overall SNR (Schmitt 1998). Optimal TE should be

equal to the T2* for grey matter at a given static magnetic field strength (Menon et al.

1995;Kruger et al. 2001).

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2.3.4 VOXEL SIZE AND SLICE THICKNESS

Increases in voxel size (Triantafyllou et al. 2005) and slice thickness(Howseman et al. 1999),

decreases the spatial resolution while increasing the SNR, and vice versa.

Collectively, optimization of some parameter will likely offset others. In determining the

optimal set of parameters for a particular study, it is crucial to weigh, the goals of the study.

In this study the BOLD MR imaging parameters were optimized to study the entire brain as

opposed to fine structures in order to map out the CVR of specific vascular territories in both

GM and WM thus sacrificing spatial resolution for SNR.

During the induction of each PaCO2 stimulus, vascular reactivity was imaged and quantified

using BOLD MRI. In the first study, segmentation algorithms were used to quantify the

BOLD response to both hypercapnia and hypocapnia globally in GM and WM.

In a second study, the CVR was quantified in the cerebral cortex according to arterial

vascular territory (left and right anterior cerebral artery (ACA), middle cerebral artery

(MCA), posterior cerebral artery (PCA)) and the sub-cortical and peri-ventricular regions in

the cerebral WM, regions known to be prone to ischemic injury

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3. CHAPTER 3

QUANTIFICATION OF BRAIN CVR TO CO2

3.1 INTRODUCTION

Cerebro-vascular reactivity (CVR) can be broadly defined as the change in cerebral blood

flow (CBF) per unit change in vasoactive stimulus. CVR is currently a semi quantitative

measure, due to a large extent to the application of inconsistent, indistinct, and often un-

measurable provocative stimuli. The inability to reliably quantify CVR makes it difficult to

discern symmetrical or global vascular abnormalities, and to generate repeatable CVR values

for specific brain regions. This is necessary to follow CVR in a given patient over time, or

compare CVR between groups.

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The easiest vasoactive stimulus to apply is a change in the partial pressure of CO2 in the

arterial blood (PaCO2). As the measure of PaCO2 is invasive, requiring an arterial blood

sample, the partial pressure of end-tidal CO2 (PETCO2) is most frequently used as a suitable

surrogate (Robbins et al. 1990b).

PaCO2 is most commonly affected by a change in ventilation. The simplest change in

ventilation is breath-holding for a standardized time, or the breath-hold index. However the

actual changes in PaCO2 during a fixed breath-hold period vary with the extent of ventilation

before the maneuver, whether the breath-hold is initiated at end inspiration or end exhalation,

the CO2 production of the subject, and more (Parkes 2006c). During the breath-hold, the

PaCO2 changes are non linear with time and are accompanied by dramatic reductions in

arterial partial pressure of oxygen (PaO2) (Parkes 2006b), which in turn affect the CBF

(Floyd et al. 2003a) as well as its measure when MRI Blood Oxygen Level Dependent

(BOLD) signal is used as a surrogate for CBF (Prisman et al. 2008b). These factors leave the

“breath hold index” as a very indirect and unreliable measure of the vaso-active stimulus.

Administering a fixed inspired concentration of CO2 and O2 via a non-rebreathing circuit also

has the appeal of simplicity, but it too is an inconsistent stimulus. Neither the PaCO2 nor the

PaO2 are simple functions of the inspired concentrations of CO2 and O2, as they are also

affected by the ventilatory response to the inhaled gases (Prisman et al. 2008a), which varies

from person to person (and likely in any one person over time). Indeed, the administration of

fixed inspired gas concentrations have been shown to not provide a reliable cerebro-vascular

stimulus (Mark et al. 2010)

Furthermore, even the administration of a reliable change in PETCO2 and PETO2 may still not

provide a suitable standard vaso-active stimulus. The relationship between PaCO2 and CBF

is not linear, but curvilinear (Ide et al. 2003d) whereas CVR has been defined as a „slope‟ of

CBF response to PaCO2. Thus for CVR to be consistent on repeated tests, the initial PETCO2

and the change in PETCO2 must remain consistent.

In this study, our aim was to standardize the provocative vaso-active stimulus with respect to

initial, and change in, PETCO2 as well as the surrogate measure of CBF, and quantify CVR In

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this initial study we performed a series of hypercapnic and hypocapnic „standardized CVR‟

test on 10 healthy male subjects aged 18 to 42 years to quantify CVR for regions of interest

(ROI) consisting of the whole brain, the grey matter (GM) and white matter (WM).

3.2 MATERIALS AND METHODS

3.2.1 ETHICS AND CONSENT

The study was approved by the research ethics board at the University Health Network,

Toronto, Ontario, Canada. Ten healthy male subjects (Age 30 ± 8.1 years, range 18 – 42

year) with no prior cerebral vascular conditions, free of medication and nicotine intake, and

abstaining from caffeine on the day of examination were recruited for this study. The entire

protocol was reviewed with each subject and informed consent was obtained.

3.2.2 MAGNETIC RESONANCE IMAGING

MR imaging was performed on a 3.0 Tesla scanner (Signa HDX; GE HealthCare,

Milwaukee, WI) with eight-channel phased array head coil. For co-registration with the fMRI

BOLD CVR measures, T1-weighted anatomical images were acquired using a three-

dimensional spoiled gradient echo pulse sequence (whole brain coverage; matrix: 256x256;

slice thickness: 2.2 mm; no inter-slice gap). BOLD MRI CVR data was acquired with a T2*-

weighted single-shot gradient echo pulse sequence with echo-planar readout (field of view:

24x24 cm; matrix: 64x64; TR: 2000 ms; TE: 30 ms; flip angle: 85; slice thickness: 5.0 mm;

inter-slice gap: 2.0 mm, number of frames: 254).

3.2.3 CONTROL OF PETO2 AND PETCO2

Subjects breathed via a sequential gas delivery manifold containing a gas reservoir on the

exhalation port to enable sequential rebreathing (Slessarev et al. 2007a). Transparent skin

tape (Tegaderm 3M St. Paul, MN) was used to secure an occlusive mask to the face and to

ensure an air tight fit. A custom built gas blender (RespirActTM

, Thornhill Research Inc.,

Toronto, Canada) used algorithms described by Slessarev et al (Slessarev et al. 2007b) to

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supply specific flows and gas blends of O2, CO2 and N2 to the manifold to attain targeted

normoxic changes in PETCO2.

We applied parallel hypercapnic and hypocapnia sequences (as opposed to mirror-image

sequences) because of the hysteresis inherent in the cerebrovascular response when PETCO2

is being raised and lowered (Ide et al. 2003c).

The hypercapnic protocol (Table 3-1) consisted of the following cyclical changes in PETCO2

- normocapnia (PETCO2 40 mmHg) for 10 s, hypercapnia (PETCO2 50 mmHg) for 45

seconds, normocapnia for 100 seconds, hypercapnia for 180 seconds, and normocapnia 110

seconds, all under normoxia (PETO2 100 mmHg).

The hypocapnic protocol (Table 3-2) consisted of the following cyclical changes in PETCO2 -

hypocapnia (PETCO2 30 mmHg) for 10 seconds, normocapnia (PETCO240 mmHg) for 45

seconds, hypocapnia for 100 seconds, normocapnia for 180 seconds and hypocapnia for 110

seconds, all under normoxia (PETO2 100 mmHg).

PETCO2 and PETO2 were monitored continuously by the RespirActTM

gas analyzers.

Respiratory data was sampled at 20 Hz, digitized and recorded (LabView, National

Instruments Corporation, Austin, TX).

3.2.4 DETERMINATION OF CEREBROVASCULAR REACTIVITY

BOLD MRI and PETCO2 data were imported into the software AFNI 12(Cox 1996d;Cox and

Hyde 1997a). BOLD images and diffusion-weighted images were automatically co-

registered to the T1-weighted anatomical dataset (Saad 2009 NeuroImage) to standardize

images and facilitate analysis of grouped data. PETCO2 data was time-shifted to the point of

maximum correlation with the whole brain average BOLD signal to compensate for temporal

error between end-tidal gas sampling and the BOLD signal time course (Figure 3-1). A line

of best fit of the BOLD time series to that of the PETCO2 was performed voxel by voxel,

using least squares, and the correlation coefficient, r, calculated. For voxels exceeding r of

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0.25, CVR was calculated as the slope of the line of best fit graphing BOLD signal vs.

PETCO2. The CVR value for a voxel was represented by assigning it a color from spectrum

ranging from neutral, through yellow to red according to the strength of positive correlations

and from neutral to dark blue for negative correlations. CVR voxels were overlaid on the

corresponding anatomical scans to generate „CVR maps‟.

3.2.5 GREY AND WM SEGMENTATION

Each of the brain slices were automatically segmented into GM and WM using statistical

parametric mapping software (SPM5, Wellcome Department of Imaging Neuroscience,

Institute of Neurology, University College, London, UK) (Figure 3-3).

Figure 3-1. Anatomical Segmentation Maps. A) Representative segmented GM (red)

anatomical map (B) Representative segmented WM (red) anatomical map.

Global CVR was calculated by averaging all voxels. The co-registered anatomical images

were automatically segmented into GM and WM using statistical parametric mapping

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software (SPM5, Wellcome Department of Imaging Neuroscience, Institute of Neurology,

University College, London, UK) permitting the calculation of CVR for each tissue type.

Voxel-based morphometry (VBM) of SPM was used to compare the local concentrations of

GM and WM between two groups of subjects. High resolution anatomical images of all

subjects in the study was first spatially normalized into the same stereotactic space

(Talairach) (Lancaster et al. 2000a) GM and WM was then segmented based on the intensity

of the image measured in each voxel. Each voxel was then assigned to a tissue class (GM or

WM) based on the prior the measured intensity probabilities derived from intensity maps

previously generated from a large number of subjects (Ashburner and Friston 2000).

3.2.6 STATISTICAL ANALYSIS

3.2.6.1 TESTING FOR DIFFERENCES IN CVR GLOBALLY

(HYPERCAPNIC CVR VS HYPOCAPNIC CVR)

A Paired t-test was used to determine if the global CVR measured at hypercapnia and

hypocapnia were significantly different.

3.2.6.2 TESTING FOR DIFFERENCES IN CVR BETWEEN AND WITHIN GREY AND WHITE

MATTER

The BOLD CVR measure is proportional to tissue blood volume, which is very different

between grey and white matter. Since we were primarily interested in the relative changes in

BOLD CVR as measured by a hypocapnic versus a hypercapnic challenge, we calculated the

normalized proportional change in BOLD CVR in each subject separately in grey and white

matter (Figure 3-2). This proportional change represents a simple measure of non linearity of

the BOLD CVR curve over the range of PaCO2 investigated (30 to 40 to 50 mmhg) that is

unaffected by a change in the scale of the BOLD CVR measure (Figure 3-3). By defining the

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BOLD CVR non linearity in this way, the difference in the degree of nonlinearity between

grey and white matter can be directly compared using a paired t-test.

BOLD CVRHypercapnia – BOLD CVRHypocapnia

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BOLD CVRHypercapnia + BOLD CVRHypocapnia

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