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Small vessel disease (SVD) is a common cause of strokes and dementia. Currently, there are no treatments; therefore, developing and validating early biomarkers of disease progression and treatment response is important for future drug trials. Though SVD pathogenesis is not well understood, findings from previous studies suggest that blood-brain barrier dysfunction and impaired cerebrovascular reactivity (CVR) contribute to the disease. The latter can be measured in vivo using a vasoactive stimulus in parallel with magnetic resonance imaging (MRI) techniques sensitive to blood flow, such as blood oxygen level dependent (BOLD) contrast, and has frequently been assessed in patients with steno-occlusive diseases. However, it is unclear if the technique is reliable when investigating cerebrovascular health in deep structures of the brain where SVD is prevalent. Therefore, this thesis aimed to assess and optimise the reliability of CVR measurements and deepen our understanding of its role in SVD pathogenesis. A systematic review was performed to provide a detailed overview of CVR MRI methodologies and clinical applications, including SVD, present in the literature, which identified several acquisition and analysis methods, a need for greater standardisation and lack of data on reliability. Specifically in SVD research, there was limited application of CVR MRI in SVD populations, little optimisation and reliability assessment of CVR in deep brain structures relevant to SVD, such as in white and subcortical grey matter. Following those findings, the effects of voxel- and region-based analysis approaches on reliability of CVR estimates were investigated using simulations and test-retest data from healthy volunteers. Voxel-based CVR magnitude estimates in tissues with high noise levels were prone to bias, whereas biases in region-based estimates were independent of noise level, but consistently underestimated CVR magnitude relative to the ground-truth mean. Furthermore, the test-retest study confirmed the repeatability of CVR estimates from a BOLD-CVR experiment with fixed inhaled stimulus, although a systematic, but small, bias was detected due to habituation to the gas challenge. The data from healthy volunteers were further used to conduct a proof-of-concept and investigate the feasibility of extracting cerebral pulsatility from BOLD-CVR data. Small-to-moderate correlations with pulsatility from phase-contrast MRI were found depending on the regions considered. CVR pulsatility was also computed in a small cohort of SVD patients: it was higher than in healthy volunteers, but no associations were found with SVD burden. It was concluded that further optimisation and validation of the technique is needed before being suitable for clinical research. Following the optimisation of the CVR MRI technique, relationships between CVR and SVD neuroimaging features, cognition, stroke severity and outcome were investigated cross-sectionally and longitudinally in a cohort of patients with mild stroke. In the cross-sectional analysis, CVR impairment in normal-appearing and damaged tissues was associated with worse SVD burden and cognition deficit. Furthermore, the longitudinal analysis showed that baseline CVR impairment predicted worsening of white matter hyperintensity and perivascular space volumes after one year. In conclusion, assessment of CVR in the brain and its deeper structures was successfully conducted in healthy volunteers and patients with SVD using MRI. However, this required appropriate optimisation of processing strategy as the latter can affect accuracy of CVR parameters and inter-study comparability. Importantly, applying the technique in a cohort of SVD patients led to the findings that CVR impairment was related to worse SVD burden and is a potential marker of SVD severity and progression.

Cerebral small vessel disease (SVD) is the breakdown of the small blood vessels of the brain, leading to many cases of stroke and dementia. The pathophysiology of SVD is largely unknown, although several mechanisms have been suggested. One such mechanism is the role of increased blood flow pulsatility into the brain, caused by vessel stiffening, leading to damage of the microvasculature. Magnetic resonance imaging (MRI) allows us to non-invasively measure blood flow and velocity using a technique called phase contrast-MRI - traditionally used with 2D slices across the vessel(s) of interest. An advanced form of phase-contrast MRI, known as 4D flow, has emerged in recent years that allows for a volume of data to be acquired, containing velocity information in all directions. However, to keep scan times practical when collecting this amount of data, spatiotemporal resolution has to be sacrificed. The main aim of this thesis was to assess 4D flow's capabilities, including comparing it to the more well-established 2D method in healthy volunteers, patients, and phantom experiments, so as to better understand its role in investigating SVD. Another aim was to learn more about the role of flow and pulsatility in SVD development in patients using data acquired in the longitudinal Mild Stroke Study 3 (MSS3). Firstly, I systematically reviewed studies that have assessed the human brain using 4D flow. Across 61 relevant studies, I found a general consensus for the current use of the technique in this context. I then optimised the Siemens prototype 4D flow sequence (N = 11 healthy volunteers), testing different parameters to find the combination that best balanced scan quality and duration. I then assessed the test-retest repeatability and intra-rater reliability of both 2D and 4D methods (N = 11 healthy volunteers), as well as differences between them. Following this, I performed the same 4D-2D comparison on SVD patients (N = 10). Absolute flow measurements using 4D flow were shown to have moderate repeatability and reliability, while flow pulsatility measurements showed acceptable repeatability and reliability. Furthermore, 2D arterial pulsatility was measured higher than with 4D, while 4D often measured higher flow rates than 2D. 4D flow was shown to be feasible when used on SVD patients, with no noticeable issues caused by potential patient movement. Flow data analysis from the longitudinal SVD study MSS3 showed that intracranial pulsatility is associated with cross-sectional SVD lesion volume but not longitudinal lesion growth, with stronger associations seen in the arteries of the neck compared to the venous sinuses.

Magnetic Resonance Imaging (MRI) has become a routine clinical procedure for the screening, diagnosis and treatment monitoring of various clinical conditions. Although MRI has highly desirable properties such as being completely non-ionizing and providing excellent soft tissue contrast which has resulted in its widespread usage across the gamut of clinical applications, it is limited by a slow data acquisition process. Several techniques have been developed over the years that have considerably improved the speed of MRI but there is still a clinical need to further accelerate MRI for many clinical applications. This thesis focuses on two recent advances in MRI acceleration to reduce the overall patient scan time. The first part of the thesis describes the development of a fast 3D neuroimaging methodology that has been implemented in a clinical Magnetic Resonance (MR) sequence which was accelerated using a combination of compressed sensing and sampling order optimization of acquired measurements. This methodology reduced the overall scan time by more than 60% compared to the normal scan time while also producing images of acceptable quality for clinical diagnosis. The clinical utility of accelerated neuroimaging is demonstrated by conducting a healthy volunteer study on eight subjects using this fast 3D MRI method. The results of the radiological diagnostic quality assessments that were carried out on the accelerated human brain MR images by four experienced neuroradiologists are presented. The results show that accelerated MR neuroimaging retained sufficient clinical diagnostic value for certain clinical applications. The second part of the thesis describes the development of an accelerated Cartesian sampling scheme for a rapid quantitative MR method called Magnetic Resonance Fingerprinting (MRF). This method was able to simultaneously generate quantitative multi-parametric maps such as T1, T2 and proton density (PD) maps in a very short scan duration that is clinically acceptable. The developed Cartesian sampling method using Echo Planar Imaging (EPI) is compared with conventional spiral sampling that is generally used for MR fingerprinting. The ability of novel iterative reconstruction techniques to improve the multi-parametric estimation accuracy is also demonstrated. The results show that accelerated Cartesian MR fingerprinting can be an alternative to conventional spiral MR fingerprinting.

Vascular cognitive impairment describes a heterogeneous condition in which cognitive decline is precipitated by underlying cerebrovascular dysfunction. Ageing, as well as vascular diseases such as hypertension, stroke, cerebral small vessel disease and cerebral amyloid angiopathy, are risk factors for vascular cognitive impairment. The precise mechanisms by which these conditions impact the cerebral vasculature to drive cognitive decline, however, are unknown. Previous research has indicated that vascular risk factors can lead to microvascular oxidative stress, inflammation and endothelial dysfunction that can lead to tissue hypoperfusion, the development of white and grey matter vascular lesions (microinfarcts and microbleeds) and cognitive impairment. It was hypothesised that ageing, a prominent risk factor for cognitive decline, would induce impairments on neurovascular coupling resulting from neurovascular unit disruption. It was further hypothesised that induction of chronic cerebral hypoperfusion would mediate neurovascular dysfunction and vascular lesion development through increased oxidative stress, resulting in cognitive decline. Finally, it was also hypothesised that neurovascular impairments resulting from ageing and chronic cerebral hypoperfusion would be exacerbated in the presence of amyloid deposition. Four studies were performed in order to test these hypotheses. Vascular risk factors can be reproduced using experimental mouse models and provide a valuable basis in which to test hypotheses and therapeutic interventions. As such, a primary aim of this thesis was to develop and validate sensitive MRI approaches that would allow the detection of vascular alterations in vivo. In the first series of studies, MRI techniques to assess resting cerebral blood flow, vessel number, vascular lesions and inflammation in experimental mice were validated using established in vivo and ex vivo techniques, so that these techniques could be used in subsequent studies for vascular assessments in vivo. Arterial spin labelling was developed to assess resting cerebral blood flow, and was able to detect reductions in blood flow following cerebral hypoperfusion that correlated well with those obtained from laser speckle imaging. Q-map imaging was able to detect reductions in vessel number in acute lesions, and in non-lesioned mice measures of vessel number correlated well with histopathological measures. Structural T2 imaging was performed in order to detect ischaemic and haemorrhagic lesions in chronically hypoperfused mice, and was validated using H&E and Perls’ staining. Finally, contrast-enhanced T2* imaging was used to detect iron oxide uptake by macrophages in the brains of hypoperfused mice, which was further validated by the identification of iron-containing macrophages in immunostained brain sections. The second study was conducted to test the hypothesis that ageing would impair neurovascular unit function and structure, and that these impairments would be exacerbated in the presence of amyloid pathology. The aim of the study was to incorporate previously developed in vivo imaging approaches in the assessment of vascular function and alterations in neurovascular unit structure in both wild type and TgSwDI mice. As predicted, ageing caused a pronounced deficit on measures of neurovascular coupling, however this was not exacerbated by accumulation of amyloid in TgSwDI mice and was not associated with alterations in baseline blood flow measured by arterial spin labelling. Structural assessment of the neurovascular unit revealed a loss of contact between astrocytic endfeet and vasculature, which was significantly associated with the impairment on neurovascular coupling, in addition to other markers of breakdown of the neurovascular unit such as loss of pericyte coverage and microglial activation. Age and thalamic vascular amyloid accumulation were also associated with an increase in the NADPH oxidase (NOX) subunit p47, indicative of increased oxidative stress. Data from this experiment indicate that ageing can profoundly impair neurovascular coupling, mediated by gliosis and loss of astrocytic contacts with vasculature. The third study aimed to test the hypothesis that chronic cerebral hypoperfusion (a prominent early feature of vascular cognitive impairment) would impair vascular function and induce the development of vascular lesions and cognitive decline. The impact of hypoperfusion on neurovascular coupling, ischaemic and haemorrhagic lesion burden and cognition was investigated in wild type and TgSwDI mice. Hypoperfusion induced deficits on neurovascular coupling, increased lesion burden and inflammation assessed with T2 and contrast-enhanced T2* imaging, and caused impairment on measures of learning and memory. Hypoperfusion was also associated with an increase in the levels of NOX2, NOX4 and 3-NT at 3 months following surgery, indicating persistent reactive oxygen species production and oxidative damage in hypoperfused mice. The findings from this study indicate that vascular dysfunction and cognitive impairment following hypoperfusion may be mediated by increased NADPH oxidase activity and resulting oxidative stress. The previous studies indicated that markers of oxidative stress were induced in response to ageing, vascular amyloid accumulation and cerebral hypoperfusion. The final study sought to determine whether increased NOX activity mediates downstream pathological effects on vascular function, vascular lesion development and cognitive decline following hypoperfusion. NOX activity was inhibited pharmacologically by administration of apocynin to hypoperfused TgSwDI mice for 3 months following surgery. Treatment with apocynin significantly restored neurovascular coupling to a level similar to sham-operated mice, and there was a trend toward reduction of ischaemic vascular lesions. However, it was unable to rescue the prominent inflammatory response or decline in cognitive ability, as apocynin-treated mice were no different on these measures to non-treated hypoperfused mice. The data indicate that whilst inhibiting NOX may have potential therapeutic value in improving vascular function, additional interventions, for example to reduce inflammation, may also be required in order to prevent cognitive decline. Overall, the work outlined within the thesis indicate that vascular risk factors of ageing, cerebral amyloid angiopathy and cerebral hypoperfusion may converge on common pathways involving oxidative stress and increased inflammation in order to drive vascular dysfunction and lead to cognitive decline. Inhibition of NOX activity was able to rescue vascular function, however the results indicate that this was not sufficient to protect against cognitive impairment, suggesting additional therapeutic targets may need to be sought in order to fully preserve vascular health and prevent cognitive decline.

Ischaemic stroke is a leading cause of death and disability in the developed world. Despite that considerable advances in experimental research enabled understanding of the pathophysiology of the disease and identified hundreds of potential neuroprotective drugs for treatment, no such drug has shown efficacy in humans. The failure in the translation from bench to bedside has been partially attributed to the poor quality and rigour of animal studies. Recently, it has been suggested that multicentre animal studies imitating the design of randomised clinical trials could improve the translation of experimental research. Magnetic resonance imaging (MRI) could be pivotal in such studies due to its non-invasive nature and its high sensitivity to ischaemic lesions, but its accuracy and concordance across centres has not yet been evaluated. This thesis focussed on the use of MRI for the assessment of late infarct size, the primary outcome used in stroke models. Initially, a systematic review revealed that a plethora of imaging protocols and data analysis methods are used for this purpose. Using meta-analysis techniques, it was determined that T2-weighted imaging (T2WI) was best correlated with gold standard histology for the measurement of infarctbased treatment effects. Then, geometric accuracy in six different preclinical MRI scanners was assessed using structural phantoms and automated data analysis tools developed in-house. It was found that geometric accuracy varies between scanners, particularly when centre-specific T2WI protocols are used instead of a standardised protocol, though longitudinal stability over six months is high. Finally, a simulation study suggested that the measured geometric errors and the different protocols are sufficient to render infarct volumes and related group comparisons across centres incomparable. The variability increases when both factors are taken into account and when infarct volume is expressed as a relative estimate. Data in this study were analysed using a custom-made semi-automated tool that was faster and more reliable in repeated analyses than manual analysis. Findings of this thesis support the implementation of standardised methods for the assessment and optimisation of geometric accuracy in MRI scanners, as well as image acquisition and analysis of in vivo data for the measurement of infarct size in multicentre animal studies. Tools and techniques developed as part of the thesis show great promise in the analysis of phantom and in vivo data and could be a step towards this endeavour.

The study of brain temperature is important for a number of clinical conditions such as stroke, traumatic brain injury, schizophrenia and birth asphyxia (for neonates). A direct method to estimate brain temperature non-invasively will allow assessment of brain thermoregulation and its variation in clinical conditions. Magnetic resonance imaging is a powerful technique widely used for diagnosis of a range of neurological conditions. All magnetic resonance procedures involve manipulation of the hydrogen nuclei in the water molecules of the human body. The resonance frequency of the water molecules is temperature dependent, thus MR thermometry is a powerful tool for non-invasive temperature measurement. Using internal reference MR spectroscopic imaging (MRSI), absolute brain temperature maps can be estimated. However a number of temperature independent factors influence MRSI data acquisition, thus a thorough validation is necessary and is the focus of this PhD study. In this PhD study using phantom (test object) studies it was shown that optimization of the MRSI pulse sequence is necessary to reduce systematic error in temperature maps and extensive in-vitro validation of MRSI temperature mapping was performed. A custom made temperature-controlled phantom was designed for this purpose and is presented in this thesis. MRSI data acquired from healthy (young and elderly) volunteers was employed to assess regional brain temperature variations and repeatability. Finally, the feasibility of employing fast echo planar spectroscopic imaging for volumetric MRSI temperature mapping will be presented in this thesis.

Time-resolved velocity information of carotid blood flow can be used to estimate haemodynamic conditions associated with carotid artery disease leading to stroke. MRI provides high-resolution measurement of such information but long scan time limits its clinical application in this area. In order to reduce scan time the MRI signal is often undersampled by skipping part of the signal during data acquisition. The aim of this work is to implement and evaluate different undersampling techniques for carotid velocity measurement on a 1.5 T clinical scanner. Most recent undersampling techniques assume spatial and temporal redundancies of real time-resolved MRI signal. In these techniques different undersampling strategies were proposed. Prior information or different assumptions of the nature of true signal were used in signal reconstruction. A brief review of these techniques and details of a representative technique, known as k-t BLAST, are presented. Another undersampling scheme, termed ktVD, is proposed to use predesigned undersampling patterns with variable sampling densities in both temporal and spatial dimensions. It aims to collect enough signal content at the signal acquisition stage and simplify signal reconstruction. Fidelity of the results from undersampled data is affected by many factors, such as signal dynamic content, degree of signal redundancy, noise level, degree of undersampling, undersampling patterns, and parameters of post-processing algorithms. Simulations and in vivo scans were conducted to investigate the effects of these factors in time-resolved 2D scans and time-resolved 3D scans. The results suggested velocity measurement became less reliable when they were obtained from less than 25% of the full signal. In time-resolved 3D scans the signal can be undersampled in either one or two spatial dimensions in addition to the temporal dimension. This allows more options in the design of undersampling patterns, which were tested in vivo. In order to test undersampling in three dimensions in high resolution 3D scans and measure velocity in three dimensions, a flow phantom was also scanned at high degrees of undersampling to test the proposed method.