Cerebral small vessel disease (SVD) is a leading cause of stroke and vascular dementia. The majority of cases are sporadic, occurring in the elderly hypertensive population. However, there also exist patients with familial disease. The most common form is Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL), caused by mutations in the NOTCH3 gene. In recent years, other genes have also been found to cause familial SVD, such as COL4A1/A2, HTRA1, FOXC1 and TREX1. Genome wide association studies (GWAS) have also revealed loci associated with sporadic SVD strokes and its related features. This thesis explores the genetic basis of SVD primarily from the angle of the 'one gene, one disease' hypothesis. We explore the phenotype of familial SVD using CADASIL as a prototype. We next adopt a candidate gene approach to rare variant discovery using high throughput sequencing (HTS) techniques in two forms: 1) a multi-gene sequencing panel to examine the presence of rare variants in a cohort of 993 presumed-sporadic, early-onset SVD stroke patients, and 2) whole genome sequencing in 118 pedigrees with suspected familial SVD. We also evaluate the prevalence of known disease-causing mutations in the general population using a cohort of whole genome sequenced non-SVD patients, and other control databases. We demonstrate that a few presumed-sporadic SVD stroke patients may in fact have familial disease that was not previously diagnosed. We show that known and novel rare variants in candidate genes are found in our cohort of familial SVD patients, and suggest a possible role for rare variants in genes associated with related phenotypes and sporadic disease in this cohort. Finally, we identify known disease-causing variants in relatively high frequencies in the population, and show that conclusions on the pathogenicity of variants based on allele frequency and functional analyses may sometimes be misguided, thus highlighting the current limitations we face in the clinical interpretation of variants identified on HTS. In recent years genetic studies have revealed that pathways in different familial diseases are likely to converge in the pathogenesis of sporadic disease. Further uncovering the genetic basis of undiagnosed cases of familial SVD may shed light on the mechanisms underlying the sporadic form of disease, and may in turn drive the identification of potential therapeutic targets.

Small vessel disease (SVD) accounts for approximately 25% of all strokes and 45% of all dementias. Although the small vessels cannot be visualised with conventional neuroimaging, the pathological changes in the cerebral white and deep grey matter secondary to SVD has been adopted as markers of SVD. These are best appreciated with magnetic resonance imaging (MRI) and includes recent small subcortical infarcts, white matter hyperintensity (WMH), lacunes, cerebral microbleeds and enlarged perivascular spaces (PVSs). There are however a number of outstanding questions regarding these surrogate neuroimaging markers of SVD and how these markers may influence clinical management. First, although a high burden of microbleeds have been associated with an increased risk of intracerebral haemorrhage (ICH) and possibly recurrent ischaemic stroke in patients with TIA or ischaemic stroke, how microbleeds should influence antithrombotic treatment use after TIA or ischaemic stroke remains uncertain. Second, the long-term prognostic implications of enlarged PVSs in patients with TIA or ischaemic stroke have not been studied. Third, although previous studies have shown possible ethnic differences in prevalence of microbleeds, whether there are any ethnic differences in prevalence of other neuroimaging markers of SVD remains unclear. Fourth, although a Total SVD Score was recently proposed to measure the global SVD burden, the prognostic value of this score in patients with TIA or ischaemic stroke has yet to be studied. Fifth, the relationships of long-term premorbid blood pressure with global SVD burden is unknown. Finally, the age and sex specific associations between renal impairment, carotid and cerebral pulsatility with burden of SVD has yet to be studied. The aim of my thesis was therefore to determine the clinical correlates, ethnic differences and long-term prognostic implications of a range of neuroimaging markers and global burden of SVD. I also aimed to determine the relationships of global SVD burden with long-term mean premorbid blood pressure, renal impairment and carotid pulsatiltiy. I have collected, collated and analysed clinical and neuroimaging data from two independent cohorts - the Oxford Vascular Study (OXVASC) and The University of Hong Kong (HKU). In particular I worked as one of the Clinical Research Fellows at OXVASC and was involved in regular recruitment, assessment and follow up of study patients. In OXVASC, 1080 predominantly Caucasians with TIA or ischaemic stroke who had a cerebral MRI performed at baseline was recruited during 2004 to 2014. I interpreted all these MRIs, specifically coding the burden of microbleeds, enlarged perivascular spaces and lacunes. I was involved in obtaining funding and developing the HKU cohort, which includes 1003 predominantly Chinese with ischaemic stroke recruited during 2008-2014 who had a cerebral MRI performed at baseline. I saw about 25% of the patients in the cohort and was involved in interpreting all of the MRIs of the cohort. All patients from both cohorts were followed-up regularly and adverse events including recurrent ischaemic stroke and ICH was determined. Presence and burden of periventricular and subcortical WMH, lacunes, microbleeds, basal ganglia and centrum semiovale PVSs was determined for all patients and the global burden of SVD estimated according to the Total SVD Score. There are several clinically relevant findings in this thesis. First, I have shown that in Caucasians and Chinese with ≥5 microbleeds, withholding antiplatelet drugs during the first year after TIA or ischaemic stroke may be inappropriate, especially early after TIA. However, the risk of ICH is likely to outweigh any benefit thereafter. Second, I have shown that TIA or ischaemic stroke patients with microbleeds on warfarin had an increased risk of subsequent ICH. However, this risk was not different from that of antiplatelet users with microbleeds. Third, I have shown that a high burden of MRI-visible basal ganglia PVSs is independently associated with an increased risk of recurrent ischaemic stroke, but not ICH. However, the prognostic value of MRI-visible centrum semiovale PVSs in the TIA or ischaemic stroke population is limited. Fourth, I demonstrated significant ethnic differences in underlying prevalence and burden of neuroimaging markers of SVD - Chinese had a greater prevalence of microbleeds, lacunes and subcortical WMH, whilst Caucasians had a greater prevalence of periventricular WMH and PVSs. Fifth, I validated the Total SVD Score and showed that the SVD Score is able to predict risk of recurrent ischaemic stroke and ICH in Caucasians and Chinese, but is unable to identify patients at high risk of ICH from those at high risk of recurrent ischaemic stroke. Sixth, I showed that mean premorbid blood pressure, especially diastolic blood pressure measurements taken 10-20 years prior to TIA or ischaemic stroke was most strongly associated with global SVD burden suggesting a latency effect of hypertension on the pathogenesis of SVD. Finally, I demonstrated age-specific associations between renal impairment, internal carotid artery pulsatility index and SVD burden.

Cerebral small vessel disease (SVD) is a distinct microvascular disorder that can lead to lacunar stroke, an important stroke subtype that accounts for a quarter of all ischaemic strokes. SVD is associated with imaging biomarkers such as white matter hyperintensities (WMH). The cause of SVD is largely unknown, although inflammation and blood-brain barrier failure via endothelial dysfunction have been implicated. Elevated plasma biomarkers of inflammation are associated with coronary heart disease and large vessel stroke but the role of inflammation in SVD is less well understood. Our hypothesis is that inflammation plays a role in SVD and we sought to examine this by reviewing the literature for evidence of this, and by conducting a brain imaging study of patients with a known inflammatory disease and reviewing the images for evidence of inflammation and SVD, and comparing findings with controls groups. Section A: This thesis begins with a systematic review and meta-analysis of 13 plasma biomarkers of four physiological processes (coagulation, fibrinolysis, endothelial dysfunction and inflammation) in lacunar stroke versus non-lacunar stroke (to control for having any stroke) and non-stroke (to compare to the general population). We sought to know if there were differences in these biomarkers between lacunar stroke and other stroke subtypes and non-stroke controls as a way of generating hypotheses for the disease mechanisms that might lead to lacunar stroke. Findings revealed differences in several biomarkers between lacunar stroke and healthy controls but only fibrinogen, D-dimer, von Willebrand factor and interleukin-6 were different (all significantly lower in lacunar stroke) between lacunar stroke and other stroke subtypes. There was heterogeneity between studies, including variations in the definition of lacunar stroke and most studies measured the biomarkers in the acute phase post stroke, which is potentially confounding. To further examine plasma biomarkers of inflammation and endothelial dysfunction in SVD, we used data from a prior study of mild stroke conducted at the Brain Research Imaging Centre, University of Edinburgh, UK. Lacunar stroke patients were compared to cortical stroke patients. The lacunar group had lower levels of tissue plasminogen activator independent of age, sex and vascular risk factors but we found no difference in the other plasma biomarkers. Section B: Non-resolving systemic inflammation is a feature of inflammatory autoimmune rheumatic diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). These patients are at increased risk of stroke but much knowledge relates to stroke in general; less is known about associations with stroke subtypes including SVD, or when in life stroke risk is greatest. Consequently, we sought to better understand the influence of inflammatory rheumatic diseases on stroke and SVD. The review and meta-analysis of cerebrovascular disease in rheumatic diseases showed an excess risk of stroke in RA, SLE, ankylosing spondylitis, gout and psoriasis over the general population. Meta-analyses of stroke subtypes (ischaemic and haemorrhagic) in RA and SLE showed an excess risk of stroke over the general population. Stroke risk across rheumatic diseases was highest in those aged < 50 years and reduced with ageing. We then requested data from NHS Lothian covering 15 years so that we could assess stroke, including stroke subtypes, among patients diagnosed with various arthropathies. We linked 6,613 rheumatology patients’ records with stroke admission records, grouped the various rheumatic diseases into the two main types of arthritis, inflammatory and non-inflammatory, and also compared the strokes in these rheumatology patients to general population data. There was no difference in stroke prevalence between inflammatory and degenerative (non-inflammatory) arthropathies, although the strokes occurred up to two decades earlier than in the general population. Section C: Lastly, we conducted MRI neuroimaging in patients with SLE and reviewed and meta-analysed diffusion tensor imaging (DTI) (an imaging technique used to assess sub-visible white matter microstructure damage) in SLE to place our findings into context. The research question here was to ascertain if patients with a known inflammatory disease had brain imaging evidence of SVD, and to compare findings to controls. We compared imaging markers of SVD and DTI between SLE patients and age-matched healthy controls and sought associations between the imaging biomarkers and plasma biomarkers of inflammation and endothelial dysfunction, measures of fatigue and cognition, and scores of rheumatic disease activity. Fifty-one patients were recruited. There was higher mean diffusivity in all white matter tracts versus controls indicating a diffuse increase in brain water mobility in SLE. Meta-analysis confirmed higher mean diffusivity in SLE patients versus controls. Fatigue in SLE was significantly higher than a normal reference range and was associated with depression, anxiety, higher body mass index, lower mean diffusivity and some blood markers of inflammation and endothelial dysfunction. The most fatigued were youngest which explained the association with lower mean diffusivity. Damage to the brain’s white matter microstructure may be accelerated in SLE as the age-related declines in the general population are normally seen much later in life. The aging pattern is consistent with inflammation-related microvascular-mediated brain damage where the inflammation is systemic in origin. Summary: This thesis has demonstrated an increase in SVD burden in the inflammatory rheumatic disease SLE and increased stroke risk at younger ages in other inflammatory rheumatic diseases. Thus, systemic inflammation as seen in inflammatory rheumatic diseases could have effects on the brain directly, including influencing stroke risk which is clinically noteworthy and would benefit from further testing in appropriately designed studies such as an inception cohort that follows inflammatory rheumatic patients from diagnosis, with regular brain imaging to track brain changes and correlates with inflammatory profiles and impact on cognition.

Lacunar stroke accounts for one quarter of all ischaemic stroke and in the long term carries a greater risk of death and disability than was previously realised. Much of our current knowledge originated from neuropathological studies in the 1950s and 1960s. In the last thirty years, brain computed tomography (CT) and magnetic resonance imaging (MRI) have revolutionised our understanding of lacunar stroke and associated features of cerebral small vessel disease (SVD), namely white matter lesions (WML), enlarged perivascular spaces (EPVS) and brain microbleeds (BMB). The purpose of the projects which led to the writing of this thesis was to improve understanding of imaging characteristics of cerebral SVD. We aimed to assess (i) clinical and imaging features which might explain misclassification of lacunar infarcts as cortical infarcts and vice versa, (ii) the proportion of symptomatic lacunar infarcts progressing to lacunar cavities and associations of cavitation, (iii) completeness of reporting of lacunar lesions in the lacunar stroke literature, (iv) definitions and detection of lacunar lesions amongst SVD researchers, (v) the relationship between WML and carotid stenosis, (vi) clinical and imaging associations of EPVS and, (vii) observer variability in the assessment of EPVS and BMB, in order to develop visual rating scales. Section one describes neuroimaging of lacunar stroke. To investigate features which might explain clinical stroke subtype misclassification (‘clinical-imaging dissociation’), I used data from a stroke study. The main factor associated with clinical-imaging dissociation was diabetes, and in patients with acute lacunar infarction, proximity of the lacunar infarct to the cortex, age, diabetes and left hemisphere location. To investigate the proportion of symptomatic lacunar infarcts progressing to cavities, I used data from two stroke studies. A fifth of patients with acute lacunar ischaemic stroke showed definite cavitation on follow-up imaging at a median of 227 days; cavitation was associated with increasing time to follow-up. To assess completeness of reporting of lacunar lesions in the lacunar stroke literature, I reviewed 50 articles from three journals with a stroke focus. There was marked variation in terminology and descriptions of imaging definitions of lacunar lesions. To assess lacunar lesion definitions and detection amongst SVD researchers, I used an online survey consisting of case-based and non-case-based questions. There was marked variation in definitions and descriptions. Cavitated lesions were detected with the highest degree of confidence. Section two describes neuroimaging of associated features of cerebral SVD. Using data from two stroke studies, I examined the relationship between WML and ipsilateral carotid artery stenosis. There was no association between carotid stenosis and WML. I tested the association of EPVS with WML and lacunar stroke subtype using data from a stroke study. Total EPVS were associated with age and deep WML; basal ganglia (BG) EPVS were associated with age, centrum semiovale (CS) EPVS, cerebral atrophy and lacunar stroke subtype. Quantification of observer variability in EPVS rating was assessed on 60 MRI scans selected from a stroke study and an ageing cohort. Intrarater agreement was good and interrater agreement was moderate. Main reasons for interrater disagreement included the visualisation of very small EPVS and the presence of concomitant WML and lacunar lesions. Observer variability in BMB rating was quantified using MRI scans from a stroke study. Interrater agreement was moderate but improved following modification of the pilot rating scale (BOMBS; Brain Observer MicroBleed Scale), which had its main effect by differentiating ‘certain’ BMB from ‘uncertain’ BMB and BMB ‘mimics’. In conclusion, neuroimaging, particularly MRI, is a valuable tool for the investigation of lacunar stroke and associated features of cerebral SVD. With recent technological advances in both CT and MRI, neuroimaging will remain central to future SVD studies, hopefully leading to a much improved understanding of this important disease.

Lacunar strokes account for approximately a quarter of all ischaemic strokes and traditionally are thought to result from occlusion of a small deep perforating arteriole in the brain. Lacunar infarcts can be up to 2cm in diameter and are found in deep brain structures such as the thalamus and internal capsule. Despite their prevalence and specific accompanying clinical syndromes, the cause of lacunar stroke and its associated vascular pathology remain unclear. Many hypotheses as to the cause exist, which fall broadly into two categories; firstly, a direct occlusion via emboli or thrombus usually from a cardiac or large artery source, microatheroma (intrinsic lenticulostriate occlusion) or macroatheroma (parent artery occlusion) all operating primarily via ischaemia. Secondly, there could be an indirect occlusion resulting from vasospasm, endothelial dysfunction or other forms of endovascular damage (e.g. inflammation). Therefore the question of whether the resulting lesions are truly “ischaemic” or actually arise secondary to an alternative process is still under debate. To clarify the chain of pathological events ultimately resulting in lacunar stroke, in this thesis I firstly undertook a systematic assessment of human lacunar stroke pathology literature to determine the information currently available and the quality of these studies (including terminology). The majority of these studies were performed in patients who had died long after their stroke making it difficult to determine the early changes, and there were few patients with a clinically verified lacunar syndrome. Therefore I adopted alternative approaches. In this thesis, I systematically looked for all potential experimental models of lacunar stroke and identified what appears at present to be the most pertinent - the spontaneous pathology of the stroke-prone spontaneously hypertensive rat (SHRSP). However, the cerebral pathology described in this model to date is biased towards end stage pathology, with little information concerning the microvasculature (as opposed to the brain parenchyma) and confounding by use of salt to exacerbate pathology. Therefore, the aim of the experimental work in this thesis was to assess pathological changes within the cerebral vasculature and brain parenchyma of the SHRSP across a variety of ages (particularly young pre-hypertensive animals) and to look at the effects of salt loading on both the SHRSP and its parent strain (the Wistar Kyoto rat - WKY). Three related studies (qualitative and quantitative histology, immunohistochemistry and a microarray study of gene expression confirmed by quantitative PCR), revealed that the presence of inflammation (via significant changes in gene expression in the acute phase response pathway and increased immunostaining of activated microglia and astrocytes) plus alterations in vascular tone regulation, (via genetic alteration of the nitric oxide signaling pathway probably secondary to abnormal oxidative state), impaired structural integrity of the blood brain barrier (histological evidence of endothelial dysfunction and significantly decreased Claudin-5 staining) and reduced plasma oncotic potential (reduced albumin gene expression) are all present in the native SHRSP at 5 weeks of age, i.e. well before the onset of hypertension and without exposure to high levels of salt. We also confirmed previous findings of vessel remodelling at older ages likely as a secondary response to hypertension (thickened arteriolar smooth muscle, increased smooth muscle actin immunostaining). Furthermore, we found not only that salt exacerbated the changes see in the SHRSP at 21 weeks, but also that the control animals (WKY) exposed to a high salt intake developed features of cerebral microvascular pathology independently of hypertension (e.g. white matter vacuolation and significant changes in myelin basic protein expression). In conclusion, via the assessment of the most pertinent experimental model of lacunar stroke currently available, this thesis has provided two very important pieces of evidence: firstly that cerebral small vessel disease is primarily caused by a non-ischaemic mechanism and that any thrombotic vessel lesions occur as secondary end stage pathology; secondly that these features are not simply the consequence of exposure to raised blood pressure but occur secondary to abnormal endothelial integrity, inflammation, abnormal oxidative pathways influencing regulation of vascular tone and low plasma oncotic pressure. Patients with an innate susceptibility to increased blood brain barrier permeability and/or chronic inflammation could therefore have a higher risk of developing small vessel disease pathology and ultimately lacunar stroke and other features of small vessel disease. Research, addressing whether lacunar stroke patients should be treated differently to those with atherothromboembolic stroke is urgently needed.

Cerebral small vessel disease (SVD) is a common cause of strokes and dementia. The pathogenesis of SVD is poorly understood, but imaging and biochemical investigations suggest that subtle blood-brain barrier (BBB) leakage may contribute to tissue damage. The most widely-used imaging method for assessing BBB integrity and other microvascular properties is dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). DCE-MRI has primarily been applied in situations where contrast uptake in tissue is typically large and rapid (e.g. neuro-oncology); the optimal approach for quantifying BBB integrity in diseases where the BBB remains largely intact and the reliability of resulting measurements is unclear. The main purpose of this thesis was to assess and improve the reliability of quantitative assessment of subtle BBB disruption, in order to illuminate its potential role in cerebral SVD. Firstly, a systematic literature review was performed in order to provide an overview of DCE-MRI methods in the brain. This review found large variations in MRI procedures and data analysis methods, resulting in widely varying estimates of tracer kinetic parameters. Secondly, this thesis focused on the analysis of DCE-MRI data acquired in an on-site clinical study of mild stroke patients. After performing basic DCE-MRI processing (e.g. selection of a vascular input function), this work aimed to determine the tracer kinetic modelling approach most suitable for assessing subtle BBB disruption in this cohort. Using data-driven model selection and computer simulations, the Patlak model was found to provide accurate estimates of blood plasma volume and low-level BBB leakage. Thirdly, this thesis aimed to investigate two potential pitfalls in the quantification of subtle BBB disruption. Contrast-free measurements in healthy volunteers revealed that a signal drift of approximately 0.1 %/min occurs during the DCE-MRI acquisition; computer simulations showed that this drift introduces significant systematic errors when estimating low-level tracer kinetic parameters. Furthermore, tracer kinetic analysis was performed in an external patient cohort in order to investigate the inter-study comparability of DCE-MRI measurements. Due to the nature of the acquisition protocol it proved difficult to obtain reliable estimates of BBB leakage, highlighting the importance of study design. Lastly, this thesis examined the relationship between quantitative MRI parameters and clinical measurements in cerebral SVD, with a focus on the estimates of blood volume and BBB leakage obtained in the internal SVD patient cohort. This work did not provide evidence that BBB leakage in normal-appearing tissue increases with SVD burden or predicts disease progression; however, increased BBB leakage was found in white matter hyperintensities. Furthermore, this work raises the possibility of a role for blood plasma volume and dietary salt intake in cerebral SVD. The work described in this thesis has demonstrated that it is possible to estimate subtle BBB disruption using DCE-MRI, provided that the measurement and data analysis strategies are carefully optimised. However, absolute values of tracer kinetic parameters should be interpreted with caution, particularly when making comparisons between studies, and sources of error and their influence should be estimated where possible. The exact roles of BBB breakdown and other microvascular changes in SVD pathology remain to be defined; however, the work presented in this thesis contributes further insights and, together with technical advances, will facilitate improved study design in the future.

Background: Cerebral small vessel disease (SVD) causes substantial cognitive, psychiatric and physical disabilities. Despite its common nature, SVD pathogenesis and molecular mechanisms remain poorly understood, and prevention and treatment are probably suboptimal. Identifying the genetic determinants of SVD will improve understanding and may help identify novel treatment targets. The aim of this thesis is to better understand genetic associations with SVD through investigating its pathological, radiological and clinical phenotypes. Methods: To unravel the genetic associations with SVD, I used three complementary approaches. First, I performed a systematic review looking at existing intracerebral haemorrhage (ICH) classification systems and their reliability, to help inform future studies of ICH genetics. Second, I performed a series of systematic reviews and meta-analyses, investigating associations between genetic polymorphisms and histopathologically confirmed cerebral amyloid angiopathy (CAA). Third, I performed meta-analyses of existing genome-wide datasets to determine associations of >1000 common single nucleotide polymorphisms (SNP) in the COL4A1/COL4A2 genomic region with clinico-radiological SVD phenotypes: ICH and its subtypes, ischaemic stroke and its subtypes, and white matter hyperintensities. Results: The reliability of existing ICH classification systems appeared excellent in eight studies conducted in specialist centres with experienced raters, although these existing systems have several limitations. In my systematic evaluation of CAA genetics, meta-analyses of 24 studies including 3520 participants showed robust evidence for a dose-dependent association between APOE ɛ4 and histopathological CAA. There was, however, no convincing association between APOE ɛ2 and presence of CAA in a meta-analysis of 11 studies including 1640 participants. Meta-analyses of five studies including 497 participants showed, contrary to an existing popular hypothesis, that while APOE 4 may increase the risk of developing severe CAA vasculopathy, there is no clear evidence to support a role of ɛ2. There were few data about the role of APOE in hereditary CAA, but in the three studies that had looked at this, there was no evidence for an association between APOE ɛ4 and CAA severity. There were too few studies and participants to draw firm conclusions about the effect of non-APOE ε2/ε3/ε4 genetic polymorphisms on CAA, but there were positive associations with TGF-β1, TOMM40 and CR1 genes in four studies. Finally, in my meta-analyses of the COL4A1/COL4A2 genomic region, three intronic SNPs in COL4A2 were associated with SVD phenotypes: significantly with deep ICH, and suggestively with lacunar ischaemic stroke and WMH. Conclusions: I have shown that while existing ICH classification systems appear to have very good reliability, further research is needed to determine their performance in different settings. For large population-based prospective studies of ICH genetics, anatomical systems are likely to be more feasible, scalable and appropriate, although they have limitations and will need to be further developed. Using systematic reviews and meta-analyses, I have confirmed a dose-related association between APOE ɛ4 and histopathological CAA, but also demonstrated that, despite popular acceptance, there is insufficient data to draw firm conclusions about the association with APOE ɛ2. I found some positive associations with CAA in other genes, which merit replication in further larger studies, and showed that there is currently insufficient data about the role of APOE in hereditary CAA. Finally, I identified a novel association between a locus in a known hereditary SVD gene – COL4A2 – and sporadic SVD. This highlights a new and successful approach for selecting candidate genes and can be expanded in future studies to include other known hereditary SVD genes.

Small vessel disease (SVD) is an important cause of stroke, cognitive decline, and age-related disability. The cause of SVD is unknown, increasing evidence from neuropathology and neuroimaging suggests that failure of the blood-brain barrier (BBB) precipitates or worsens cerebral SVD progression and its failure is associated with SVD features such as white matter hyperintensities (WMH), perivascular spaces (PVS) and lacunar infarcts. The BBB change mechanism may also contribute to other common disorders of ageing such as Alzheimer's disease (AD). Magnetic resonance imaging (MRI) has revolutionised our understanding of SVD features. The MRI contributes to better understanding of the SVD pathophysiology and their clinical correlates. The purpose of this project was to better understand the pathogenesis of SVD, which involves improved understanding of BBB structures and pathophysiology and accurate measurement of cerebral SVD imaging characteristics on MRI scans. We aimed to assess (1) structures related to the BBB and factors that affect the BBB; (2) efficient and consistent WMH measurement method; (3) effect of stroke lesions on WMH and cerebral atrophy progression; (4) development and optimisation of computational PVS measurement method; (5) the relationships between PVS and SVD, blood markers, and BBB permeability. Section one describes structures and pathophysiology of the BBB. I reviewed the BBB structural and functional components from the view of neurovascular unit, PVS, and junctional proteins. The PVS part was done in a systematic search. I also reviewed some common stimuli for BBB permeability including inflammation and ischemia. Ischemic triggers for the BBB permeability were summarized systematically. Based on the literatures above, I summarized changes in junctional proteins in ischemia, inflammatory pain and AD models. Section two describes accurate measurement of WMH progression and atrophy. I used data from 100 patients who participated in a stroke study about BBB permeability changes in lacunar versus cortical stroke. To find a most efficient and consistent WMH measurement method, we tested several computational methods and effect of common processing steps including bias field correction and intensity adjustment. To avoid the effect of artefacts, I did a systematic search about artefacts and tested methods of image segmentation to avoid WMH artefacts as much as possible. To investigate the effect of stroke lesions on WMH and atrophy progression, I did the WMH, atrophy segmentation and stroke lesion measurements in a subgroup of 46 patients with follow-up scans, and showed that stroke lesions distorted measurement of WMH and atrophy progression and should be excluded. Section three describes development and optimization of a computational PVS measurement method, which measures the count and volume for PVS based on a threshold method using AnalyzeTM software. We tested the observer variability and validated it by comparison with visual rating scores. We investigated the associations between PVS results with other SVD features (WMH, atrophy), risk factors (hypertension, smoking and diabetes), blood markers, and BBB permeability. In conclusion, MRI is a valuable tool for the investigation of cerebral SVD features and BBB permeability. Exclusions of artefacts and stroke lesions are important in accurate measurement of WMH. PVS are important features of BBB abnormalities, and they correlate and share risk factors with other SVD features, and they should be considered as a marker of SVD and BBB permeability. Further systematic histological and ultrastructural studies of BBB are desirable in understanding the BBB regarding to the different parts of the cerebral vascular tree.

Cerebral small vessel disease (SVD) is a vascular neurodegenerative disease which is the leading cause of vascular dementia and causes 20% of strokes. 20-30% of those over 80 show signs of the disease as white matter hyperintensities on MRI scans, doubling their risk of stroke and trebling their risk of dementia. Sporadic SVD is thought to be caused by hypertension but 30% of sufferers are normotensive and an alternative hypothesis implicates loss of integrity of the blood brain barrier (BBB). To investigate this, I studied brains from normotensive people with early stage SVD and found reduced capillary endothelial claudin-5 (a BBB tight junction protein), more oligodendrocyte precursor cells (OPCs; the precursors to myelinating oligodendrocytes), and more microglia/macrophages compared to controls. Furthermore, in a relevant rat model of spontaneous SVD, the Stroke Prone Spontaneously Hypertensive Rat (SHRSP; disease model; DM) I found that reduced endothelial claudin-5 was the earliest change, appearing at 3 weeks of age, followed by OPC proliferation, appearing at 4 weeks, and then increased number of microglia/macrophages, appearing at 5 weeks. Importantly, all these changes occurred at a young age (< 5 weeks), before any measurable hypertension. These changes were confirmed in an ex vivo slice culture model (i.e. removing blood flow), ruling out direct damage by leakage of blood components through an impaired BBB and suggesting an inherent endothelial cell dysfunction as the primary cause, with secondary BBB defects. This hypothesis of endothelial dysfunction is supported by increased endothelial cell proliferation in both human SVD tissue and the DM rats, and lower levels of endothelial nitric oxide synthase (eNOS) in brains of DM rats. To study this further I isolated primary brain microvascular endothelial cells (BMECs) from DM and control rats and found that those from DM rats formed less mature tight junctions (less membranous claudin-5) than control BMECs. I also found that conditioned media (CM) from DM BMECs causes OPCs in culture to proliferate more and mature less. This indicates that the endothelial dysfunction is inherent to the endothelial cells, rather than induced by other cell types, and through secreted factors causes OPC changes mirroring what is seen in vivo. Using an antibody array, I identified HSP90α as a candidate secreted factor and showed that it is necessary (by blocking the protein in CM) and sufficient (by adding recombinant HSP90α) to induce the maturation phenotype in OPCs, but not the proliferation phenotype. The idea that endothelial dysfunction causes SVD begs the question of what causes endothelial dysfunction, especially in our inbred DM rat strain. To establish this, I reanalysed sequencing data of the DM and control rats from a previously published study, searching for mutations which lead to truncated proteins in genes expressed in brain endothelial cells. We confirmed the candidate gene Atp11b, a phospholipid flippase, was mutated as predicted. I found that knocking down Atp11b using siRNA in a control endothelial cell line caused endothelial dysfunction and a loss of tight junction maturity, and that CM from these cells causes OPCs to proliferate more and mature less, mirroring what we see in primary DM BMECs and suggesting that Atp11b has a key function in promoting normal endothelial function. Furthermore, I showed that knocking down Atp11b causes cells to secrete increased levels of HSP90α. I propose a mechanism whereby ATP11B regulates the retention of HSP90α within endothelial cells, which in turns regulates eNOS levels and activity, as has been shown previously. In summary, this work shows that there are many pre-symptomatic changes which occur in the brain in the development of SVD in DM rats, and that these are ultimately caused by endothelial dysfunction. As these changes are similar to those found in spontaneous human SVD, I propose that endothelial dysfunction is a key mechanism of human SVD, which may in the future lead to new therapies.

Cerebrovascular disease (CVD) can result in cerebral small vessel disease (cSVD) and structural brain changes such as decreased cortical volume, brain atrophy and cerebral infarcts which are major causes of stroke and dementia. CVD is also associated with increased depression and depressive symptoms in later life. Midlife vascular disease and adult socioeconomic status (SES) are well established risk factors but less is known about the effect of factors from earlier in life on CVD and depressive symptoms in later life. A series of systematic reviews of current literature examining early life factors and stroke, cSVD and depression following stroke are presented at the beginning of this thesis. These reviews found that childhood IQ, education and childhood SES were associated with stroke and cSVD in later life. The reviews also found that education level was associated with depression following stroke. However few of the studies adjusted for vascular risk factors and adult SES. Therefore this thesis aimed to investigate associations between birth and childhood factors and cerebrovascular disease and depressive symptoms, after adjustment for vascular risk factors and adult SES, in four community dwelling cohorts: the Stratifying Resilience & Depression Longitudinally (STRADL) cohort (n=280, 45% male, mean age= 62.1 (SD=4.1) years), the Dutch Famine Birth cohort (n= 151, 44% male, mean age 67.6 (SD=0.9) years), the Lothian Birth Cohort 1936 (LBC 1936, n= 865, 50% male, mean age 72.7 (SD=0.7) years), and the Simpson cohort (n=130, 31% male, mean age 78.5, (SD=1.5) years). This Thesis first examined associations between (i) cSVD burden (ii) total and regional brain volumes and (iii) self-reported symptoms of depression and anxiety measured using the Hospital Anxiety and Depression Scale. All analyses were adjusted for age, sex, hypertension, smoking behaviour, adult SES and cognition. Neither cSVD nor brain volumes were associated with symptoms of anxiety. Higher white matter hyperintensity volumes, having one or more cerebral infarct and increased cerebral atrophy were associated with increased depressive symptoms independent of vascular risk factors and adult SES. Secondly, this thesis examined associations between birth and childhood factors and cSVD burden and total and regional brain volumes. Each cohort was analysed individually and then all available data meta-analysed. All analyses were adjusted for age, sex, hypertension, smoking behaviour, adult SES and other early life factors. Meta-analysis found that increasing birth weight was associated with decreased risk of lacunes across all cohorts. Placental weight, which was only available for the Simpson cohort, was associated with decreased risk total cSVD, WMH severity and volume and cerebral infarcts. In the LBC 1936 and Simpson cohort increasing childhood and premorbid IQ and more years of education were associated with fewer cortical infarcts. The association between premorbid and childhood IQ and infarcts was independent of education level. Across three cohorts low education level was associated with more microbleeds. These findings suggest that factors other than traditional vascular risk factors may contribute to cSVD and structural brain changes in later life. Thirdly, this thesis examined associations between birth and childhood factors and self-reported symptoms of depression and anxiety measured using the Hospital Anxiety and Depression Scale (HADS) and the Quick Inventory of Depressive Symptoms (QIDS-16). All analyses were adjusted for age, sex, hypertension, smoking behaviour, adult SES and cognition In the Dutch Famine Birth Cohort people born before the famine had lower scores of depression and anxiety on the HADS compared to those exposed to famine in early gestation and those conceived after the famine. In the LBC 1936 increasing ponderal index was associated with lower depressive symptoms, increasing childhood and premorbid IQ were associated with lower symptoms of anxiety and depression. Lower educational attainment and some indicators of childhood SES were associated with higher symptoms of depression and anxiety. Overall results suggest that early life factors, particularly childhood IQ, may contribute to structural brain changes and symptoms of depression and anxiety in later life, independent of vascular risk factors and other early life factors. Efforts to understand factors which may contribute to late life health, from the earliest stages of life, are important and may be used to inform changes in social policy. The effect sizes and potential impact of these findings suggest that larger sample sizes with more vascular disease and more depression are needed to robustly test these associations.