Cerebral blood flow distribution,

collateral function and pulsatility

in healthy and in patients

with symptomatic carotid stenosis A magnetic resonance imaging approach

Laleh Zarrinkoob

Department of Pharmacology and Clinical Neuroscience & Department of Radiation Sciences

Umeå 2019

This work is protected by the Swedish Copyright Legislation (Act 1960:729) Dissertation for PhD ISBN: 978-91-7855-036-4 ISSN: 0346-6612 New Series Number: 2023 Cover design courtesy of Anders Wåhlin and Lars Stenberg, showing the Circle of Willis illustrated by 4D phase-contrast magnetic resonance imaging. Electronic version available at: http://umu.diva-portal.org/ Printed by: CityPrint i Norr AB Umeå, Sweden, 2019

توانا بود هر که دانا بود

Mighty is the one who has knowledge

-Ferdowsi

A tribute to my father

i

Table of Contents

Abstract ....................................................................................................... iii

List of Abbreviations .................................................................................... v

Original papers ........................................................................................... vii

Populärvetenskaplig sammanfattning ......................................................... ix

Introduction .................................................................................................. 1

Background .................................................................................................. 3

A short history of the cerebral arteries ............................................................................3

Normal cerebral blood flow ............................................................................................. 4

Circle of Willis and cerebral collateral circulation ......................................................... 6

Blood pressure and arterial stiffness ............................................................................... 9

Cerebral arterial pulsatility ............................................................................................ 10

Carotid artery stenosis and hemodynamic stroke ........................................................ 11

Cerebral vascular imaging and cerebral blood flow imaging ..................................... 14

Magnetic resonance imaging .......................................................................................... 16

Phase-contrast magnetic resonance imaging ................................................................ 16

Research rationale ...................................................................................... 19

Aims ............................................................................................................ 21

Materials and Methods ............................................................................... 22

Ethical considerations .................................................................................................... 22

Subjects and patients ...................................................................................................... 22

Magnetic Resonance Imaging ........................................................................................ 27

Post-processing and blood flow rate analysis .............................................................. 29

Statistics .......................................................................................................................... 33

Results ........................................................................................................ 34

Normal tCBF and its relative distribution .................................................................... 34

tCBF changes due to age................................................................................................. 34

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tCBF changes due to anatomical variation, carotid artery stenosis and CEA ............ 35

Arterial BFR and its pulsatility changes due to age ..................................................... 36

Arterial BFR changes due to carotid stenosis ............................................................... 38

Collaterals before and after CEA ................................................................................... 40

Discussion .................................................................................................. 42

tCBF and aging ............................................................................................................... 42

Normal cerebral blood flow and lateralized cerebral blood flow ............................... 43

Cerebral collateral function ........................................................................................... 44

Cerebral hemodynamics normalize after CEA ............................................................. 46

PI increase and the dampening of pulsations decrease due to aging .......................... 47

Technical considerations and clinical perspectives ...................................................... 48

Conclusion.................................................................................................. 50

Acknowledgements ...................................................................................... 51

References .................................................................................................. 53

iii

Abstract

Background: For the detection and treatment of early cerebral vascular disease it is of paramount importance to first understand the normal physiology of the cerebral vasculature, and subsequently, to understand how and when pathology can develop from that. This is especially important as the population above 65 years of age is increasing and aging itself is an established risk factor for the development of cerebral vascular disease. This, however, is not always an easy task, since there is a subtle balance and overlap between age-related physiological and pathophysiological changes in the arterial system. Atherosclerotic changes that lead to the development of carotid artery stenosis are responsible for about one fifth of all ischemic strokes. Today, the current state of evidence and the algorithm for carotid revascularization is mainly focused on the degree of carotid stenosis and not on its impact on cerebral hemodynamics. One reason for this is the lack of a non-invasive method, that allows for repeated investigations and provides accurate and reliable results to study cerebral hemodynamic changes. The overall aim of this thesis was to explore and develop a comprehensive approach to investigate the cerebral blood flow distribution, collateral function and pulsatility in healthy subjects and in patients with symptomatic carotid stenosis using a phase-contrast magnetic resonance imaging (PCMRI) platform. The thesis is based on four scientific papers (papers I—IV).

Methods: In papers I and II, 49 healthy young (mean 25 years) and 45 healthy elderly (mean 71 years) subjects were included. 2D PCMRI was used to assess cerebral blood flow rate (BFR), pulsatility index (PI) and dampening factor (DF) in 15 cerebral arteries and in the ophthalmic arteries (OA). Thirty-eight patients (mean 72 years) with symptomatic carotid stenosis were included in paper III. Nineteen of these patients (mean 71 years) underwent carotid endarterectomy (CEA) (paper IV). 4D PCMRI was used for BFR assessment in papers III and IV. BFR, its distribution and collateral routes, was measured in 17 cerebral arteries and in the OA. The BFR on ipsilateral side (with symptomatic stenosis) was compared to the contralateral side (papers III and IV). BFR laterality was defined as contralateral BFR minus ipsilateral BFR in paired arteries and, BFR was compared before and after CEA (paper IV).

Results: On average, in healthy subjects, 72% of the total cerebral blood flow (tCBF) was distributed through the anterior circulation and 28% through the posterior circulation. The distribution was symmetrical and not affected by age, sex, or brain volume (paper I). Aging resulted in lower BFRs, increased pulsatility and reduced dampening capacity in cerebral arteries. Anatomical variations in the circle of Willis resulted in an asymmetrical distribution of blood flow (papers I and II). In patients with carotid stenosis, a lower BFR was found in the internal

iv

carotid artery (ICA) and its branches on the ipsilateral side. The anterior cerebral artery territory was bilaterally, primarily, supplied by the contralateral ICA. In addition to the supply through the ICA, the middle cerebral artery (MCA) territory on the ipsilateral side was secured by collateral supply from the OA and the posterior communicating arteries, seen as retrograde flow in those arteries. Despite these compensations, BFR in ipsilateral side MCA was lower, and this laterality was more pronounced in patients with severe carotid stenosis (≥70%). After CEA, the distribution of BFR going into the cerebral arteries was found to be symmetrically distributed. Total CBF increased postoperatively in patients with collateral recruitment preoperatively (n=9). The BFR laterality in MCA observed prior to CEA, was found only in the group of patients with collateral recruitment preoperatively (paper IV). The degree of stenosis did not differ between the groups with and without collateral recruitment.

Conclusions: This thesis provides a new and comprehensive approach to mapping and quantifying normal cerebral blood flow and pulsatility. By presenting the distribution of tCBF going into cerebral arteries, instead of using absolute values, the effect of age could be neutralized and the results can be applicable when describing healthy cerebral blood flow, regardless of age. 4D PCMRI made it possible to describe the altered blood flow distribution and collateral ranking in patients with carotid stenosis prior to CEA and its normalization after the procedure. Our findings highlight the importance of BFR quantification for understanding cerebral hemodynamics in patients with carotid stenosis. 4D PCMRI technique is a promising clinical tool for investigations of cerebral hemodynamics in patients with stroke.

v

List of Abbreviations

2D PCMRI Two-dimensional phase-contrast magnetic resonance imaging

4D PCMRI Four-dimensional phase-contrast magnetic resonance imaging

ACA Anterior cerebral artery

ACA1 A1 segment of anterior cerebral artery

ACA2 A2 segment of anterior cerebral artery

ACoA Anterior communicating artery

ASL Arterial spin labeling

BA Basilar artery

BFR Blood flow rate

BMI Body mass index

BP Blood pressure

CAS Carotid artery stenting

CBF Cerebral blood flow

CBV Cerebral blood volume

CEA Carotid endarterectomy

CNS Central nervous system

CPP Cerebral perfusion pressure

CS Carotid stenosis

CT Computed tomography

CTA Computed tomography angiography

CVR Cerebral vascular resistance

CW Circle of Willis

DF Dampening factor

DSA Digital subtraction angiography

DSC Dynamic susceptibility contrast

EC-IC Extracranial–intracranial

ECG Echocardiography

ECST European Carotid Surgery Trial

HE Healthy elderly

HR Heart rate

HY Healthy young

vi

ICA Internal carotid artery

MAP Mean arterial pressure

MCA Middle cerebral artery

MMSE Mini mental state exam

MRA Magnetic resonance angiography

MRI Magnetic resonance imaging

mRS Modified Rankin Scale

NASCET North American Symptomatic Carotid Endartecerctomy Trial

NIHSS National Institutes of Health Stroke Scale

OA Ophthalmic artery

PC VIPR Phase-contrast vastly undersampled isotropic projection

reconstruction

PCA Posterior cerebral artery

PCA1 P1 segment of posterior cerebral artery

PCA2 P2 segment of posterior cerebral artery

PCMRI Phase-contrast magnetic resonance imaging

PCoA Posterior communicating artery

PCT Perfusion computed tomography

PET Positron emission tomography

PI Pulsatility index

PP Pulse pressure

ROI Region of interest

SD Standard deviation

SPECT Single-photon emission computed tomography

tCBF Total cerebral blood flow

TIA Transient ischemic attack

TOF Time of flight

TTP Time to peak

VA Vertebral artery

VENC Velocity encoding

Xe-CT Xenon-enhanced computed tomography

vii

Original papers

This thesis is based on the following papers, which are referred to by Roman numerals in the text.

I. Zarrinkoob L, Ambarki K, Wåhlin A, Birgander R, Eklund A, Malm J. Blood flow distribution in cerebral arteries. Journal of Cerebral Blood Flow & Metabolism 35: 648–654, 2015. *

II. Zarrinkoob L, Ambarki K, Wåhlin A, Carlberg B, Birgander R, Eklund A, Malm J. Aging alters the dampening of pulsatile blood flow in cerebral arteries. Journal of Cerebral Blood Flow & Metabolism 36(9): 1519–1527, 2016. *

III. Zarrinkoob L, Wåhlin A, Ambarki K, Birgander R, Eklund A, Malm J.

Blood flow lateralization and collateral compensatory mechanisms in patients with carotid artery stenosis. Stroke, in press. Epub ahead of print. DOI: 10.1161/STROKEAHA.119.024757, 2019 *

IV. Zarrinkoob L, Wåhlin A, Ambarki K, Eklund A, Malm J. Quantification

and mapping of cerebral hemodynamics before and after carotid endarterectomy. A 4D PCMRI study. In manuscript, 2019

*Reprinted with permission

viii

ix

Populärvetenskaplig sammanfattning

Bakgrund

Världens befolkning blir allt äldre och hög ålder är en etablerad riskfaktor för att utveckla stroke. Stroke är ett globalt hälsoproblem och är den tredje vanligaste orsaken till död i världen. I Sverige nyinsjuknar cirka 25.000 personer av stroke varje år och sjukdomen leder till fler vårddygn jämfört med de flesta andra sjukdomar. Varje minut får hjärnan cirka 600–800 milliliter blod, så kallad total cerebralt blodflöde (tCBF). Blodet når hjärnan via fyra pulsådror, två främre blodkärl (karotisartärerna) och två bakre blodkärl (vertebralisartärerna). Blodflödet till hjärnan minskar lite grann med stigande ålder och gränsen mellan vad som är normal och sjuklig minskning av blod till hjärnan är oklar. Vidare, så är fördelningen av tCBF i hjärnans blodkärl inte välstuderad, särskilt inte i de mest yttersta grenarna av kärlträdet.

Med stigande ålder så blir pulsådrorna styvare och förmågan att dämpa hjärtats pulsationer sämre. Detta kan leda till att hjärnan mottar ett ökat pulsatilt blodflöde som kan skada dess småkärl. Hos flera åldersrelaterade neurologiska sjukdomar har det föreslagits att det finns ett samband mellan tillståndet och pulsatilt flöde som skadar hjärnans småkärl. Hit hör sjukdomar som småkärlsinfarkt, Alzheimers sjukdom och normaltryckshydrocefalus.

För att kunna upptäcka och behandla vaskulär sjukdom i hjärnan behövs det kun-skap om hjärnans normala blodförsörjning, kärlanatomi och fysiologi. Kunskap om pulsatilt blodflöde och normal fördelning av tCBF kan vara av betydelse, ef-tersom avvikande mönster och tecken till kompensationsmekanismer kan tala för begynnande kärlsjukdom i hjärnan. Kollateraler fungerar som kompensations-mekanism och är de kärl som övertar blodförsörjningen till hjärnan när huvudkärlet är avstängt eller förträngt. Trots att kunskapen om kollateralers funktion i hjärnan funnits sedan lång tid tillbaka, så har det inte funnits bra metoder som har kunnat kvantifiera dess blodförsörjning.

Förträngning i halspulsådern (karotisstenos), orsakad av åderförkalkning, räknas till storkärlssjukdom och är orsaken till ca 20% av alla hjärninfarkter. Utan behandling återinsjuknar drygt 10% redan första veckan. Karotiskirurgi är etablerad behandling för patienter som haft ett strokeinsjuknande och har en förträngning som är ≥50%. Ingreppet innebär att förkalkningen tas bort. Effekterna av en förträngning på hjärnans blodflöde undersöks ofta inte före operationen, och en orsak till detta kan vara bristen på en icke-invasiv metod som

x

möjliggör upprepade undersökningar och ger ett noggrant och pålitligt resultat av hjärnans blodflöde.

Det övergripande syftet med denna avhandling var att undersöka och utveckla tillvägagångssätt för att beskriva hjärnans blodflöde, dess pulsatilitet och kollateralfunktion hos friska och hos patienter med karotisstenos som gett upphov till stroke eller TIA. För att undersöka detta använde vi oss av faskontrast magnetisk resonanstomografi (PCMRI). 2D PCMRI kan mäta blodflöde i milliliter per minut i alla kärl som har en diameter på minst 1.5 millimeter. 4D PCMRI är en nyare variant av PCMRI-tekniken och användes i studie III och IV. 4D PCMRI och 2D PCMRI ger båda information om blodflöde, flödesriktning, flödeshastighet och kärldiameter. Fördelen med 4D PCMRI är att undersök-ningen endast tar 10 minuter, och då kan hela kärlträdet analyseras och man kan i efterhand bestämma i vilka kärl man vill mäta.

Metoder och resultat

De två första studierna (I och II) var baserade på friska personer. Fyrtionio yngre personer (medel 25 år) och fyrtiofem äldre personer (medel 71 år) undersöktes. Samtliga undersöktes med 2D PCMRI. Blodflöden mättes i hjärnans blodkärl och vi undersökte hur ålder, kön och anatomiska kärlvarianter påverkade blodflöden. Utöver detta undersökte vi pulsatilt blodflöde och dämpning av blodflödespulsationer i hjärnans blodkärl. Resultaten i studie I och II visade att blodflödet i hjärnans blodkärl minskade med stigande ålder. Kön påverkade inte blodflödet. Stigande ålder ledde också till ökad pulsatilt blodflöde och minskad dämpningskapacitet. Fördelningen av tCBF var i genomsnitt 72% genom de främre blodkärlen och 28% genom de bakre blodkärlen. Fördelningen var symmetrisk mellan höger och vänster hjärnhalva och påverkades inte av ålder eller kön. Däremot påverkade anatomiska kärlvarianter fördelning av blodflödet. I studie III och IV undersöktes patienter med karotisstenos med 4D PCMRI. I studie III ingick trettioåtta patienter (medel 72 år) med strokesymptom och karotisstenos. Nitton av dessa patienter (medel 71 år) genomgick karotiskirurgi och fick utföra 4D PCMRI undersökning efter kirurgin och beskrivs i studie IV. Resultatet från dessa båda studier visade att karotisstenos ger en asymmetrisk fördelning av tCBF. Kollateraler var aktiverade och försökte kompensera för det minskade blodflödet som sågs i det förträngda halskärlet och dess grenar. Trots kompensationer via kollateraler så kunde man ändå se ett lägre blodflöde till vissa kärl i hjärnan. Efter karotiskirurgi var fördelningen av tCBF i hjärnans kärl normaliserat, dvs symmetriskt. Vi kunde också se att de patienter som hade aktiverade kompensationsmekanismer via kollateraler före karotiskirurgi, hade lägre blodflöde i vissa kärl och lägre tCBF före kirurgi jämfört med efter kirurgi.

xi

Slutsatser

Vi använde oss av PCMRI som plattform för att beskriva ett nytt sätt att studera och kvantifiera hjärnans blodflöde. Referensvärden för unga och gamla av hjärnans blodflöde och fördelning av tCBF beskrivs och detta är nödvändig kunskap för att kunna studera strokepatienter. Om man använder sig av fördelning av tCBF i hjärnans kärl (”procent av totalt blodflöde”) så kan man beskriva blodflödet hos en patient utan att resultatet påverkas av ålder. Vidare, så studerade vi hur en karotisstenos påverkar blodflödet i hjärnans kärl. Vi fann att blodflödesfördelningen är störd och att kompensationsmekanismer inte alltid räcker till. 4D PCMRI genomgår snabb teknisk utveckling, framförallt på analyssidan, och vi tror att tekniken har en plats i utredningen av strokepatienter, till exempel före karotiskirurgi för att förbättra omhändertagandet av denna patientgrupp.

1

Introduction

Since aging is an established risk factor for the development of cerebral vascular disease, 1 knowledge about the cerebral vascular anatomy and normal cerebral blood flow physiology is of paramount importance to understand, detect and treat early stages of cerebral vascular disease.

Total cerebral blood flow (tCBF) is approximately 15% of the cardiac output and can be measured by adding the quantified blood flow rates (BFR) in the cervical arteries, i.e., the two internal carotid arteries (ICA) and the two vertebral arteries (VA). 2-6 The segmental distribution of tCBF in the cerebral arteries of healthy subjects has only been reported in a small number of studies. 5, 6 Information about normal cerebral blood flow distribution can be of clinical importance, as deviations from the normal flow can suggest the presence of compensatory mechanisms (collateral activation) and vascular pathology. Although the importance of collaterals in relation to cerebral arterial stenosis or occlusion has been known since the pioneering studies done by Thomas Willis, 7 the magnitude of the collateral supply is not well studied. 8

Further, with increasing age, arteries stiffen and the dampening function of the arteries becomes less efficient, leading to an increasingly pulsatile blood flow to the cerebral arteries. 9 This increasingly pulsatile blood flow can damage the microvasculature in the brain. 10 The pathophysiology of several age-related diseases such as lacunar infarcts, normal pressure hydrocephalus, mild cognitive impairment and Alzheimer´s disease 10-12 may be linked to increased pulsatile stress in the brain microvasculature.

Atherosclerotic changes leading to the development of carotid stenosis is responsible for about one fifth of all ischemic strokes. The degree of stenosis and plaque morphology, which can lead to embolism, is the main etiology for stroke caused by carotid stenosis. 13, 14 Carotid endarterectomy (CEA) is the main treatment option in cases of symptomatic carotid stenosis ≥50%. 15 The term hemodynamic stroke is often used to describe ischemic strokes caused by cerebral hypoperfusion that can be a result of systemic causes (e.g., cardiac failure or systemic hypotension) or regional causes (e.g., large artery stenosis or occlusion). However, the impact of a stenosis on cerebral hemodynamics in a standard clinical setting has not been investigated. One reason for this is that previous methods for studying cerebral hemodynamics in patients with carotid stenosis have failed to identify additional patient groups that could benefit from carotid revascularization. 16 Another reason can be the lack a non-invasive method for

2

studying cerebral hemodynamics that is both accurate, reliable and that allows for repeated investigations.

By using phase-contrast magnetic resonance imaging (PCMRI), blood flow rate (BFR) can be quantified in mL/min in arteries down to a diameter of 1.5 mm. 17 Further, a more recent implementation of the PCMRI technique, i.e., 4D PCMRI, can measure BFR in all 3-flow directions. 4D PCMRI technique provides whole brain coverage and simultaneous measurement of cerebral arteries with a reasonable scan time.

This thesis is focused on the exploration and development of a comprehensive approach to investigate the cerebral blood flow distribution, collateral function and pulsatility in healthy subjects and in patients with symptomatic carotid stenosis, using PCMRI as a platform.

3

Background

A short history of the cerebral arteries Thomas Willis was the first person to coin the term “Neurology”, and he did so nearly 400 years ago. Many believe that he was the first person to illustrate the arterial circle at the base of the brain, i.e., the circle of Willis (CW). This is an error, as the circle had been described at an even earlier date. 7

The knowledge of the existence of a vascular network around the brain goes back to the time of Aristotle (384−322 BC), who provided one of the earliest known descriptions of the cardiovascular system. 18 Further, Hippocrates (460−370 BC) used the term apoplexy, meaning ”a striking away”, when describing lesions in the carotid arteries that ultimately led to contralateral hemiplegia. 19

Another Greek physician, Herophilos (335−280 BC), often named the “father of anatomy”, made remarkable progress in the field of neuroanatomy. 20 In addition to providing a description of the cranial nerves, ventricles, meninges and cerebellum, he described the Rete Mirabile (“wonderful net”). 20 The Rete Mirable, found in some non-human vertebrates, is a complex of arteries and veins that lie close to each other, and its functions include acting as a countercurrent exchanger of heat and ions. Later, the Rete Mirabile was incorrectly described in humans by the Greek physician Galen (129−201). He described it as a vascular network of the brain and assumed that it was the origin of the intracranial segments of the internal carotid arteries. 21

Later, the Persian physician Zakaria (865−924) wrote in his large encyclopedia: “[The internal carotid arteries], once penetrated within the skull, wonderfully part into a formation similar to a network and then from that network they join again and give origin to two arteries of the same size as their precursors, which spread within the brain”. 18

The Swiss physician Johann Jakob Wepfer was probably the first person to describe the cerebral blood supply. In his book Treatise de Apoplexiae (1658) he described the association between pathological changes in cerebral arteries and cerebral ischemia.

While the anatomy of the cerebral vascular system was well known at that time, the physiology was not well known. Thomas Willis (1621−1675) was the first person to recognize the importance of the arterial anastomoses of the circle for maintaining collateral blood flow to the brain. 22 In his book, Cerebri anatome, Willis wrote that the arteries within the circle are open for each other so that the

4

blood can find other passages in case of a stop in an artery. “…if the Carotids of one side should be obstructed, then the vessels of the other side might provide for either Province…” Further, he wrote that in case of a stop in both carotids, the brain could be supplied by the vertebral arteries. 23 Willis’ physiological discovery of the cerebral arteries is still valid, and today it is well known that good cerebral collateral function is correlated to a better stroke outcome. 24 Hopefully, this thesis will add additional knowledge to the field of cerebral collateral vasculature which Thomas Willis once described.

Normal cerebral blood flow Total cerebral blood flow (tCBF) is approximately 15% (616−781 mL/min) of the cardiac output and can be measured by adding the quantified blood flow rates (BFR) in the cervical arteries i.e., the two internal carotid arteries (ICA) and the two vertebral arteries (VA). 2-6 Cerebral blood flow (CBF) at the tissue level (usually referred to as perfusion) can be measured by several different modalities and varies with age and sex. It also depends on whether it is measured in gray or in white matter and if it is reported in mL/min/100 g or mL/min/100 mL. 25 CBF is reported to be approximately 50−55 mL/min/100 g or 49−62 mL/min/100 mL. 25-29 The difference between the two values is given by the modalities that study the brain volume and the density of the human brain tissue, which is approximately 1.04−1.06 g/mL. 30

Cerebral autoregulation denotes the ability of the vascular bed in the brain to maintain a constant CBF even when cerebral perfusion pressure (CPP) is changed, i.e., CPP equals mean arterial pressures (MAP) minus intracranial pressure (ICP) (CPP=MAP-ICP). That is, CBF is controlled by homeostatic regulation (autoregulation), based on CPP and cerebral vascular resistance (CVR) (CBF=CPP/CVR). 31 This response to altered CPP is accomplished by performing changes in the diameter of the arterioles leading to appropriate changes in cerebral vascular resistance. 32, 33 This can be tested by administering acetazolamide 34 or increasing carbon dioxide concentrations 35 and observing the vascular response by measuring CBF or BFR/blood flow velocity. If the subject can be proven to respond to the vasodilatory stimuli, we will know that the cerebral reserve capacity is functioning as it should. Alternatively, in the case of no response, the autoregulatory system is impaired. 36

In his landmark paper published in 1959 reviewing 7 studies containing several groups of subjects and patients, Lassen showed that a stable CBF is maintained by means of autoregulation over a wide range of MAP, namely approximately between 60 and 150 mmHg. 31 Figure 1 shows the classical cerebral autoregulation curve and CBF plateau over the MAP range for static autoregulation. Hypertension is known to shift the curve towards higher MAP, also called a “right

5

shift”, 32 while the autoregulation can be impaired or completely lost in the presence of cerebral pathology, e.g., after a stroke or traumatic brain injury. 36 Chronic hypo- and hypertension have been found to be related to reductions in CBF, which in turn is known to be associated with cognitive decline and late-life depression. 37 Despite years of research in the field of cerebral autoregulation, authors are still not in agreement regarding the range of the curve, as well as the different methods for studying cerebral autoregulation. 32, 37-39

To better understand the pathophysiological changes that occur during the development of cerebral vascular disease, we need to improve our current knowledge about the healthy cerebral vascular system. In healthy subjects, tCBF and its distribution is dependent on factors such as age and anatomical variations of CW, which have important implications for the interpretation of arterial blood flow in disease states. 3, 5 An age-related annual decrease in tCBF has been estimated to be approximately 3 mL/min. 3 Further, tCBF is highly correlated to brain volume and decreases with increasing age. This can partly be explained by decreases in cerebral volume due to aging. 26

Figure 1. Classical cerebral autoregulation curve. A stable cerebral blood flow is maintained by autoregulation over a wide range of mean arterial pressures (∼60−150 mmHg). 31, 32

Due to increasing life expectancies around the world, the global burden of diseases affecting the cerebral vascular system is increasing, and it is rapidly becoming an important global health issue. Aging seems to be a main risk factor for cerebral vascular disease. 1, 40 It is therefore of great importance to evaluate the effects of healthy aging in order to give a better understanding of patients with

6

cerebral vascular disease. Several neurological diseases are linked to aging and disturbed cerebral blood flow, i.e., cerebral small vessel disease, 41 cognitive impairment, 42 Alzheimer's disease 43, 44 and vascular headaches. 45 Therefore, there is an increasing interest in the determination of cerebral blood flow as part of understanding the pathophysiology behind these diseases.

Circle of Willis and cerebral collateral circulation The cervical arteries (the right and left ICAs and the right and left VAs) form the arterial network at the base of the brain, i.e., the CW. The two ICAs, middle cerebral arteries (MCA), anterior cerebral arteries (ACA) and anterior communicating artery (ACoA) form the anterior circulation. The two VAs, basilar artery (BA) and posterior cerebral arteries (PCA) form the posterior circulation. Finally, the posterior communicating arteries (PCoA) connect the anterior and the posterior circulations (Figure 2). Collateral blood flow in cerebral arteries can provide perfusion through an alternative route and might offset cerebral ischemic injury. Cerebral collateral circulation can be divided into three main groups: I. The collaterals within the CW (primary collaterals) (Figure 2). 46 These work as a collateral connection between the right and left hemisphere (ACoA) and as an anterior-posterior connection (PCoA). II. The leptomeningeal arteries (secondary collaterals), which are small arterial connections between the terminal branches of MCA, ACA and PCA at the cortical surface. 46 III. The collaterals between the internal and external carotid arteries (secondary collaterals), such as the ophthalmic arteries (OA) and middle meningeal arteries. 46

The function of collaterals as an alternative route to provide blood flow depends on several factors: anatomical variations of collaterals (Figure 3), location of stenosis/occlusion in relation to the collateral route (Figure 4), systemic arterial pressure, capacity for autoregulatory dilatation and chronicity of the vascular condition. All of these factors can affect the extent of collateral circulation in an individual. 47, 48 Some of these factors will be discussed in the following section.

The presence of collaterals varies between individuals, and collateral recruitment is highly variable due to anatomical variations in CW. 49 The anatomical morphology of the CW has been studied in autopsy material using different radiological modalities, and the circle has been consistently found to be incomplete in approximately 50% of cases. 49-52 Hypoplasia (incomplete development), duplication, fenestration (segmental duplication) and missing parts of the CW are among the many variations that have been found. 51 Figure 3 A−D demonstrates the most common anatomical variations in the CW. It has been shown that the diameter of a communicating artery is inversely proportional to the risk of stroke, since decreased blood flow from the original feeding artery has to be compensated by a functioning collateral circulation. 54

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Figure 2. The circle of Willis. The arterial network of the brain forming the connection between the right and left and anterior-posterior vascular territory. (The figure presents automatically labeled arteries by flow identification method based on 4D phase-contrast magnetic resonance imaging, courtesy of Tora Dunås) 53

Moreover, an incomplete CW has been related to the occurrence of future anterior circulation stroke events. 55

The location of the stenosis or occlusion determines which collateral routes that can be activated and used. For example, a stenosis or occlusion in the carotid artery will first activate the primary collaterals (ACoA and PCoA). 56-58 In the case of an occlusion in MCA, the primary collaterals will not be able to provide blood flow to the MCA territory (Figure 4B), and the blood flow to MCA territory will be dependent on leptomeningeal collaterals. 59-61 Several studies have looked at the interindividual variations of primary and secondary collaterals in relation to ischemic events. In the case of carotid atherosclerosis, the recruitment of

8

collaterals from leptomeningeal and external carotid arteries has been linked to a poor hemodynamic status, while lack of leptomeningeal collaterals in acute stroke settings is related to poor cerebral blood flow in the ischemic core. 59, 62, 63

While the collaterals within the CW are ready to use in cases of acute cervical artery occlusion, other collaterals may function over time. The system does not function in the same way due to an acute obstruction compared with a slowly progressive stenosis, since the arteries need time to adapt and enlarge to maintain perfusion. Angiogenesis, which is considered to be microvascular development at the periphery of an ischemic region is sometimes referred to as “tertiary collaterals”. 64 Still, angiogenesis in human cerebral arteries is not well known, and there is uncertainty regarding the pathophysiological factors, which lead to its development. 47

Although the importance of collaterals in cases of cerebral arterial stenosis or occlusion has been known since the time of the studies done by Thomas Willis, 7 the magnitude of their supply is not well studied. 8, 65

Figure 3. Anatomical variations in the circle of Willis. A:Hypoplasia or missing ACoA; B: A1 segment of ACA; C: PCoA, D: P1 segment of PCA (i.e., the P2 segment of PCA arrives from a large PCoA that has its origin from ICA). Note that these variations can coexist and can, in the case of "C" and "D", do so bilaterally. 51

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Figure 4. Carotid artery occlusion/stenosis (A) and middle cerebral artery occlusion (B). In the case of a carotid artery occlusion, reversed BFR can be found in the ipsilateral ACA, PCoA and OA (red arrows). 57, 58 In the case of an MCA occlusion (B), the primary collaterals within the circle of Willis cannot supply the brain with blood as shown in (A) and the brain is mainly dependent on collaterals from the leptomeningeal arteries. 59, 60

Blood pressure and arterial stiffness It has been established that pulsatile flow delivered by the left ventricle is attenuated with distance along an artery. 66 Pulse pressure is at its highest in the aorta and large arteries, and it basically disappears in the arterioles and the capillary network in healthy young subjects. 66 The system is influenced by cardiac stroke volume, arterial stiffness, and early wave reflections, and it is changed by aging. 40, 67 Elastic arteries, i.e., the aorta and its proximal branches, have an important role in dampening pressure oscillations produced by the left ventricle in order to provide a continuous and almost non-pulsating flow to the recipient organs. 66 The compliance of the large central arteries refers to their ability to distend during systole and recoil during diastole. This is called the Windkessel effect, hence, continuous blood flow will be delivered to the organs throughout the entire cardiac cycle. 68

An increase in the systolic, diastolic and mean blood pressures has been observed in healthy subjects when they reach the age span of 30−49 years. This increase is thought to be due to an increased peripheral vascular resistance. With further aging, systolic blood pressures continue to rise, while diastolic blood pressures decline somewhat. 69 These changes lead to an increase in pulse pressure (PP) (the difference between systolic and diastolic blood pressure). Increased PP has been linked to the stiffening of large arteries 69, 70 and an increased risk of cardiovascular events. 71, 72

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The dampening ability of elastic arteries can be described by using different terms (compliance, distensibility, stiffness). 66 Arterial stiffness can be measured using direct or indirect methods, which differ in clinical feasibility. The main non-invasive ways to describe arterial stiffness are: 1. pulse wave velocity, 2. analysis of the pressure-wave contour and 3. direct measurement of the arterial lumen cross-sections’ area and diameter change during the cardiac cycle. 73

Cerebral arterial pulsatility Insufficient dampening of the cardiac pulsatile flow due to increased arterial stiffness exposes highly-perfused organs, such as the brain, to pulsatile stress and, possibly, to microvascular damage. 66, 74 The term pulse wave encephalopathy is sometimes used when describing how increases in pulsatile flow leads to microvascular damage in the brain. 10 Leukoariosis, 12 lacunar infarcts, 75 cerebral small vessel disease, 41 normal pressure hydrocephalus, mild cognitive impairment and Alzheimer´s disease 10, 11 may all be linked to a common pathophysiology of increased pulsatile stress in the brain microvasculature.

Pulsatility index (PI), assessed using Gosling’s pulsatility index, 76 has been used as a way of describing distal vascular resistance. 75, 77 However, looking at the equation, PI is not a pure index for describing distal vascular resistance. 77 PI is calculated by taking the ratio between the pulsatile component of blood flow velocity and the mean blood flow velocity. Since both the central and the peripheral vascular systems are integrated into producing the mean blood flow and the pulsatile blood flow, PI should depend on both changes in the aorta and in the arterioles. An increase in PI can therefore be due to increased aortic stiffness or increased cerebral vascular resistance. PI in the cerebral arteries increases with age. 78 With respect to the equation, this can be explained by an increased systolic blood flow velocity, decreased diastolic blood flow velocity and decreased mean cerebral blood flow velocity with increasing age. 5, 79 The physiological explanation for this could be central stiffness, i.e., a reduced distensibility of the aorta due to changes occurring in the media and intima, where the elastic lamina gets replaced by thinning, splitting and fragmentation of the elastic fibers. The degeneration of the elastic fibers is associated with an increase in collagen fibers and deposition of calcium. 80 In addition, the distal vascular resistance increases with age due to vascular remodulation. 81 Using PI, the dampening capacity of arteries can be measured by applying Gosling’s dampening factor (DF), which is the ratio between the PI in a proximal artery and a distal artery. 76 This can be a way to increase our understanding of how the cardiac pulsatile flow is transmitted into the cerebral arteries and how it is affected by different physiological and pathological factors.

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Carotid artery stenosis and hemodynamic stroke Approximately 20% of ischemic strokes are due to carotid artery disease, athero-sclerosis being the most common pathology affecting the carotids. 13, 14, 82 Patients with symptomatic carotid stenosis are at an increased risk of future ischemic events due to distal plaque embolization. 83, 84 In addition, hemodynamic disturb-ances, most notably cerebral hypoperfusion and insufficient perfusion pressures, can also contribute to stroke risk. 85-87

The management of carotid stenosis remains a cornerstone of stroke prevention, and the available treatment options are best medical treatment, carotid endarterectomy (CEA) (Figure 5) and carotid artery stenting (CAS) (Figure 6). 16 Extensive research has been done in the field of management of carotid stenosis, especially since the introduction of CEA more than half a century ago as a therapeutic option for the prevention of stroke. 88 Carotid endarterectomy became a part of routine clinical therapy in the 1990s after the results from the North American Symptomatic Carotid Endarterectomy Trial (NASCET) 89 and the European Carotid Surgery Trial (ECST) became available. 90 Based on pooled data from those trials, a clear benefit from surgery was seen for patients with ≥70−99% stenosis and, to some extent, benefit was also seen for patients with 50−69% stenosis, when compared to best medical therapy. 91 In addition, the benefit of CEA also depends on the timing of the surgery. Currently, most available guidelines recommend that surgery be performed within 2 weeks after the ischemic event. 92

While the strongest individual determinant of stroke risk is the degree of stenosis, 93, 94 the clinical manifestations of carotid artery disease are highly variable. Some patients may be diagnosed with an asymptomatic carotid stenosis, while others have the corresponding stenosis combined with a devastating cerebral infarction. It is also common that patients have varying degrees of stenosis bilaterally in the carotids or have other intracranial arterial stenoses. Therefore, decision making between treatment options can sometimes be challenging. 95

There is irrefutable evidence that the degree of stenosis and plaque morphology, which can lead to embolism, are the main risk factors for a future ischemic event in patients with carotid stenosis. In addition to those risk factors, hemodynamic disturbances also contribute to patient risk and should be taken into consideration when investigating patients with carotid stenosis. 96, 97

The term hemodynamic stroke is often used to describe ischemic strokes caused by cerebral hypoperfusion that can be a result of systemic causes (e.g., cardiac failure or systemic hypotension) or regional causes (e.g., large artery stenosis or

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occlusion). 62 Clinical symptoms related to cerebral hypoperfusion or hemo-dynamic transient ischemic attacks can be limb shaking, loss of consciousness or retinal claudication. Examples of precipitating events can be rising from supine position, exercise and the administration of antihypertensive medication. 62 However, a recent study suggests that hypoperfusion symptoms are not related to stroke risk or cerebral hemodynamic status in vertebrobasilar disease, as assessed by PCMRI. 97

To differ between embolization and hypoperfusion as possible causes of a stroke can be a difficult task, since there is no gold standard method for investigating stroke events due to hypoperfusion. In addition, it has been suggested that impaired washout due to hypoperfusion increases the risk of distal embolization, that is, emboli and hypoperfusion can co-exist in patients with symptomatic carotid stenosis. 85 Decades of perfusion studies have shown that chronic cerebral hypoperfusion (often referred to as "misery perfusion") remains a risk factor for future stroke events. 62, 98-100 However, the evidence for effective treatment options in hemodynamic stroke is sparse, and there is no clear benefit from any medical or interventional therapies (e.g., extracranial-intracranial (EC-IC) bypass surgery). 98, 101-103 Patients suffering from an ischemic stroke or a transient ischemic attack (TIA), should have, regardless of cause (embolic or hemodynamic), both antihyperlipidemic and antithrombotic treatment. 62 The management of blood pressure in this setting might be different from traditional stroke treatment since aggressive blood pressure lowering can increase stroke risk in patients with cerebral hypoperfusion. 104, 105

Regarding carotid stenosis, studies have shown that the degree of stenosis correlates poorly with the cerebral hemodynamic status, and the CBF is mainly dependent on the patency of the collaterals. 87, 94 The arteries within the CW are considered to be the main collateral pathway (Figure 2) in the case of a carotid stenosis or occlusion, 56, 58 and recruitment from the external carotid arteries or leptomeningeals can be a sign of a poor hemodynamic state. 62, 63 While the role and efficacy of CEA in stroke prevention for patients with symptomatic carotid stenosis has been established by large randomized trials, 15, 90 studies have not been able to identify which patients with asymptomatic carotid stenosis who will most likely benefit from CEA. 16 Offering CEA or CAS to these patients is no longer considered as optimal management. The European Society for Vascular Surgery recommends that patients with a 60−99% asymptomatic carotid stenosis who present with a clinical or imaging risk (e.g., the morphology of the stenosis, embolus detection using transcranial Doppler or symptoms of cerebral hypoperfusion) of a cerebral vascular event may be considered for CEA. 16 It is still uncertain which patients that will most likely benefit from CEA.

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Figure 5. Carotid endarterectomy. Carotid arteries in a patient with a symptomatic right sided carotid artery stenosis (A). An atherosclerotic plaque involving the common carotid artery and internal carotid artery (B). Photograph with permission from the patient and courtesy of Kerry Filler (who performed the surgery) and Maria Persson (who took the photograph).

Common carotid artery

External carotid artery

Internal carotid artery

Plaque inside the common and internal carotid artery

A

B

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Figure 6. Carotid artery stenting. Digital subtraction angiography image showing an internal carotid artery stenosis in left carotid artery (white arrow in image A). Image B and C shows the internal carotid artery after revascularization with the stent in place at the site where the stenosis was located. Image with permission from the patient and courtesy of Mats Cronqvist (who performed the stenting).

Cerebral vascular imaging and cerebral blood flow imaging There are several diagnostic imaging methods available for investigating the cerebral vascular system. Digital subtraction angiography (DSA) remains the gold standard method for investigating cerebral arterial pathologies, and especially for the detection of cerebral aneurysms. 106, 107 The technique works by imaging while there is no contrast in the blood and then subtracting the image after the injection of iodine contrast in an artery or vein. The final image consists of the vascular system filled with contrast and its washout. 107 DSA is an invasive, catheter-based method that requires iodinated contrast media and hospitalization at least over the day. 108 Due to its higher risk of complications and advances in non-invasive vascular imaging techniques, DSA is today mostly used for interventional purposes and is not routinely used as a diagnostic tool. 109

Doppler ultrasonography, computed tomography angiography (CTA) and magnetic resonance angiography (MRA) (mentioned under the magnetic resonance imaging (MRI) section), allow for direct and indirect assessment of the cerebral circulation. These methods are used in daily clinical practice for the visualization of cerebral arteries. Doppler ultrasonography is a non-invasive

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method and can be performed in a bedside manner. Using Doppler ultrasound, blood flow velocity can be measured in cm/s, and by designating the area or diameter of an artery, the blood flow can be measured in mL/min for cervical arteries. 107 In daily practice, Doppler ultrasound is commonly used for the investigation of atherosclerotic plaques in the carotid arteries. 110, 111 Doppler ultrasonography or transcranial Doppler can be used to investigate blood flow velocity in cerebral arteries. These methods are mainly limited to the proximal part of the CW. In addition, they are operator-dependent and not all subjects can be investigated due to the absence of a temporal bone window. 112 CTA is a feasible method for the investigation of the cerebral vasculature. The imaging time is short and the availability is high − therefore ideal in acute settings. However, this method lacks temporal resolution and provides no information about the actual flow rate in a specific artery. In addition, this method is considered invasive due to the use of ionizing radiation and iodinated contrast media.

Over the last five decades, there have been major advances in the development of perfusion imaging methods. The Kety-Schmidt method is the reference method for the measurement of global cerebral blood flow, which can be quantitatively measured, based on Fick’s principle. 29 This method, introduced in the 40s, is based on measuring the concentration of inhaled nitrous oxide in arterial blood and in venous blood, and in this way we can find out the tissue perfusion. 29 The Kety-Schmidt mathematical model is used in other perfusion techniques such as Xenon-enhanced CT (Xe-CT) and positron emission tomography (PET). 113 PET, Xe-CT and single-photon emission tomography (SPECT) perfusion computed tomography (PCT), MRI dynamic susceptibility contrast (DSC), arterial spin labeling (ASL) are examples of imaging methods that investigate blood flow at the tissue level. PET is a quantitative method that uses radioactive tracers, injected intravenously or inhaled, for the measurement of CBF, cerebral blood volume (CBV) and cerebral metabolism. 114 This method has mainly been used in stroke patients for hemodynamical assessment, e.g., in patients with carotid stenosis and carotid occlusion, and for the selection of patients with carotid occlusion for (EC-IC) bypass surgery. 115, 116 Xe-CT has been used for brain perfusion measurements for approximately 40 years, where Xenon is inhaled as contrast material which dissolves into the blood stream and easily crosses the blood-brain-barrier. 117 The study can be repeated within 20 minutes, making it possible to monitor the hemodynamic state of the patient. However, a significant drawback to the method is that it requires patient cooperation in order to perform the examination properly. 117 SPECT is an imaging technique using gamma rays that works with several different radioactive tracers. The images represent the distribution of radionucleotides within the brain, and it is mainly evaluated qualitatively. 113 PCT is being used more and more frequently in acute stroke settings as CT scans have become more widely available and the imaging time is short. The main use of the method is to provide information about the "tissue at

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risk". 113 Regarding the MRI-based perfusion studies, there are methods with exogenous and endogenous contrast enhancements. ASL uses magnetically labeled arterial blood water as a diffusible flow tracer and, therefore, there is no need for gadolinium. The different methods for studying cerebral vasculature and cerebral blood flow all have their advantages and disadvantages, and there are a number of different assumptions that have to addressed for them to work properly. 118, 119 Some of these methods are invasive, due to the use of ionizing radiation, radionuclide injection and/or they have a complex modality. Due to the lack of a gold standard method for perfusion imaging, comparing results from different modalities has been challenging. Further, perfusion imaging methods investigate the CBF at a specific brain region and the collateral flow routes cannot be quantified or visualized.

Magnetic resonance imaging MRI is a non-invasive method because it does not use ionizing radiation. It functions through the interaction between nuclear spins and an external magnetic field. Nuclear spin is a fundamental property of all elementary particles, and in the human body, the highly abundant hydrogen nuclei (protons) have this magnetic property. For imaging using MRI, the external magnetic field aligns the spins in the body, creating a net magnetization 120 A second magnetic field is then used to excite the spins, pushing the net magnetization into a transverse plane where it moves around the main magnetic field at the magnetic field strength-dependent Larmour frequency. In the excited state, an electric current is induced that will produce detectable signals in the receiver coils. Using additional gradient fields that produce local variations in the precession frequency of the spins, spatial positions are coded into the induced signals. The detected signals can then be reconstructed into an image. 121

The vascular system in the brain can be visualized by the MRA technique. Two imaging techniques, time of flight (TOF) 122 and PCMRI, 2 can create bright blood images by using a gradient-echo pulse sequence. TOF, a non-contrast technique, is based on imaging flow-related enhancement of spins entering an imaging volume. The blood entering the imaged area is unsaturated and generates a higher signal than the background tissue (stationary tissue) that is saturated by rapid repeated radio frequency pulses. 123

Phase-contrast magnetic resonance imaging PCMRI is a non-invasive MRI sequence that can quantify blood flow rate (mL/min) and its direction in the cerebral vascular tree with submillimeter resolution. 2, 17 The method has been available since the 1980s for in vivo applications and today it is available on most MR systems. 124-126 The method is

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based on the principle that a spin moving in a magnetic gradient field obtains a different change in phase of the magnetization in contrast to stationary spins. 2 A bi-polar gradient field rewinds stationary spins while there is a remaining phase shift in the moving spins, and the phase shift is proportional to the velocity. Vessels have different flow velocities, and by using appropriate velocity encoding gradients (VENC), it is possible to measure flow in a specific artery or vein. The VENC is set prospectively for each specific vessel that is going to be investigated based on the expected maximum velocity in that vessel. If the VENC is set lower than the actual maximum velocity that is found in an investigated vessel, aliasing may occur. This can be corrected, in some cases, in the data post-processing phase. 127 Using electrocardiogram (ECG) or pulse oximetry-based gating, time-resolved flow can be obtained. The scan can be gated prospectively or retrospectively. 128

2D phase-contrast magnetic resonance imaging In 2D PCMRI, one encoding direction is commonly used. While the subject is inside the MRI scan, a preselection of a location on the desired artery has to be performed. This is done by placing a perpendicular plane to the flow direction (explained more in detail in the methods section). 127 The post-processing of flow measurements using the 2D PCMRI technique is shown in Figure 7. By placing a region of interest (ROI) around the artery where BFR will be measured, BFR in mL/s will be obtained over the imaging frames representing the cardiac cycle. 17

4D phase-contrast magnetic resonance imaging In recent years, the 4D PCMRI imaging technique has been developed and seems to be a promising tool for vascular imaging. 128 The technique provides, similar to 2D PCMRI, both functional and morphological information about the vascular system in a non-invasive manner. The method has been used in cardiovascular and cerebral vascular investigations in different pathological conditions such as cerebral aneurysms, aortic aneurysms, and congenital heart disease. 129, 130 Using 4D PCMRI, with the phase-contrast vastly isotropic projection reconstruction (PC VIPR) acquisition, flow can be measured in the large cerebral arteries with high isotropic spatial resolution, using a single scan. The scanning time is reasonable (approximately 10 minutes) and there is no need to place perpendicular imaging planes at the scanner. 129 These developments make 4D PCMRI a promising technique for clinical use in the near future. However, the postprocessing time is still demanding and needs a skilled operator, which is why the method is still not ready for clinical use in the field of vascular neurology.

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Figure 7. 2D PCMRI imaging and blood flow rate quantification in internal carotid artery. Magnitude image (A) and phase image (B) at one point during the cardiac cycle. The magnitude image is used as an anatomical landmark for placing the region of interest (the red circles), in this case for measuring flow in the two internal carotid arteries and the two vertebral arteries at the level of 2nd-3rd cervical vertebrae (see Figure 10 A). The phase image is used for flow measurements. By placing a region of interest around the arterial boundary, the blood flow rate in that artery can be quantified over the cardiac cycle (C). Abbreviation: BFRsyst: systolic blood flow rate; BFRdiast: diastolic blood flow rate; BFRmean: mean blood flow rate.

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Research rationale

Extensive knowledge is needed regarding normal cerebral blood flow physiology to understand pathophysiological changes in the cerebral vascular system. Therefore, for the detection and treatment of early stages of cerebral vascular disease, it is of paramount importance to understand the normal physiology of the cerebral vasculature and subsequently, how and when pathology can develop from that. This is especially important as the population above 65 years of age is increasing and aging itself is an established risk factor for the development of cerebral vascular disease. This, however, is not always an easy task as there is a subtle balance and an overlap between age-related physiological and pathophysiological changes in the arterial system. Non-invasive, feasible methods are needed that can identify critical thresholds and that can distinguish between healthy vascular systems and what should be considered as pathological, in order to guide clinical decision making.

Further, the anatomy of the cerebral vascular system is highly variable between individuals and these variations can affect the outcome in various vascular diseases. Although the importance of collaterals in case of cerebral arterial stenosis or occlusion is well known, the magnitude of their redistribution is not well studied. The current state of evidence and the algorithm for decision making prior to CEA and CAS are based on the degree of carotid stenosis in a patient with symptomatic carotid stenosis. The impact of the stenosis on the cerebral hemodynamic function is often not a part of an algorithm for decision making. Since CEA and CAS are not without risk, there is a need for a non-invasive imaging technique that can provide a more informative description of the collateral system and predict blood flow distribution intraoperatively and postoperatively. Doing so, we can improve our understanding of the hemodynamic changes that may occur after CEA and CAS, and of the risks and benefits of the operative procedure and of the selection of patients.

The new advancements in stroke treatment put high demands on cerebral vascular imaging for clinical decision making. Methods should be easy to use repetitively, be non-invasive and give accurate information about the vascular condition. Cerebral perfusion has been investigated for more than a half century. However, a method that can feasibly quantify the blood flow in the large cerebral arteries has been lacking.

In a recent advancement of the PCMRI technique, i.e., 4D PCMRI, BFR can be measured in all 3-flow directions. 4D PCMRI provides whole brain coverage and simultaneous measurement of cerebral arteries within a reasonable scan time. In addition to this, the direction of flow and its pulsatility can be described. This

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method could be developed into a promising tool that can allow the evaluation of cerebral hemodynamics in large cerebral arteries. There is obvious potential with this novel approach of flow measurement in cerebral arteries, but scientifically verified clinical applications in the neurological field are missing.

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Aims

The overall aim of this Ph.D. project was to explore and develop a comprehensive approach to investigate the cerebral blood flow distribution, collateral function and pulsatility in healthy subjects and in patients with symptomatic carotid stenosis by using a PCMRI platform.

The specific aims were:

• To provide reference data for blood flow rate, tCBF distribution and pulsatile blood flow (including its dampening capacity) in the main cerebral arteries in healthy individuals with regard to age, sex and anatomical variations in the circle of Willis (papers I and II).

• To describe the hemodynamic disturbances in cerebral arteries caused by symptomatic carotid stenosis, its compensatory pattern of collateral flow (paper III) and its normalization after carotid surgery (paper IV).

• To develop and evaluate 4D PCMRI as a new approach for analyzing

cerebral arterial hemodynamics (papers III and IV).

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Materials and Methods

Ethical considerations Oral and written information was given to all included subjects and patients. Written consent was obtained from all participants. All studies were performed in accordance with the Declaration of Helsinki.

The research project was approved by the Regional Medical Ethics Board in Umeå, Sweden (papers I-II: Dnr 2010-381-31M, papers III-IV: Dnr 2011-440-31M).

Subjects and patients The enrolled subjects and patients in all four studies are listed in Table 1. For papers I and II, all subjects were shared. For papers III and IV, 19 patients were shared.

Papers No. of subjects (n) Age (years) Sex (f/m)

I HE (n=45), HY (n=49) 71±4, 25±2 50/44 II HE (n=45), HY (n=49) 71±4, 25±2 50/44 III CS (n=38) 72±6 11/27 IV CS (n=19) 71±6 2/17

Table 1. Overview of healthy subjects and patients enrolled in the four studies. Abbreviations: f: female, m: male, CS: carotid stenosis. Age is represented as mean ± standard deviation.

Study population for papers I and II The first and second papers were based on the same group of subjects. They were included during 2011 through an advertisement in the local newspaper. The final population fulfilled our criteria for being vascular and neurologically healthy based on history and clinical examination. A healthy subject was defined as having no previous history of hypertension, cardiac disease, renal disease or peripheral vascular disease. No medications affecting the central nervous system (CNS), and no previous disease in the CNS were allowed. Exclusion criteria were blood pressure ≥160/90 mmHg, mini mental state exam (MMSE) <28 points, 131 ventricular hypertrophy detected on ECG, cardiac arrhythmia and previous myocardial infarction. Subjects with contraindications for MRI examination (e.g., pacemaker, claustrophobia) were excluded as well. The recruitment process and the reasons for exclusions are shown in Figure 8.

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The subjects were recruited into two groups based on age: healthy young (HY, defined as age 20−30 years) and healthy elderly (HE, defined as age 64−80 years) Cerebral and vascular features of the two groups can be seen in table 2.

Figure 8. Study population for papers I and II. One hundred and eleven subjects were included after a short anamnesis prior to physical examination. Ten subjects were excluded after physical examination. In total 7 subjects were excluded after the MRI examination. The final study population consisted of 94 healthy subjects.

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Cerebral and Vascular features

HE (n=45)

HY (n=49)

p-value

Systolic BP (mmHg) 140±14 124±10 <0.001

Diastolic BP (mmHg) 84±7 73±6 <0.001

HR (bpm) 69±8 65±12 0.53

MAP (mmHg) 103±9 91±7 <0.001

PP (mmHg) 57±13 51±9 0.01

Table 2. Vascular features (papers I and II). Values are mean ± standard deviation.

Study population for papers III and IV Patients with carotid stenosis were included during 2012−2015 from a larger prospective stroke project. Patients with symptomatic carotid stenosis who were investigated for carotid endarterectomy (CEA) at Umeå University Hospital were offered to participate and to be investigated with magnetic resonance imaging, including 4D PCMRI before and after CEA. Patients could be included in papers III and IV if modified ranking scale (mRS) 132 was <3 and MMSE was ≥24 points. Patients with severe aphasia, previous ischemic events or other neurological diseases affecting the central nervous system were excluded. Further, patients with atrial fibrillation, contralateral carotid artery occlusion or contraindications for MRI examination were also excluded. All patients included in papers III and IV were investigated with Doppler ultrasound measurement and the degree of stenosis was determined by translating highest peak systolic velocity into degrees of stenosis. 133 Thirty-one patients in paper III and 15 patients in paper IV underwent CTA. In these patients, the CTA images were used for grading the carotid stenosis. The carotid stenosis was graded according to NASCET (Figure 9). 134

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Figure 9. NASCET method for grading carotid artery stenosis. The carotid stenosis was graded according to NASCET, using the algorithm shown in the figure. Picture courtesy of Rebecka Johansson.

In total 38 patients (11 women), with a mean age of 72±6 years with symptomatic carotid stenosis ≥50% with or without contralateral stenosis, were included in paper III. These patients were investigated with MRI within a median of six days (range 2–60 days) from symptom onset.

Nineteen patients with symptomatic carotid stenosis ≥50%, with or without contralateral stenosis, were included in paper IV. Carotid artery endarterectomy was performed within a mean of 14 days (range 4−98) after the ischemic event. MRI examination was performed before and after CEA. The MRI examination before CEA was performed within a mean of 7 days after the ischemic event (range 2–23) and after CEA within a mean of 497 days (range 109–1074 days) in 18 patients. In one patient the postoperative MRI was performed three days after CEA. Table 3 presents the clinical and vascular features, respectively, for the patients included in papers III and IV.

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Table 3. Clinical and vascular features for the included patients in papers III and IV. Hypertension, diabetes mellitus and hyperlipidemia were based on the medical history at arrival.

Clinical features

Patients

paper III

(n=38)

Patients

paper IV

(n=19)

Age years (mean and range) 72 (58–80) 71 (58–79)

Sex (F/M) (11/27) (2/17)

BMI (kg/m2±SD) 27±4 26±4

MMSE (points±SD) 27±2 27±2

mRS (median and range) 0 (0–2) 0 (0–2)

NIHSS (median and range) 1 (0–6) 0 (0–2)

Hypertension (%) 79 84

Diabetes mellitus (%) 24 32

Hyperlipidemia (%) 55 74

Ever smoker (%) 72 74

Subacute ischemic lesion on MRI (%) 47 53

Vascular features

Mean degree of symptomatic stenosis (%) 76±14 74±13

Mean degree of contralateral stenosis (%) 39±27 42±28

Unilateral stenosis 50–69% (n) 11 5

Unilateral stenosis 70–99% or occlusion (n) 15 7

Bilateral stenosis >50% (n) 12 7

Systolic BP (mmHg) 138±17 135±16

Diastolic BP (mmHg) 71±10 72±11

Heart rate (bpm) 69±10 68±11

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Magnetic Resonance Imaging

All subjects and patients for papers I-IV were investigated using the same 3-Tesla MRI scanner (GE Discovery MR 750, Waukesha, WI, USA) with a 32-channel head coil. For papers I and II, a 3D TOF sequence was used for the identification of vascular pathology, anatomical variations in the CW and PCMRI measurement planning. For papers I and II, arterial BFR was assessed with 2D PCMRI while for papers III and IV the patients were investigated with 4D PCMRI.

2D PCMRI for papers I and II The following parameters were used for collecting the 2D PCMRI data: repetition time/ echo time: 9/5 ms; slice thickness 3 mm (cerebral arteries), slice thickness 5 mm (cervical arteries); flip angle: 15 degrees; field of view: 180×180 mm; acquisition matrix: 512×512; in-plane resolution: 0.35×0.35 mm six views-per-segment and two averages. Velocity encoding (VENC): 20 cm/s – 120 cm/s. Highest VENC (120 cm/s) was set for middle cerebral artery and lowest for middle meningeal artery (20 cm/s). Retrospective cardiac gating was used, generating 32 images representing an average cardiac cycle. The total duration of the MRI examination was approximately 1 h. While the patient was in the MRI scanner, one operator (K. A.) selected a location, based on source images of three-dimensional time-of-flight data in the axial, sagittal, and coronal directions, where the perpendicular PCMRI measurement was placed (Figure 10 and Table 4).

4D PCMRI for papers III and IV The following parameters were used for collecting the 4D PCMRI data: The number of radial projections: 16000, imaging volume: 220x220x220 mm;

reconstruction matrix size: 320x320x320, voxel size: 0.7x0.7x0.7 mm3, VENC for the control group: 110 cm/s; VENC for the patient group: 110 cm/s and 40 cm/s; repetition time/echo time: 6.5/2.7 ms; flip angle: 8°; bandwidth: 166.67 kHz. Retrospective cardiac gating was used and 20 images were generated representing an average cardiac cycle. For investigation of the main cerebral arteries, VENC 110 cm/s was used and for investigation of OA, VENC 40 cm/s was used. The scan time was approximately nine minutes for each VENC setting.

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Figure 10. 2D PCMRI planes for the investigated cervical (A) and cerebral (B) arteries (papers I and II). Using maximum intensity projection method, the planes of the cerebral arteries were set perpendicular to the artery in a reconstructed MRA. (A): Anatomical and MRA location for internal carotid arteries and vertebral arteries. (B): (1, 2) the right and left M1 segment of MCA; (3, 4) ACA; (5, 6) PCA; (7, 8) distal MCA; (9, 10) and OA. The planes for BA and distal ACA are described in Table 4.

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Brain volume calculation Investigation of intracerebral pathology and calculation of brain volume was performed using a sagittal T1-weighted sequence, which was processed with the VBM8 toolbox (http://dbm.neuro.uni-jena.de/vbm.html) with default parameters in the SPM8 software (http://www.fil.ion.ucl.ac.uk/spm/) using Matlab R2013b (The MathWorks Inc., Natick, MA, USA). This method is based on adaptive maximum, and tissues are classified into classes such as white matter, gray matter and cerebrospinal fluid. Brain volume was computed by adding the volumes of gray and white matter. 135

Post-processing and blood flow rate analysis The investigated arteries were the same for papers I and II. Figure 10, demonstrates how the planes were selected using a TOF image (papers I and II) and Figure 11 describes the anatomical locations where BFR was measured using 4D PCMRI. Table 4 demonstrates the locations where BFR was measured for specific arteries in papers I-IV. In total 15 cerebral arteries and OAs were investigated in papers I and II and 17 cerebral arteries and OAs were investigated in papers III and IV. In papers I, III and IV, the blood flow rates were presented in mL/min. In paper II, PI and DF were calculated for all investigated arteries. For all subjects and patients in all four studies, BFR measurements were performed on deidentified images and the observers were blinded for the age, sex and clinical features of the included subjects/patients.

For papers I and II, all post-processing analysis was performed manually using Segment v1.8 software (http://medviso.com) on de-identified images. A circular or elliptical ROI was drawn to delineate the lumen of an artery using the magnitude images. The largest cross-sectional area during the cardiac cycle was chosen for placing one ROI for all 32 cardiac phases, and the ROI was kept constant during the cardiac cycle (Figure 7 A). The blood flow velocity (cm/s) in an artery of interest was computed in each voxel of the phase image inside the ROI area. For calculating the blood flow rate (mL/min), the average velocity during the cardiac cycle was multiplied by the cross-sectional area (ROI). The flow measurement was performed by one investigator (L.Z). For calculating tCBF, the mean flow rates of the two ICA and the two VA were added. To estimate average perfusion, tCBF was divided by the brain volume multiplied by 100 and expressed in mL/min/100 mL. 136

The morphology of the CW was investigated (papers I and II) in 3D TOF images using the Sectra IDS7 software (Sectra AB, Linköping, Sweden). All images were analyzed by one investigator (L.Z.) and 25% of the images were investigated and validated by a senior neuroradiologist (R.B.). The two investigators had a 100%

30

concordance. An incomplete CW was defined as the absence or hypoplasia of an artery forming CW. Hypoplasia was defined as an arterial diameter <0.8 mm. 51

For paper I, interclass correlation coefficient was used to investigate post-processing test-retest variability. 137 BFR in 10 randomly selected patients (5HY) were investigated by another investigator (K.A.). Measurement of BFR was done in ICA, MCA, ACA and PCA on the left side in addition to BFR measurement in BA. The interrater variability was >0.9.

For papers III and IV, flow analysis was performed in Matlab (The Mathworks, Natick, MA) using software developed in-house for calculating BFR. 138 tCBF was calculated by adding the BFR from the two ICA and the two VA here as well. Blood flow rate was calculated by two independent investigators (L.Z. and A.W.) in all arteries (Figure 11 and Table 4) except for OA. In ACA2, the right and left arteries were often close to each other and therefore manual segmentation was used. The mean BFR from the two investigators was used, and if the difference in BFR was >20%, a consensus measurement was performed. This was the case for approximately 9% of all measurements in both papers.

In paper IV, collateral recruitment was investigated and defined as: retrograde BFR in PCoA or OA on the ipsilateral side of the stenosis. A substantial increase in contralateral ACA BFR that completely supplies ACA territory bilaterally together with an ipsilateral ACA BFR of 0 mL/min or retrograde BFR, (see Figure 4 A for anatomy and comparison). In addition, we investigated if leptomeningeal collateral supply was present in any patient by defining leptomeningeal supply as an increased BFR in ipsilateral ACA2 and PCA2 by >22 mL (mean SD of ipsilateral and contralateral side) before CEA and its normalization, <22 mL, after CEA. Primary collaterals were defined as collateral routes through the ACoA and PCoA, and secondary collaterals were defined as collaterals through OA and the leptomeningeal arteries.

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Location for BFR measurements

Arteries Papers I and II

(2D PCMRI)

Papers III and IV

(4D PCMRI)

ICA/VA Cervical vertebrae 1–2 C3−C4/V4

MCA M1, if short, the sum of

two M2

M1, if short, the sum of

two M2

MCAdist Sylvian fissure Not measured

BA Superior to AICA Superior to AICA

ACA/ACA1 After branching off from

ICA (A1)

After branching off from

ICA (A1)

ACAdist/ACA2 Pericallosal, A3 After ACoA, A2

PCA1 Not measured Proximal to the aperture

of PCoA

PCA/PCA2 Distal to the aperture of

PCoA

Distal to the aperture of

PCoA

PCoA Not measured Anywhere along the artery

OA Proximally, after

branching off from ICA

Proximally, after

branching off from ICA

Table 4. Investigated arteries in papers I-IV. All paired arteries were measured bilaterally at the same level. For papers I and II, the internal carotid arteries and vertebral arteries were measured at cervical level 1-2. For papers III and IV, the internal carotid arteries were measured at segment C3−C4 and the vertebral arteries at segment V4. Note that distal ACA was measured at A3 for papers I and II and at A2 for papers III and IV.

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Figure 11. 4D PCMRI planes for the investigated cerebral arteries in papers III and IV (ophthalmic arteries are not shown).

Pulsatility index

Pulsatility index (PI) was calculated using Gosling´s pulsatility index equation accordingly: 76

BFRsyst, BFRdiast and BFRmean are the systolic, diastolic and mean BFR, respectively.

This equation is traditionally used for describing PI using Doppler ultrasonography, and the PI is calculated based on velocity (cm/s) measurements. In paper II, the PI was based on BFR measurements in mL/min. Since the cross-sectional area of the measured artery was kept constant during the cardiac cycle, and assuming a laminar flow profile, PI calculation based on BFR or velocity should not differ from each other. In addition, the maximum area of the artery was used for placing the ROI for all 32 phases. For investigation of the pulsatile flow dampening, Gosling’s dampening factor was used accordingly: 76

PI =BFRsyst - BFRdiast

BFRmean

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As shown in the equation, the PI of a proximal feeding artery, i.e., ICA, was divided by its distal branch, i.e., MCA. Proximal feeding artery was set as ICA and VA. Branches of ICA were defined as MCA, MCAdist, ACA, ACAdist, and OA. Distal branches of VA were defined as BA and all P2 segments of PCA that arrived from posterior circulation, i.e., from a P1 segment.

Statistics

The data in all papers were analyzed using PASW Statistics (version 18, 22, 25) (IBM, Chicago, IL, USA). Flow analyses for arteries were performed in all four papers, and if an artery was either invisible or hypoplastic and its blood flow was not measurable, the flow of that artery was set as 0 mL/min.

For paper I, we used a one-sample Kolmogorov-Smirnov test without the Lilliefors correction for testing the normality of the BFR distribution. Initially we compared the mean BFR between right and left arteries (when applicable) using a paired t-test. To avoid redundancy and to limit the number of investigated arteries, the mean BFR of right and left paired arteries was presented in paper I. If an artery was missing due to technical reasons, BFR in the contralateral artery was used. For paper II, the mean PI of the right and left arteries was used. Using an independent t-test, the effect of age was analyzed. For correlations between tCBF and brain volume, a bi-variate correlation analysis was used.

For papers III and IV, a paired t-test was used to compare mean BFR between symptomatic and contralateral sides and to compare before and after CEA. An independent t-test was used to compare BFR in cerebral arteries in patients with and without subacute ischemic lesions. A paired t-test was used to analyze the laterality of BFR in ICA, MCA, ACA2 and PCA2. Laterality was calculated as contralateral BFR-ipsilateral BFR (eg., MCAcontralateral-MCAipsilateral). In paper III, we dichotomized the patients into two subgroups to investigate how the degree of stenosis affects the cerebral arterial BFR: patients with moderate carotid stenosis (<70%) went into one group and patients with severe carotid stenosis (≥70%) went into the other group. 15, 139An independent t-test was performed to compare differences in BFR between subgroups of patients, while a paired t-test was used to compare ipsilateral/contralateral differences. Significance threshold was set as p<0.05.

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Results

Normal tCBF and its relative distribution The mean tCBF for healthy subjects was 717±123 mL/min (paper I). The relative anterior/posterior distribution of tCBF in CW for healthy subjects was 72%/28% and was not affected by age (p=0.7) (Figure 12). The distribution of tCBF was even and symmetrically distributed for paired cerebral arteries (p=0.08) (Figure 12) (paper I). There was no difference in tCBF between sexes (women: 724±131 mL/min; men: 709±114 mL/min, p=0.65) and the anterior/posterior distribution of tCBF was not affected by sex (p=0.7) (paper I).

tCBF changes due to age In HY subjects, the tCBF was 771±122 mL/min, which was higher than the 657±94 mL/min found in HE subjects (p<0.001) (paper I). Brain volume (HY: 1294±116 mL; HE: 1171±98 mL, p<0.001) and mean CBF (HY: 60±8 mL/min/100 mL; HE: 56±7 mL/min/100 mL, p=0.04) were also reduced with age. A linear correlation was found between tCBF and brain volume (r=0.60, p<0.001) (paper I).

Figure 12. Mean ± standard deviation for the relative distribution of tCBF in circle of Willis for all healthy subjects based on right side (r) and left side (l).

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tCBF changes due to anatomical variation, carotid artery stenosis and CEA A complete configuration of CW was found in 51% of the subjects (48/94) (paper I). Missing or hypoplastic A1 segment of ACA (A) and fetal type PCA (B) affected the distribution of tCBF (Figure 13). These anatomical variations caused an asymmetry in ICA inflow (missing A1, Figure 13 A) and affected the anterior/posterior distribution (missing P1, Figure 13 B).

Figure 13. Mean ± standard deviation for the relative distribution of tCBF in circle of Willis for subjects with missing or hypoplastic A1 segment of ACA (A) and fetal-type PCA (B) on ipsilateral (i) and contralateral (c) side.

tCBF was found to be 562±113 mL/min prior to CEA and increased to 635±133 mL/min after CEA (p<0.01) (paper IV). Further, tCBF was compared between subgroups of patients with (n=9) and without (n=10) collateral recruitment (575±144 mL/min and 550±81 mL/min, respectively; p=0.65). In the subgroup without recruited collaterals, tCBF increased to 564±68 mL/min after CEA (p=0.43). However, for the group with collateral recruitment, tCBF increased to 714±145 mL/min after CEA, p<0.01. After CEA, tCBF increased in patients with collateral recruitment when compared to the subgroup without recruited collaterals (p=0.01).

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Figure 14 demonstrates the relative distribution of tCBF into the cerebral arteries before and after CEA (paper IV). Carotid stenosis causes an unsymmetrical distribution of tCBF in the ICAs and in the cerebral arteries (Figure 14 A).

An increased BFR was also found in ipsilateral ICA (p<0.001), MCA (p<0.01), ACA1 (p<0.01) and PCoA (p=0.03) after CEA. In addition, BFR in contralateral ACA1 and BA decreased after CEA (p<0.01, for both). These changes resulted in a postoperative symmetrical inflow and distribution of tCBF into the CW (p>0.42) (Figure 14 B).

Figure 14. Mean ± standard deviation for the relative distribution of tCBF on ipsilateral (i) and on contralateral side (c) in circle of Willis for patients with carotid stenosis, before (A) and after (B) carotid endarterectomy. The degree of stenosis was 75±12% on the ipsilateral (symptomatic) side and 42±28% on the contralateral side.

Arterial BFR and its pulsatility changes due to age A decreased BFR was found in all cerebral arteries due to aging (Table 5) (paper I). Except for BFR in PCA, which was lower in men than in to women (p=0.04), there was no difference in cerebral arterial BFR due to sex (p>0.28). Cerebral arterial PI increased (not for VA) with increasing age (Table 5) (paper II). A progressive dampening of pulsations was found along the cerebral arterial tree for both HY and HE subjects (Figure 15). The dampening of pulsations was significantly higher in HY than in HE for all cerebral arteries, i.e., more pulsations were left in the distal part of the cerebral arteries (MCAdist, ACAdist, PCA) in HE as compared to HY (p≤0.001 for all cerebral arteries) (paper II).

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Arteries HY

(BFR) HE

(BFR) p-values

HY (PI)

HE (PI)

p-values

ICA 276±47 236±41 <0.001 0.84±0.13 0.96±0.15 <0.001

MCA 161±31 131±23 <0.001 0.72±0.11 0.89±0.12 <0.001

MCAdist 46±15 40±12 0.02 0.65±0.10 0.84±0.13 <0.001

ACA 88±18 75±15 <0.001 0.67±0.12 0.88±0.14 <0.001

ACAdist 33±11 27±8 0.01 0.58±0.11 0.77±0.12 <0.001

VA 109±23 90±17 <0.001 1.07±0.22 1.11±0.18 0.31

BA 162±44 128±28 <0.001 0.71±0.12 0.88±0.12 <0.001

PCA 58±13 51±10 0.004 0.64±0.10 0.85±0.13 <0.001

OA 10±4 11±5 0.36 1.10±0.26 1.20±0.22 0.04

Table 5. Mean blood flow rate in mL/min and pulsatility index ± standard deviation for each artery in healthy young (n=49) and healthy elderly (n=45) subjects. Bold values signify p-values <0.05

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Figure 15. Alteration of pulsatile flow in percentage for cerebral arteries in young (A) and elderly (B) healthy subjects. Gosling’s dampening factor was used to describe the dampening of pulsatile flow along the cerebral arterial tree (i.e., PIdistal/PIproximal), starting with 100% pulsation in the feeding arteries (ICA and VA). The pulsations were dampened while traveling along the cerebral arteries.

Arterial BFR changes due to carotid stenosis For patients with carotid stenosis, a decreased BFR was found in ipsilateral (stenotic) ICA compared with contralateral side ICA, in addition to a lower or reversed BFR in all branches of ipsilateral ICA compared with the contralateral side (Table 6) (paper III). The lower BFR in MCA and ACA2 found on the ipsilateral side had a linear correlation to tCBF (r=0.70, p<0.001 and r=0.67, p<0.001, respectively).

A retrograde BFR was found in ipsilateral ACA in seven patients with carotid stenosis (mean BFR of -53±44 mL/min). In the patients with anterograde BFR (n=31), 13 patients had an asymmetric BFR in the ACAs, that is, a contralateral BFR contribution that was more than double than that of the ipsilateral side. The mean BFR, found by adding the ACA BFR on ipsilateral and contralateral sides, was 77±19 mL/min, (ACAipsilateral+ACAcontralateral)/2) (paper III).

Comparing the ipsilateral side with the contralateral side, we found that in patients with moderate carotid stenosis, a decreased BFR was found only in the ipsilateral ICA (178±61 mL/min vs. 231±57 mL/min, p<0.05). In patients with severe carotid stenosis, a decreased BFR was found in ipsilateral ICA (111±90 vs. 277±108, p<0.001), MCA (121±32 vs. 143±38, p<0.01), ACA1 (23±62 vs. 134±81, p<0.001), ACA2 (58±21 vs. 65±23, p=0.04), OA (-4±14 vs. 6±6, p<0.01) and

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PCA1 (83±55 vs. 50±37, p=0.01) (paper III). Further, we compared how the degree of stenosis affects the BFR in a specific artery (Table 7) (paper III). We found that in patients with severe carotid stenosis, when compared with patients with moderate carotid stenosis, BFR was lower in ipsilateral ICA and ACA1, and higher in the contralateral ACA1, ipsilateral PCA1, PCA2 and BA.

Arteries Ipsilateral BFR (mL/min)

Contralateral BFR (mL/min)

p-value

tCBF 568±102 NA

ICA 134±87 261±95 <0.001

MCA 121±32 140±36 0.001

ACA1 35±58 119±72 <0.001

ACA2 56±19 61±20 0.03

BA 156±58 NA

PCA1 73±49 48±33 <0.01

PCA2 62±25 56±20 0.11

PCoA -5±28 10±28 0.03

OA -2±12 6±6 0.001

Table 6. Mean blood flow rate ± standard deviation in mL/min for cerebral arteries based on ipsilateral and contralateral side. Bold values signify p-values <0.05.

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Arteries

Carotid stenosis <70%

BFR (mL/min)

(n=13)

Carotid stenosis ≥70%

BFR (mL/min)

(n=25)

(p-value)

tCBF 565±79 569±113 0.89

ICA-ipsi 178±61 111±90 0.01

ICA-contra 231±57 277±108 0.10

MCA-ipsi 120±32 121±32 0.95

MCA-contra 132±31 143±38 0.34

ACA1-ipsi 59±43 23±62 0.04

ACA1-contra 90±39 134±81 0.03

PCoA-ipsi 2±15 -8±33 0.21

PCoA-contra 9±22 10±31 0.91

ACA2-ipsi 53±12 58±21 0.37

ACA2-contra 55±11 65±23 0.09

OA-ipsi 1±8 -4±14 0.22

OA-contra 4±7 6±6 0.27

BA 134±38 168±64 0.04

PCA1-ipsi 54±24 83±55 0.03

PCA1-contra 45±25 50±37 0.59

PCA2-ipsi 52±14 67±29 0.04

PCA2-contra 49±14 59±21 0.09

Table 7. Mean blood flow rate ± standard deviation in mL/min for cerebral arteries. Comparison between the two subgroups based on the degree of symptomatic carotid stenosis, <70% and ≥70%. Bold values signify p-values <0.05.

Collaterals before and after CEA We identified four main patterns of collateral pathway recruitment in 9 patients (paper IV). Primary collaterals: ACoA (n=5) and PCoA (n=3), and secondary collaterals: OA (n=3) and the leptomeningeal pathway (n=1).

The degree of carotid stenosis was 79±12% in the recruited collateral group and 71±10% in the non-recruited group, (p=0.15). In the subgroup with recruited collaterals, ipsilateral ICA BFR was lower (78±23 mL/min) than in the subgroup without collateral recruitment (181±49 mL/min) (p=0.001). After CEA, there was no difference in ICA BFR between the two subgroups (p=0.85).

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According to the definition in the methods section, one patient had a leptomeningeal collateral pathway through PCA2. In this patient a BFR of 150 mL/min was found in PCA2 on the symptomatic side and 64 mL/min on the contralateral side, before CEA. After CEA the BFR was equalized, i.e., the BFR on in PCA2 was 77 mL/min on the ipsilateral side and 70 mL/min on the contralateral side, while the BFR in ipsilateral MCA increased from 53 mL/min before CEA to 164 mL/min after CEA (168 mL/min on the contralateral side after CEA).

Figure 16 demonstrates the BFR laterality in MCA for the whole group and in the patients with and without collateral recruitment. In patients with collateral recruitment, MCA laterality was larger prior to CEA than after CEA (41.9 mL before vs. 8.4 mL after, p<0.01). While in the subgroup of patients without collateral recruitment, there was no difference in MCA laterality before and after CEA (3.6 mL before vs. -4.8 mL after, p=0.2). When comparing the subgroup with recruited collaterals with the subgroup without collateral recruitment, a higher MCA laterality was found prior to CEA (p<0.01). This laterality was not found after CEA (p=0.2). Regarding BFR laterality in ACA2 and PCA2 in the subgroups, there was no difference before or after CEA (p>0.05).

Figure 16. Middle cerebral artery (MCA) mean blood flow rate laterality± standard deviation before and after carotid endarterectomy (CEA) in subgroups with and without recruited collaterals. * indicates p-value <0.01.

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Discussion

In summary, this thesis provides a new and comprehensive approach that describes cerebral hemodynamics from a physiological and a pathophysiological perspective. In papers I and II, we provided reference data for cerebral arterial BFR, its distribution and pulsatility in healthy subjects. These data were described with regard to age, sex and anatomical variations. Knowledge about the physiological factors that affect cerebral blood flow is of paramount importance when interpreting pathological changes in cerebral hemodynamics. In papers III and IV, patients with a symptomatic carotid stenosis were investigated. In these two studies, we examined the disturbed blood flow distribution in cerebral arteries, ranked the importance of collaterals, and quantified the collateral supply (paper III) before and after CEA (paper IV). These results provide a new way to map and investigate cerebral hemodynamics and can have implications for the vascular investigation of patients with carotid stenosis.

tCBF and aging We found an age-related decrease in tCBF, CBF and cerebral volume in paper I, which is in line with previous studies. 3, 6, 140, 141 tCBF decreased by 15% when comparing the HY (mean 25 years) with the HE subjects (mean 71 years). This decrease gave a decline of ~2.7 mL/min/year, which was mainly explained by a reduction in brain volume (approximately 10%). As the subjects were healthy, the remaining 5% of its reduction should have some other age-related explanation. Some possible explanations for this could be decreased cerebral metabolism, 142,

143 small artery disease 144 or large artery disease, 40 all of which had not manifested clinically in the HE subjects (paper I).

Due to tCBF being highly related to cerebral volume, blood flow going into the brain would be more adequately expressed in units of CBF (mL/min/100 mL) rather than in mL/min. The mean CBF in HE was slightly higher (56 mL/min/100 mL) than in previous studies (~49 to 51 mL/min/100 mL). One possible explanation for this could the inclusion of non-healthy subjects in those studies, which could have led to a lower tCBF. 26, 136, 141 Another explanation could be the anatomical level at which the tCBF had been measured; we added the BFR from the two ICAs and VAs, whereas other studies have used ICAs and BA, which limits the possibility of measuring the entire blood flow going into to the cerebellum. 3, 141, 145 In paper I, the reported BFR values represented mean values of BFR measurements in an artery measured over a time span of several minutes. As the subjects were healthy and the measurements were unprovoked, the mean CBF in paper I should be on the linear portion of the autoregulation curve (Figure

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1). Based on this, hypoperfusion may be defined as mean global CBF <42 mL/min/100 mL, which is 2 SD below the mean CBF value found in paper I.

While aging does not cause vascular disease by itself, it is a main risk factor for the development of vascular disease due to structural and functional changes in the vascular system. 146 These changes can disturb the cerebral autoregulation. One such example is hypertension, which is usually associated with increased age, and which is known to shift the autoregulation curve towards higher mean arterial pressures, called a “right shift” (see Figure 1, page 5 for autoregulation curve). 32, 33 That is, small decreases in cerebral perfusion pressure could result in cerebral hypoperfusion in patients with hypertension. While higher values of tCBF are has been shown to be inversely related to lower numbers of white matter lesions, 147 hypoperfusion and hypertension are related to the development of cerebral white matter lesions, which can be early signs of cerebrovascular disease. 148, 149

Normal cerebral blood flow and lateralized cerebral blood flow The anterior/posterior ratio of tCBF distribution was found to be ~3:1 in healthy subjects, with a symmetrical distribution between the right and left hemispheres (Figure 12) (paper I). Decreases in BFR between 12% and 21% were found in individual cerebral arteries, and the findings were attributable to the effects of aging (Table 5), a finding confirmed by other PCMRI studies. 5, 6 However, the distribution of tCBF in cerebral arteries was not affected by physiological factors such as brain volume, age or sex. Therefore, distribution as a method may have the potential to be useful in clinical settings in the future and may serve as a screening tool for cerebral vascular disease (paper I). Using this approach, one has to take the anatomical variations of the CW into account, as they can affect the anterior/posterior distribution as well as inflow symmetry between the ICAs (Figure 13). 145 However, the anatomical variations did not affect the distribution of blood flow in the distal branches (MCAdist and PCA). BFR quantification of these arterial segments could therefore be a useful approach for studying disturbances in BFR going into the cerebral arterial territories.

A mean tCBF of 568 mL/min was found in patients with symptomatic carotid stenosis, which was lower than HE (paper I) and elderly patients with vascular disease. 26, 150 Furthermore, a high correlation was found between tCBF and BFR in ipsilateral MCA and ACA2 on the ipsilateral side (paper III). That is, a low tCBF was seen as a low BFR in those two arteries. This correlation may be explained by reduced BFR due to the stenosis in the ipsilateral ICA, causing a post-stenotic pressure drop and reduced perfusion pressure, resulting in decreased BFR in MCA and ACA2. In these patients, systemic factors such as heart failure, aggressive antihypertensive treatment and intraoperative hypotension during

44

CEA could potentially be risk factors for developing cerebral hypoperfusion and future ischemic events. 104 However, the exact role of cerebral hypoperfusion in the development of ischemic stroke in patients with carotid stenosis is still unclear. Although numerous studies have shown that hemodynamic compromise is related to increased stroke risk, a gold standard method for investigating hemodynamic compromise is missing, and there is no established treatment for this condition. 96, 151 In papers III and IV we showed that collateral and distribution analysis, as assessed by 4D Flow, have a potential as diagnostic tools during the preoperative workup of patients with carotid stenosis prior to revascularization to assess their hemodynamic status.

We used the laterality approach to describe blood flow distribution in cerebral arteries in patients with symptomatic carotid stenosis, and found disturbed cerebral hemodynamics (Table 6 and Figure 14 A) (papers III and IV). The mean BFR in ipsilateral (stenotic) ICA was decreased (49% lower) when compared to the contralateral side (Table 6). Furthermore, a decreased, or in some cases reversed, BFR was found for all of the branches of the stenotic ICA. Although the carotid stenosis was the cause of the decreased BFR in those arteries, the degree of stenosis was not always related to CBF and BFR in cerebral arteries. 87, 94 A carotid stenosis of 70−80% degree has often been described as a potentially hemodynamically significant stenosis. 89, 139, 152 Spencer’s curve 153 has widely been used in the literature on cerebral Doppler ultrasonography to describe the effect of a stenosis on blood flow velocity. The flow model that was described by Spencer et al. is based on a short symmetrical arterial stenosis without bifurcations and with stable blood pressure and viscosity. This model is still relevant, as it demonstrates that the relation between flow and the degree of stenosis is non-linear, and that blood flow velocity will start to fall dramatically when the lumen diameter approaches a 70−80% reduction. 153 According to Poiseuille's law 154, the resistance of a vascular segment (R) is defined by its radius (r), length (l) and the blood viscosity (η), and blood flow rate is equal to Q=ΔP/R, where R=(8ηl/r4 π). This equation describes vascular resistance in relation to tube diameter and fluid viscosity, and we can see that the length of the stenosis also affects the cerebral hemodynamics. Further, the ability to recruit collaterals varies between patients, which is another reason why cerebral hemodynamics cannot be accurately predicted by only investigating the carotid stenosis. The role of collaterals for the preservation of cerebral blood flow will be discussed below.

Cerebral collateral function The main collaterals that help preserve the cerebral perfusion are thought to be the primary collaterals within the CW, while the recruitment of secondary collaterals is linked to a poor hemodynamic status. 62, 63 In paper III, the patients were dichotomized into two subgroups based on the degree of symptomatic

45

carotid stenosis (<70% = moderate stenosis and ≥70% = severe stenosis) (Table 7). Severe carotid stenosis resulted in BFR laterality and activated collateral routes. However, we found no difference when comparing the BFR in ipsilateral MCA and ACA2 between patient groups with moderate vs. severe carotid stenosis. That is, a severe stenosis does not necessarily give a lower BFR in MCA and ACA2 than a more moderate stenosis does. This finding supports the statements in the discussion above, suggesting that the degree of carotid stenosis alone should not be used to predict cerebral hemodynamics 87, 94, and the reason for this could be due to the presence of collateral compensatory mechanisms. 155 We identified four important collateral routes in patients with carotid stenosis (papers III and IV).

1. In cases of carotid stenosis, ACA1 works as an important collateral route, not only for the MCA territory, but also for maintaining bilateral blood supply to the ACA territories. The BFR in ACA1 on the ipsilateral side of the carotid stenosis was decreased or reversed, and the BFR on the contralateral side ACA1 was significantly higher. TCD studies often refer to collateral supply via ACA if reversed blood flow is seen in the ipsilateral A1 segment of ACA. 57 By quantifying the BFR in ACA1 as in paper III, we could show that 20 patients out of 38 had retrograde (n=7) or asymmetrical (lower in ipsilateral ACA1) BFR in the ACAs. Therefore, it is a crude binary simplification to only look at the retrograde flow as collateral supply. Further, the mean BFR reaching ACA territory (77 mL/min) was comparable to the mean BFR in ACA in HE found in paper I (75 mL/min). We concluded that ACA is the main collateral route in case of a carotid stenosis.

2. A reversed blood flow in PCoA serves as collateral supply from the posterior to the anterior circulation. However, due to its wide anatomical variation, there is interindividual variation between patients in the degree recruitment of PCoA as a collateral route. 156 This is probably one explanation why there was no difference in the PCoA recruitment between patients with moderate and severe stenosis.

3. The BFR in OA was reversed or not detectable in almost 70% of the patients. While OA is considered as a collateral source of blood supply, it has also been associated with a poor cerebral hemodynamic status. 63, 157 In addition, since reversed BFR was only seen in patients with severe carotid stenosis, OA may function as an indicator of a high degree of carotid stenosis (or low BFR/pressure in ICA). The mean contribution from OA was -2 mL/min in the whole group of patients and -4 mL/min in patients with severe stenosis (paper III). In HE subjects (paper I), the BFR in OA was 11 mL/min. Thus, a reversed BFR in OA was found to add approximately 15 mL/min to the cerebral BFR.

46

4. Leptomeningeal arteries can serve as collaterals in the presence of carotid stenosis. Using 4D PCMRI, we could indirectly quantify the occurrence of collateral supply through posterior leptomeningeal arteries to the MCA territory (paper IV). This was demonstrated by observing that BFR was more than twice as high in the ipsilateral PCA2 than in the contralateral side prior to CEA, and that BFR was normalized after CEA. In addition, the BFR in ipsilateral MCA increased by 2.5-fold after CEA when compared with the time before CEA. Historically, due to the anatomical location, the quantification of the leptomeningeal supply has been difficult to perform and is not well studied. 8 Our approach demonstrates a rather easy way to study the leptomeningeal supply in the presence of carotid stenosis.

The above-mentioned collateral recruitments helped compensate for a low BFR in the stenotic carotid artery. However, the compensation was incomplete. Significant laterality was found in MCA, i.e., about 15% lower BFR in the ipsilateral MCA (paper III), which can be interpreted as hypoperfusion in the MCA territory.

Cerebral hemodynamics normalize after CEA In patients with symptomatic carotid artery stenosis, the tCBF was found to increase by 15% after CEA. The distribution was found to be symmetrical in all cerebral arteries (Figure 14 B) (paper IV). Interestingly, when comparing the subgroups that had vs. those that did not have collateral recruitment, an increase was only seen in patients with collateral recruitment (24% vs. 3% increase) (paper IV). Since the patients were not investigated directly after CEA, cerebral hyperperfusion should be excluded as an explanation for the increased tCBF. 158. Instead, the increased perfusion should be interpreted as a sign of improved cerebral hemodynamics after CEA in a hemodynamically stable postoperative state.

Decreased cerebral blood flow in patients with symptomatic carotid stenosis prior to undergoing CEA has been shown in previous studies using MRI technique. 159,

160 However, decreased cerebral blood flow has not previously been related to the activation of collateral recruitment. A way to understand collateral recruitment as a sign of disturbed hemodynamics is by understanding the post-stenotic pressure drop (discussed above) that occurs due to a severe stenosis in the carotid artery. The pressure drop will cause a pressure difference in the CW, which will cause a driving force for collateral flow towards the stenotic ICA. 161

The MCA laterality, seen in patients with collateral recruitment, was normalized after CEA (Figure 16). These two findings, i.e., increased tCBF and normalized

47

BFR in MCA after CEA, suggest that patients with collateral recruitment may have a hemodynamic component in addition to the embolic.

Intraoperative cerebral monitoring techniques (electroencephalography, Doppler ultrasound, stump pressure measurement) have not been able to identify patients who need intraoperative shunting or have postoperative risks such as hyperperfusion syndrome. 162 Since CEA and CAS are not without risk; there is a need for a clinical screening tool that can accurately identify patients who have a high risk of perioperative complications prior to surgery/intervention. 4D PCMRI for BFR quantification, as in papers III and IV, may provide useful information about patients that can be at risk of hypoperfusion during CEA or CAS or hyperperfusion after revascularization. 163

PI increase and the dampening of pulsations decrease due to aging Increased arterial pulsatility, which has been described in several studies using PI, is related to brain atrophy, reduced regional brain volume and higher numbers of white matter lesions. 79, 164 Several chronic neurological disorders, such as mild cognitive impairment, 44 Alzheimer’s disease 165, and normal pressure hydrocephalus, 166 have been related to increased arterial pulsatility. While the BFR in cerebral arteries was found to decrease with increasing age (paper I), PI was found to be increased (paper II). An increased PI, i.e., low mean BFR and/or high pulsatile BFR, can be interpreted as an increased distal vascular resistance or an increased stiffness in large arteries (aortic stiffness). Both of these changes can lead to an increased pulsatile flow to the brain. 9, 164

We used Gosling’s dampening factor when describing the dampening of pulsatile flow along the cerebral arteries, using a proximal-to-distal approach. 76 Pulsatile blood flow was dampened when using this approach in both the HY and the HE. However, this dampening was reduced with increased age, leading to an augmented pulsatile blood flow reaching the distal arterial segments in HE subjects (paper II). Since the subjects in paper I were healthy and without manifest vascular disease, we concluded that increased age can result in augmented pulsatile blood flow reaching the brain prior to the development of vascular disease. This result is in agreement with studies showing age-related cerebral vascular changes, and it is important when trying to understand the development of cerebral vascular pathology prior to clinical signs of vascular disease. 167, 168

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Technical considerations and clinical perspectives Due to recent advances in the field of treatment in vascular neurology, there is a need for developing reliable methods that can investigate the vascular system repeatedly and without risk or discomfort to the patient. 169, 170

There are several radiological methods available for investigating the cerebral vascular system. Choosing the appropriate technology depends on the specific aim of the measurement. While perfusion techniques using, e.g., MRI, CT or PET, can investigate a disturbed CBF, the collateral pathways cannot be quantified or visualized by these methods. Further, some methods, e.g., CTA, are fast and therefore ideal in acute settings, but CTA lacks temporal resolution and is therefore not optimal for flow measurements. Using Doppler ultrasound, a surrogate marker of blood flow (velocity), the cerebral vascular system can be measured repetitively and in a bedside manner. However, it is limited to the proximal parts of the CW.

PCMRI technique provides us with both functional and morphological information about the vascular system in a non-invasive manner. 130 The measurement is time-resolved and the data are collected by cardiac gating and reconstructed to describe flow over an average cardiac cycle. There is a risk of overestimating BFR with PCMRI, because of partial volume effects, especially when investigating small arteries. 171 Still, a good agreement is shown when comparing flow measurements with a phantom. 17

Whole brain coverage can be achieved in about 10 minutes by using 4D PCMRI, and BFR can be quantified without anatomical limitations, which has been a drawback with other methods. 130 The cerebral vascular system can be investigated by performing a single measurement. Since the artery of interest can be identified and investigated during the post-processing phase, pre-selection is generally not needed. However, the disadvantage of performing only one scan is that a single VENC setting is used for all arteries, instead of an individual and optimized VENC setting for each investigated artery, as in 2D PCMRI. 2D PCMRI is an established method for blood flow rate evaluation. 17, 172 4D PCMRI was recently validated against 2D PCMRI in a study showing 4D PCMRI to be accurate across a wide range of BFR and arterial lumen diameters. 173 The post-processing time is still demanding and needs a skilled operator. However, we have introduced a post-processing tool that automatically recognizes and analyzes BFR in cerebral arteries – a tool which can shorten the postprocessing time significantly. 53

Further research and development of the 4D PCMRI technique may ultimately result in a clinical tool that is useful for investigating patients with cerebral

49

vascular disease. One clinical scenario could be to investigate BFR during the preoperative medical workup for a stenting operation of an intracerebral arterial stenosis. By using 4D PCMRI, clinical decisions can be made based on BFR quantification instead of using other methods that are either surrogate markers of BFR (Doppler ultrasonography) or invasive (DSA). Furthermore, the best treatment for patients with asymptomatic carotid stenosis is not completely clear. 16 Due to its non-invasive nature, 4D PCMRI allows for repetitive investigation of the cerebral vascular system before and after carotid revascularization and can provide a new way to study the relationship between carotid stenosis and cerebral hemodynamics. Further, in contrast to perfusion imaging studies that cannot investigate the collateral routes, or Doppler ultrasonography which is only a surrogate marker of BFR, 4D PCMRI can simultaneously quantify BFR in cerebral arteries without anatomical restrictions. 174, 175

50

Conclusion

This thesis provides a new comprehensive approach to mapping and quantifying normal cerebral blood flow and pulsatility. By presenting the distribution of tCBF in the cerebral arteries instead of using absolute values, the effect of age could be neutralized, and the result is applicable for describing healthy cerebral blood flow regardless of age. A symmetrically distributed tCBF was found in the cerebral arteries, with an anterior/posterior ratio of approximately 3:1 regardless of age and sex. However, this was affected by anatomical variations within the CW. Further, the pulsatility of BFR increased with aging and the dampening of pulsations was less pronounced in elderly.

We found that a symptomatic carotid stenosis, especially if severe, caused disturbed distribution of tCBF, i.e., a laterality with lower BFR in the ipsilateral side to the stenosis. The compromised BFR in the stenotic carotid artery was mainly compensated by contralateral ICA, which mainly supplied the ACA territory bilaterally. Despite collateral activation, BFR in ipsilateral MCA was lower compared to contralateral side, especially in patients with severe carotid stenosis. However, when comparing BFR in ipsilateral MCA between patient groups with moderate and severe carotid stenosis, we did not find any difference. MCA laterality, seen in patients with collateral recruitment prior to CEA, was not found after surgery, pointing towards a hemodynamic etiology of the stroke event in addition to the embolic.

4D PCMRI technique may be a feasible method for hemodynamic investigations, and collaterals could be quantified in a way that has been previously impossible due to anatomical difficulties. With further development of this method, especially the post-processing part, it can become a promising clinical tool for the investigation of cerebral hemodynamics in patients with stroke.

51

Acknowledgements

Jag är väldigt glad över att ha fått möjligheten att slutföra min avhandling och vill därför tacka alla er som har stöttat mig, inspirerat mig och sett till att jag gått i mål. Jag vill i synnerhet tacka:

Alla toppenfina personer som ställt upp som deltagare i mina studier. Utan er så hade detta inte varit möjligt.

Min huvudhandledare, Jan Malm, som introducerat mig till forskningsvärlden, föreningsvärlden, stroke, akut neurologi, Berghems pizzeria, Ali Baba och andra intressanta saker. Du har en central och inspirerande roll för mig som läkare och som forskare.

Anders Eklund, min bihandledare, som alltid kommer med den klokaste och mest genomtänkta kommentaren. Du ser det vi andra missar. Vilken tur jag haft som har fått ta del av dina tankar och din kunskap under dessa år.

Monsieur Khalid Ambarki, min bihandledare och vän. Tack för all din tid och allt du har lärt mig med tålamod och entusiasm. Det är mycket jag inte hade kunnat om det inte var för din skull. Detta inkluderar även att använda Excel på franska. Merci!

Anders Wåhlin, medförfattare och glädjespridare. Din ödmjukhet, kunskap och öga för design får mig alltid att häpna. Och hur kommer det sig att du aldrig har en dålig dag?

Kristin Nyman, forskningssköterska och vän. Tack för alla timmar du lyssnat på mig oavsett om det handlar om forskning eller om kolikbarn. Du har varit min kloka vän hela vägen.

Richard Birgander, den multibegåvade neuroradiologen och medförfattaren som senaste åren oftast svarat på mail cyklandes på andra sidan jordklotet.

Tora Dunås, Sara Qvarlander (statistik-hjältinnan), Jenny Larsson, Madelene Holmgren och resten av vår härliga forskargrupp för trevliga och lärorika dagar tillsammans.

Bo Carlberg, för ditt värdefulla bidrag som medförfattare.

Anders Hildén och Johan Jacobsson, båda mina före detta chefer, kliniska förebilder och vänner, som alltid sett till att jag fått tid till forskning.

52

Rebecka Johansson, Kerry Filler, Mats Cronqvist och Maria Persson för snygga bilder till min avhandling.

Ann-Catrine Larsson för all din hjälp med MR-undersökningarna och Umeå Centre for Functional Brain Imaging som möjliggjort MR-undersökningarna.

Per Jonasson och Lars-Owe Koskinen för all kunskap som ni delat med er inom neurovaskulär anatomi, fysiologi och främst patofysiologi.

Elias Johansson, för lärorika karotis-kaffestunder.

Jörgen Andersson, hur snabbt kan man få hjälp när det trasslar? Tack!

Mina eminenta neurodamer: Helena Fordell, Cecilia Lindgren, Tara Raouf, Camilla Brorsson och Jadranka Plese för att ni ständigt delar med er av era kunskaper om hjärnan och ryggmärgen.

Michael Haney, Roman A´roch och Magnus Hultin som välkomnat mig på min nya institution.

Carina Olofsson för din ständiga kommentar: ”Blir du klar nångång?”. Tack! Hade nog inte blivit klar utan den kommentaren.

Alla kollegor som gör jobbet till världens bästa jobb, i synnerhet min mycket kompetenta kliniska handledare, Marie Hanes som också är en förebild.

Min underbara familj: Andreas, Dina, Sofia och hela den stora norsk-iransk-svenska släkten, i synnerhet min mamma Farideh, mina gudföräldrar Mona och Lars och mother- and father-in-love Ingrid och Torleif. Ett stort tack till underbara Dina för ditt tålamod. Äntligen är jag klar med mina ”läxor”. Andreas, utan dig hade jag inte fixat detta.

Och till sist men inte minst mina härliga vänner, i synnerhet Elin Lidman, Magnus Andersson s.k. Mr. Anderson och Arni Hafstad för all hjälp och trevligt häng dessa dagar.

53

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