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White matter (WM) makes up a critical component of the CNS, composed primarily of myelinated axons. Herein, axons and the myelin sheath form a dynamic and communicative axo-glial unit which acts to regulate and support axonal function. The myelin sheath not only electrically insulates axons allowing for rapid and efficient axonal conduction and complex neuronal circuitry but also supports axonal viability. The loss or dysregulation of the myelin sheath is detrimental to axons as well as overall brain function. Furthermore, this is a key hallmark, not only, of classical WM diseases including multiple sclerosis but also is observed in brain ageing and dementias including Alzheimer's disease. Where, it is considered a key driver of cognitive impairment and disability. Thus, the need to have robust and reliable means of modelling WM pathology is critical, not only to determine how WM specifically responds to pathology but furthermore to identify therapeutic targets and agents that act to preserve WM integrity. The results of which could have widespread clinical implications and benefits. This thesis will examine this concept in two very distinct models; one utilising acute ex-vivo modelling, the other chronic and in-vivo, predominately these models will be assessed both functionally using electrophysiology and structurally using microscopy. Although unrelated, the observations made in these models will hopefully give insights into some critical considerations and the potential future of modelling WM pathology. As well as identifying new potential therapeutics which may be successful in the preservation of WM clinically.

Systematic Review Abstract Background: Multiple Sclerosis (MS) is a complex neurodegenerative condition. People with MS (pwMS) frequently encounter healthcare professionals (HCPs) and healthcare services. Understanding the healthcare experiences of pwMS is essential, as often, their needs are unmet. This can impact the management of disease, long-term health outcomes, and future healthcare access. Objectives: This qualitative systematic review aimed to understand pwMS' healthcare experiences across an international context. Method: A systematic search of Embase, MEDLINE, and CINAHL was conducted, followed by a forward citation search. Twenty-five papers met the full-text screening criteria. The papers were summarised and critically evaluated, and a thematic synthesis was conducted. Results: Six main themes were generated; pwMS felt there was a lack of person centred information from HCPs, holistic care options, and attention to inclusion and diversity. PwMS felt dismissed and abandoned by HCPs and experienced a range of difficult emotions both due to their MS and healthcare experiences. PwMS highly valued peer support. Conclusions: The findings from the review indicate that pwMS would like increased person-centred information about their MS, attention to inclusion and diversity, and a comprehensive range of care options. The findings highlight the emotional impact of living with MS, the healthcare experience, and the importance of peer support. Based on these findings, clinical implications for HCPs and clinical psychologists are suggested. Empirical Paper Abstract Objective: I aimed to explore people with Multiple Sclerosis's (pwMS) narrative experiences of outpatient neurology appointments, particularly concerning their relationships with their neurologists. Design: I interviewed twelve participants with Multiple Sclerosis (MS). Participants' interviews were analysed using narrative analysis. I identified story summaries, subject positions, turning points. I used positioning theory to consider the patient doctor relationship. Results: Three main narrative types were identified: processing, frustration, and acceptance. The analysis identified participants' differing positions as both a person with MS and their peers with MS. Within their narratives participants positioned their neurologists as either 'heroes' or 'villains'. Analysis of turning points highlighted that for participants, the COVID-19 pandemic and having something they could practically do for their MS changed how participants spoke about their MS and their neurology appointments. Conclusion: The findings indicated the different experiences of pwMS in outpatient neurology appointments and how their needs change across the care pathway. The findings also tell us about the importance of the patient-doctor relationship and the impact this relationship can have on a person's experience of MS. Practical suggestions are made for clinical psychologists working with neurologists in multi disciplinary teams (MDTs).

Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system (CNS) targeting the myelin sheath and oligodendrocytes (OL). Destruction of both can lead to the loss of underlying neurons and functional decline in patients. Myelin regeneration (remyelination) can occur in early stages of the disease but often fails with disease progression, in part due to impaired oligodendrocyte progenitor (OPC) differentiation. However, to date there are no treatments available that can therapeutically enhance remyelination. Intriguingly, remyelination is boosted in areas of inflammation. IL-1β, a potent pro-inflammatory mediator, was found in MS lesions but conversely, was also shown to enhance CNS repair in an animal model of MS. However, its function in OPC biology, myelination and remyelination remains incompletely understood. This project aims to determine the function of pro-inflammatory IL-1β signalling on glial cells in non-injury context and after damage. Mixed and pure glial cultures were used to investigate the impact of IL-1 signalling on OPC biology in context of other CNS glial cells and in isolation. IL-1β exposure enhanced differentiation of OPC and increased myelin basic protein (MBP) production indicating OPC differentiation into mature OL but did not affect OPC proliferation. In order to investigate the role of IL-1 signalling on glial cells after myelin damage mice deficient of IL1 signalling were subjected to focal demyelination and oligodendrocyte lineage cells investigated. Absence of IL-1 signalling did not affect OPC differentiation or OL numbers in the damaged area but enhanced the number of proliferating OPC early after demyelination. We here show OPC response to IL-1β varies depending on the context. As IL-1β was found in MS lesions, this could be important for the outcome of the response after myelin damage. Further analysis will uncover the underlying mechanisms, which might prove valuable for the development of novel MS therapies targeting IL-1 signalling.

Multiple sclerosis (MS) is an inflammatory demyelinating neurodegenerative disease of the human central nervous system (CNS). MS research has traditionally focused on the white matter, but the importance of grey matter pathology is increasingly acknowledged, as neuronal loss and cortical atrophy correlate with disability progression, while MS therapeutics fail to significantly impact grey matter neurodegeneration. Evidence from other neurodegenerative diseases suggests that the structural and functional compromise of synapses may occur prior to irreversible neuronal demise, and previous work from our lab reveals a selective vulnerability of inhibitory synapses in MS grey matter. Microglia, the resident myeloid cells in the CNS parenchyma, are candidate effectors of MS-associated synaptic loss, as they are reported to eliminate synaptic connections during development and contribute to degeneration-associated synaptic compromise. The present PhD thesis asks how microglia contribute to synaptic pathology in the MS grey matter using post-mortem human tissue and two mouse models of grey matter demyelination and remyelination. The motor cortex is investigated, as previous results from our lab show the specific functional compromise and structural demise of motor cortical inhibitory synapses and inhibitory neurons. I demonstrate that the cuprizone model of global toxic demyelination recapitulates this pattern of pathology, as it produces a selective loss of cortical inhibitory synapses early in demyelination, appearing before signs of axonal damage and in the absence of neuronal loss. During cortical remyelination the density of inhibitory synapses returns to control levels, suggesting recovery of lost synaptic structures. Excitatory synapses remain unaffected throughout demyelination and remyelination in this model. As cortical demyelination in the cuprizone model proceeds in the absence of overt inflammation and peripheral immune infiltration, a second model of cortical demyelination is also investigated. The focal experimental autoimmune encephalomyelitis (EAE) model recapitulates aspects of autoimmune cortical demyelination by the intracortical administration of a proinflammatory cytokine mixture in mice with clinical signs of EAE. Findings in the focal EAE model agree with the human post-mortem and cuprizone results, and show the selective loss of cortical inhibitory synapses and inhibitory neurons during autoimmune cortical demyelination, while excitatory synapses and excitatory neurons remain unaffected. Furthermore, using the cuprizone model, I interrogate the role of microglia in the early vulnerability of inhibitory synapses during demyelination. I find evidence of microglial engulfment of inhibitory synaptic components early during cortical demyelination, but not at maximal demyelination, and that it corresponds to the extent of inhibitory synaptic loss. Findings in the cuprizone model are corroborated by evidence of elimination of inhibitory synaptic elements by microglia in post-mortem cortical samples from donors with progressive MS. In the cuprizone model, microglia also appear to engulf excitatory synaptic components even though excitatory synapses are not affected by demyelination, thus questioning the role of microglia in inhibitory synaptic pathology. To investigate whether microglia actively contribute to the observed inhibitory synapse loss or passively phagocytose already discarded synaptic materials, I deplete microglia pharmacologically during cuprizone-induced demyelination. Microglial depletion prevents the loss of cortical inhibitory synapses during demyelination, while excitatory synapses remain unperturbed. Following depletion, engulfment of inhibitory and excitatory synaptic components is diminished. Overall, the results of the present thesis suggest that the inhibitory network is selectively vulnerable during cortical demyelination, in the presence or absence of autoimmunity. Microglia are required to execute inhibitory synapse loss, while excitatory synapses may have superior compensatory mechanisms that allow them to withstand demyelinating insults and microglial engulfment. The spontaneous recovery of inhibitory synapses during remyelination suggests that early synaptic pathology may be a therapeutic target in MS to prevent subsequent neurodegeneration.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system where neurodegeneration often leads to irreversible disability. Clinical and pathological heterogeneity is the rule, with genetic (e.g. HLA-DRB1*15 status) and environmental factors playing important roles. The mechanisms underlying such heterogeneity remain elusive. Astrocytes, which are involved in MS pathogenesis, are an attractive candidate to contribute to such variation in MS pathology due to their complex functions in health and disease. Recent progress has also challenged the conventional view that astrocytes represent a homogenous population, indicating that astroglial diversity may underscore MS heterogeneity. In this thesis, we undertake a quantitative neuropathological analysis of GFAP+ and ALDH1L1+ astrocyte subpopulations in the motor cortex of a large cohort of human post-mortem MS cases genotyped at the HLA-DRB1*15 locus. We find that GFAP+ and ALDH1L1+ astrocytes represent distinct subpopulations that differ in their distribution along the motor cortex and in their relationships with MS pathological outcomes. GFAP+ astrocytes show a greater extent of reactivity in the MS motor cortex, especially in areas of demyelination and in younger HLA- DRB1*15+ cases. In contrast, MS ALDH1L1+ astrocytes associate with neuronal density and macrophage/microglial inflammation, a relationship not seen in cases carrying the HLA-DRB1*15 allele. These astrocyte subpopulations also vary in their relationship with surrogate markers of blood-brain barrier dysfunction with decreasing perivascular GFAP+ astrocyte expression and increasing perivascular ALDH1L1+ astrocyte expression in MS. Given the histopathological differences between the two astrocyte markers, we also perform proteomics using laser capture microdissection coupled to mass spectrometry to probe the deep proteome of GFAP+ and ALDH1L1+ astrocyte subpopulations. Protein expressions show clear differences between GFAP+ and ALDH1L1+ astrocytes. In MS cases, GFAP+ astrocytes abundantly express immune-related proteins compared to ALDH1L1+ astrocytes, which associate with protein expression changes related to synaptic vesicle clustering and voltage-gated potassium channels. Immune-related proteins in astrocyte subpopulations are impacted by the carriage of the HLA-DRB1*15 allele. Altogether, our data implicate astrocyte subpopulation diversity as a contributing factor of MS heterogeneity. We suggest that GFAP+ astrocytes have relatively detrimental roles, whilst ALDH1L1+ astrocytes have relatively protective roles in MS. This thesis highlights the pressing need to understand the role of astrocyte diversity in MS so that selective targets amenable to therapeutic modulation may be identified and halt the devastating outcomes of MS.

Multiple sclerosis (MS) is a highly prevalent demyelinating autoimmune condition; the mechanisms regulating its severity and progression are unclear. The IL-17-producing Th17 subset of T cells has been implicated in driving MS and the mouse model, experimental autoimmune encephalomyelitis (EAE). However, the differentiation and regulation of Th17 cells during EAE remain incompletely understood. Although evidence is mounting that the antimicrobial peptide cathelicidin profoundly affects early T cell differentiation, no studies have looked at its role in longer term T cell responses. The aim of this project was to investigate cathelicidin expression in EAE and MS, determine which cells produce cathelicidin and elucidate how T cells respond to cathelicidin during disease. Here I show that cathelicidin drives severe disease in EAE. Cathelicidin is expressed during disease, and released from neutrophils, microglia and endothelial cells throughout disease. Mice lacking cathelicidin are protected from severe EAE, but surprisingly have equivalent central nervous system leukocyte infiltration as wildtype mice. It's interaction with CD4+ T cells increases T cell activation and potentiates Th17 differentiation in lymph nodes at disease onset. Interestingly, in the spinal cord cathelicidin increases Th17 to exTh17 plasticity and IFN-γ production during peak disease. In addition, we show that cathelicidin is produced by the same cell types in the active brain lesions in human MS disease. More recent data shows CD8+ T cells have decreased granzyme B expression upon cathelicidin exposure in the lymph nodes and central nervous system, that cathelicidin alters neutrophil function in EAE, and that neutrophils affect T cell activation in MS, however, these mechanisms are less understood. We propose that cathelicidin directs T cells to become inflammatory cytokine-producing, highly primed and activated, with enhanced pathogenicity which in turn drives severe autoimmune disease. These data contribute to a novel understanding of how neutrophil-derived peptides can orchestrate adaptive immunity during long-term inflammation.

Multiple sclerosis (MS) is a chronic, immune-mediated demyelinating disease of the central nervous system (CNS), characterised by focal areas of demyelination, neuroinflammation and ultimately neurodegeneration. There is an endogenous regenerative process termed remyelination, in which oligodendrocyte progenitor cell populations are recruited to the lesioned area, differentiate into oligodendrocytes and re-wrap myelin sheaths around axons. Unfortunately, remyelination ultimately fails in MS patients, in part due to a failure of oligodendrocyte differentiation, leading to an accumulation of disability and disease progression. Although there is no cure for MS, there has been an expansion in the number of diseases modifying therapies over the last few decades which aim to prevent relapses in MS patients. However, there are currently no approved therapies which aim to promote remyelination in MS patients. Although CD4+ T cells are involved in the pathogenesis of MS, they are also involved in CNS remyelination. Regulatory T cells have been shown to promote oligodendrocyte differentiation and remyelination, however T helper (Th) 17 cells impair endogenous remyelination. The roles of the other CD4+ T cell subsets have not yet been elucidated, and the aim of this PhD thesis was to evaluate the regenerative capacities of other effector T cells in CNS remyelination. Th1 cells have been implicated in the pathogenesis of MS and in animal models of immune-mediated demyelination, associated with macrophage-induced demyelination in lesions and impaired oligodendrocyte differentiation. Th22 cells are a more recently discovered CD4+ T cells subset, characterised by the secretion of IL-22, which have been detected in the cerebrospinal fluid of MS patients during both relapse and remission phases. Knowledge of the roles of Th22 cells in the CNS is limited, but Th22-secreted factors have been shown to reduce the severity of immune-mediated demyelination in the CNS. The Th22-secreted factor, IL-22, has also shown regenerative effects in other tissues by inducing proliferation of resident stem cell populations. This led to the following hypotheses: 1) Th1 cells negatively impact OPC differentiation and myelination, 2) Th22 cells promote OPC differentiation and myelination, and 3) IL-22 promotes OPC proliferation, differentiation and remyelination. The results in this PhD thesis show that both Th1 and Th22 cells promote OPC differentiation into oligodendrocytes in vitro through secreted factors. Results suggest that Th1 cells also enhance the rate of myelination, whereas Th22 cells do not impact myelination but decrease the axonal density of organotypic brain slice cultures ex vivo. Interestingly, the re-stimulation of OPC or brain slice cultures with Th1-secreted factors limits the extent of oligodendrocyte differentiation. Recombinant IL-22 does not have any effect on OPC proliferation, differentiation or myelination in vitro or ex vivo. The IL-22 receptor complex was not expressed in the CNS following lysolecithin-induced demyelination, indicating that IL-22 signalling is not relevant in this context. This work further elucidates the regenerative roles of CD4+ T cells in the CNS and opens up different avenues of future research to investigate these further, hopefully for a remyelinating therapy for MS patients.

This thesis broadly concerns quality of life (QoL) and wellbeing in people with a diagnosis of Multiple Sclerosis (MS). It begins with a systematic review into the effects of menopause on QoL in MS, followed by an empirical study exploring the experience of attending an online Acceptance and Commitment Therapy (ACT) group for people with MS, and its impact on QoL and wellbeing. The thesis concludes with a discussion relating to how findings might impact on future research and clinical practice. Firstly, a systematic review investigated the potential dual-impact of menopause on MS. Hormone reduction during perimenopause can cause symptoms of depression and anxiety in the general population, sometimes triggering first-episode psychosis and major depression. Perimenopause in MS may lead to additional difficulties: reproductive hormones have anti-inflammatory properties, reductions of which can impact on MS severity and course, further impacting mental health. This review attempted to investigate this and assess how many studies approach the matter from a psychological perspective. Evidence of dual-impact of menopause was observed in the majority of the 13 papers included in this review. Only four of the 13 studies employed psychological measures; none approached the matter from a purely psychological perspective. Secondly, people with MS were asked about their experience of attending an online ACT group. Reasons for engaging were explored, along with maintenance and attrition processes. Possible mechanisms behind changes in the way people experienced their diagnosis were discussed, and general feedback was summarised. Findings suggested that the group was generally well-accepted. ACT concepts appeared to facilitate psychological flexibility and bring about acceptance, which positively impacted wellbeing. Lastly, both studies' findings and implications were brought together and discussed in the final chapter, followed by reflections on the research process.

Multiple Sclerosis (MS) is a neurodegenerative inflammatory disease characterised by demyelination and axonal loss in both, white and grey matter. It affects motor, sensory, cognitive and language functions. Language impairments have only recently been studied as a clinical manifestation, with word retrieval deficits as the most common symptoms. Anomia is also the most self-reported language feature between people with MS and even subtle deficits can affect communicative participation and quality of life. This thesis investigated the extent and nature of anomia in people with Relapsing-Remitting (RR) MS through behavioural and imaging analyses and evaluated the use of a word retrieval software-based treatment as a form of self-management of anomic symptoms. In order to explore the scope of anomia in the context of cognitive, linguistic and speech production skills, 151 participants with RR MS were assessed using general cognition tasks and a bespoke picture test which focused on accuracy and latency. Next, in order to understand the factors involved in the anomic symptoms, a wide array of neuropsychological and communication assessments were conducted with the RR MS participants (n=21). Later, the efficacy of a novel word retrieval software-based treatment for anomic symptoms was examined in participants (n=13), which focused on combined accuracy and speed intervention. Finally, grey matter (GM) volumes of 105 participants with RR MS were assessed and compared with healthy individuals as well their relationship with verbal fluency tests outcomes, as a means to a better understanding of the neural nature of verbal fluency in RR MS. Results showed that participants with RR MS often present with anomic symptoms characterised as word retrieval inaccuracy and delayed latency. It was established that anomia could not be fully explained by speech deficits such as dysarthria and that difficulties in naming retrieval may have stemmed from a disruption in the systems of working memory and speed of information processing, and deficits in the semantic access, search and/or memory store. Furthermore, MRI on GM volumes suggested that low scores in verbal fluency tasks showed a general decline in information processing skills. Finally, we observed that word retrieval therapy produced gains in naming accuracy and latency and these could be generalised to connected speech task. However, the speeded therapy did not give an advantage for improving confrontational naming. In conclusion, anomia is a common symptom in RR MS and could be described as a cognitive-communication disorder rather than a pure language deficit. Targeted early interventions could help to improve or maintain language abilities in people with RR-MS which may enhance their quality of life.

Parkinson's is a progressive neurodegenerative condition characterized by a variety of motor and non-motor symptoms that can lead to increasing disability and complex psychosocial challenges. While medical treatments can help people living with Parkinson's to manage symptoms, they do not fully address many challenges associated with the condition, including balance issues, stigma and unpredictability. Art and exercise are increasingly being recognized as therapeutic for people living with Parkinson's, and dance interventions have been demonstrated to improve several motor and non-motor symptoms and quality of life (QOL) in a number of small studies. Outside of laboratory and clinical settings, dance programs for people living with Parkinson's have been implemented in communities worldwide, yet more research is needed to characterize their effects and understand their broader impact. This thesis aims to assess the effects and impact of dance using mixed methods. First, a systematic review of randomized controlled trials was conducted to assess the efficacy of dance for people living with Parkinson's and the quality of existing evidence. Then, the feasibility of implementing a dance program for people living with Parkinson's and conducting an intervention trial in Northern Ireland was assessed and preliminary analyses were conducted on dance's effects on motor and non-motor outcomes. Based on feedback from participants in our feasibility study and in the literature that dancing can have an immediate, short-term effect on mobility, mood and energy, a follow up study was conducted to investigate the short-term effects of dance on several motor and non-motor outcomes. The final participatory study explored the lived experience of dancing in people living with Parkinson's and Multiple Sclerosis and its impact on the body, self and relationships with others using a range of qualitative methods, including semi-structured interviews and observations analyzed using a phenomenological approach. This thesis concludes that dance has the potential to impact movement of the body, one's state of mind, relational health, and the quality and movement of life in people living with Parkinson's. Many people living with Parkinson's and other chronic illnesses who dance seem to make it a part of their lives due to its holistic impact on several dimensions of health and the passion that develops from the practice.

Multiple sclerosis (MS) is an immune-mediated, neurodegenerative disease of the central nervous system (CNS) characterised by focal demyelination (lesions) and axonal loss. Clinical presentation is heterogeneous; yet the relapsing-remitting MS (RRMS) sub-type is defined by acute clinical episodes, which distinguish it from continuously worsening disability in progressive sub-types. Early intervention with disease modifying therapies (DMTs) may prevent neurodegeneration. Current DMTs, however, generally target inflammation, are expensive and are limited by accompanying adverse effects. In vivo biomarkers are thus warranted for early stratification of patients, longitudinal observation of therapeutic response and evaluation of novel treatments. Magnetic resonance imaging (MRI) plays a key role in MS diagnosis; yet the predictive power of conventional MRI biomarkers, such as T2-w lesion load and brain atrophy, to determine prognosis and disease severity in early disease is weak. Advanced MRI techniques such as magnetisation transfer imaging (MTI) and diffusion-weighted imaging (dMRI), which probe tissue microstructure non-invasively, may be more sensitive to MS neuropathology, and show promise as putative biomarkers of underlying MS pathology. Quantitative MTI has been widely applied in MS studies. Poor intra- and inter-site reproducibility of the derived magnetisation transfer ratio (MTR) metric and the time-consuming protocols required for fully quantitative MTI have restricted clinical usage of MTI. MTsat, a semi-quantitative MTI measure, is less influenced by variance in the MTR signal caused by T1 relaxation and B1 inhomogeneities, and thus may be a better prognostic biomarker than MTR. Combining quantitative MTI with dMRI measures may also improve specificity to the heterogeneous biological processes which occur in MS at diagnosis. In this work, I assessed MTI literature in RRMS through systematic review and meta-analyses. I compared the reproducibility of MTsat with MTR in healthy volunteers. I also assessed the reproducibility of the MRI aggregate g-ratio, derived from combined MTsat and dMRI-derived neurite orientation dispersion and density imaging (NODDI) measures. I validated the use of MTsat in a clinical cohort of people recently diagnosed with RRMS, before therapeutic intervention, and evaluated the relationship between MTI biomarkers and clinical disability. We substantiate the MRI g-ratio as a biomarker in RRMS by comparing it with an established blood biomarker of axonal damage, neurofilament. Finally, I determine the longitudinal evolution of MTsat, compared with MTR, and g-ratio in early RRMS.

Background The benefits of exercise and physical activity for people with multiple sclerosis are well recognised, as are the challenges faced by many in adhering to these activities over the long term. Adherence is considered by some to be the single most important modifiable factor affecting outcome. With years lived with disability increasing and healthcare resources limited, the need to develop and implement effective and acceptable interventions and support people with adherence to physical activity is pressing. Aim This integrative summary presents work from three related areas regarding exercise and physical activity in people with multiple sclerosis. The body of work represents a systematic and rigorous approach to the topic and comprises outputs from three streams of work: 1) Web-based intervention studies; a systematic review and a qualitative study exploring the participant's experience. 2) Evaluation of a home-based standing frame programme; a randomised controlled trial evaluating the clinical and cost-effectiveness, a qualitative study exploring experiences of using the standing frame, and production of a series of four short films to present these experiences using both images and the voices of the people involved. 3) A systematic review of adherence to exercise interventions in people with multiple sclerosis that presents recommendations regarding trial design in this area. The Behaviour Change Wheel is used as a "reflection framework" to facilitate integration of my current work and consider areas for future study. The reflection process has helped highlight important 'threads' that bring my work together and has informed the development of two concepts that provide a fresh perspective in this field. Conclusion This summary draws together work from a variety of studies encompassing original research and systematic reviews, which demonstrate the creation and interpretation of new knowledge in the field of physical activity in people with multiple sclerosis.

In the past fifteen years two new antibody-mediated Central Nervous System (CNS) diseases, have been identified as distinct from multiple sclerosis (MS): Aquaporin-4-antibody (AQP4-Ab) and the more recently discovered myelin oligodendrocyte glycoprotein antibody-associated disorder (MOGAD). Although conventional brain MRI may help distinguish MS from antibody-mediated diseases, the use of quantitative and non-conventional imaging may give more pathological information and explain the clinical differences. This thesis comprises a clinical study, describing the main clinical characteristics of MOGAD, and a cross-sectional MRI study that compares structural, quantitative and functional brain MRI findings across MOGAD, NMOSD-AQP4, MS and healthy controls. The principal clinical finding is: • MOGAD patients have a good recovery from visual and physical disability compared to NMOSD-AQP4, although some of them can be left with sphincter, erectile dysfunction, and cognitive impairment. The main MRI study findings are: • In MOGAD most of the white matter lesions tend to resolve, but patients are left with grey matter damage, and this may be related to persistent white matter lesions. Despite the greater residual disability, NMOSD-AQP4 patients showed a relative sparing of deep grey matter volumes. • Non-lesional tissue fractional anisotropy (FA) was only reduced in MS, although focal reductions were noted in NMOSD-AQP4 patients, reflecting mainly optic nerve and corticospinal tract pathways. In MOGAD there is no abnormal non-lesional tissue FA. • Disability (EDSS) in NMOSD-AQP4 is associated with NAWM FA alone, with deep grey matter atrophy and female sex in MS, and with brainstem volume in MOGAD. Visual acuity was associated with optic chiasm volume in NMOSD-AQP4 disease and with thalamic volume and female sex in MOGAD. • in MOGAD, despite the good visual recovery, and the absence of structural and microstructural tissue damage, there is a reduced functional connectivity in the primary visual cortex. In NMOSD-AQP4 there is a broader involvement of the large brain networks (visual, salience, sensorimotor, default mode network), possibly related to the higher level of disability (and tissue damage), while in MS, a preferential involvement of subcortical network was found (i.e., basal ganglia). • Cognitive performance is influenced by fatigue and depression in MOGAD and NMOSD-AQP4 but not in MS. Low cognitive score is associated with low deep grey matter volume in MOGAD and MS, while is mainly driven by cortical volume in NMOSD-AQP4. The clinical and MRI studies from this thesis, represent some of the earliest studies directed at better understanding the MRI changes behind the clinical recovery and outcome in MOGAD. The comprehensive clinical and MRI approach, investigating not only physical disability, but also the often-neglected cognitive function, added novelty to this body of work and can open to future larger studies.

The most tractable strategy to delay or prevent the progressive phase of MS is to promote endogenous remyelination; doing so restores nerve conduction and prevents demyelinated axons from degenerating. The rate-limiting stage in this process is differentiation of oligodendrocyte progenitor cells (OPCs) into mature, myelinating, oligodendrocytes. In animals, agonism of the retinoid X receptor (RXR)-γ enhances remyelination in this way. Metformin also promotes remyelination, this time by overcoming an age-associated block to the responsiveness of OPCs to pro-differentiation factors. In this PhD I show that bexarotene, a non-selective agonist of the RXR receptor, promotes remyelination in people with relapsing remitting multiple sclerosis. Converging evidence from electrophysiology and neuroimaging in a phase II clinical trial (CCMR One) demonstrate that this occurs in demyelinated lesions and is greatest in lesions located in grey matter regions of the brain. I additionally conducted analyses which suggest an age-dependency of bexarotene's remyelinating effect and led a follow-up sub study of the trial participants which showed the treatment effect afforded by bexarotene is sustained, years after treatment. Unfortunately, bexarotene was poorly tolerated, and so the legacy of this trial will likely be one of shaping the framework for future assessments of remyelinating therapies. As such, I designed, obtained funding and secured approvals for a clinical trial to test the remyelinating effect of the combination of metformin with clemastine (CCMR Two), implementing lessons from CCMR One in the trial design; this is due to commence participant recruitment in 2021. In exploring treatments for progressive forms of MS, not limited to the process of remyelination, I worked with the MS Society to develop and implement a rigorous, expert-led, evidence-based approach to the selection of licensed drugs for repurposing and testing in clinical trials of people with progressive MS. I reviewed the preclinical and clinical literature for a list of compounds and condensed these into a database of summary documents. These were presented to a panel of experts and people affected by MS, ultimately leading to four treatments being recommended for immediate testing in progressive MS trials: R-α-lipoic acid, metformin, the combination treatment of R-α-lipoic acid and metformin, and niacin. Regrettably, much of my research has been halted by the COVID-19 pandemic. In this PhD I had additionally sought to evaluate electrophysiological techniques for quantifying remyelination and neuroprotection - multifocal VEP and saccadometry - in cohorts of people with MS and in the setting of the CCMR Two trial. Instead, when these studies were delayed, I embraced an opportunity to help the Cambridge COVID-19 research effort, working on the RECOVERY trial in the first wave, and leading my own project to analyse enrolment to treatment trials, ultimately describing the barriers to, and implications of, low recruitment rates in advance of the subsequent waves. Consequently, further exploratory research with electrophysiology and the CCMR Two trial will be the subject of post-doctoral research.

White matter damage can occur in a variety of neurological diseases where it contributes significantly to impairment of brain function. Dysregulation of ion gradients across membranes is a common phenomenon across many diseases and can be damaging due to increased metabolic demand, oedema and activation of destructive enzyme cascades. Breakdown of ionic gradients can occur under many circumstances. For example, loss of energy supply to ion pumps causes disruption early in stroke progression. Activation of ion channels through voltage or ligand gating contributes greatly to complications such as intracellular calcium overload in many diseases. This thesis investigated the contribution of different mechanisms of ionic alterations to white matter damage in ischaemic stroke and multiple sclerosis as well as to establish potential treatment options. Particular attention was paid to consequences of glutamate receptor activity. Using the oxygen glucose deprivation model of ischaemia, in the mouse brain slice, it was found that ischaemia-induced potassium release is greater in cortical grey than in corpus callosum white matter. This distinction is dependent on release of vesicular glutamate, as demonstrated by abolition by calcium free media and glutamate receptor antagonists. Variation of ischaemia-induced acidification of grey and white matter was also investigated but no difference between the two tissues was found. A new acute model of demyelination was established, using direct application of cuprizone to the isolated mouse optic nerve, and protection afforded by glutamate receptor antagonists was determined. Antagonism of AMPA and NMDA receptors acted in synergy to provide greater protection than block of each alone. This effect may allow treatment at lower concentrations to limit adverse side effects. When applied separately, NMDA receptor antagonists provided more protection than AMPA receptor antagonists, suggesting this channel contributed more to the injury progression. The same combination of glutamate antagonists was then investigated in the OGD model of ischaemic stroke but provided no more protection than AMPA receptor antagonist alone.

Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS), characterised by focal areas of myelin destruction (demyelination), oligodendrocyte death with variable extent of axon damage contributing to neurodegeneration. The aetiology of MS is not fully understood by the scientific community, with considerations of a primary inflammatory root with an autoimmune component as well of a degenerative disorder with secondary inflammation under investigation. Demyelination leads to conduction block of action potentials and to loss of metabolic and structural support offered by the oligodendrocytes, contributing to axonal vulnerability to damage and may in the long-term lead to neurodegeneration, the main driver of permanent disability in MS patients. Regeneration of the myelin sheaths (remyelination) around axons can help protect demyelinated axons from damage, restore nerve conduction and thus function. Remyelination is carried out by oligodendroglia, but in MS, this process is inefficient and promoting remyelination represents a therapeutic goal due to the hopes to achieve prolonged neuroprotection. In the healthy brain, interstitial tissue oxygen levels are low. Thus, CNS cells, particularly oligodendrocytes and their precursors habitually exist in a physiological low oxygen environment. To withstand shifts in the oxygen tension, such as mild hypoxia, adaptive mechanisms take place in the brain, via the hypoxia inducible factors (HIFs) and its target genes, that help sustain physiological functions with changes in metabolism, angiogenesis, cell proliferation and cell death. In humans and animal models of MS, neuropathological and imaging studies show the presence of tissue hypoxia and evidence of tissue adaptations to this hypoxia. Oligodendrocytes and their precursors are especially susceptible to hypoxia. Remyelination, thus occurs within this hypoxic environment, but we do not understand whether hypoxia worsens tissue repair or whether the tissue adaptations aid remyelination, similarly to the regenerative effects of hypoxia pre-conditioning in other organs and diseases. To test this hypothesis, I used two experimental paradigms: 1) exposure to hypoxia during remyelination and 2) exposure to hypoxia prior to demyelination, as pre-conditioning. To study remyelination, in the absence of an autoimmune response, I used stereotactic injection of lysophosphatidylcholine (LPC) into the mouse corpus callosum as a focal model of demyelination, exposed mice to mild hypoxia (10% normobaric oxygen) and assessed remyelination efficiency at 10- and 15-days post lesion (Dpl) by electron microscopy. Firstly, I found that demyelinating lesions at 3 Dpl are hypoxic, as assessed using the oxygen sensitive immunohistochemical probe, pimonidazole, and this hypoxia persisted during remyelination at 10 and 15 Dpl. I also found that exposure to additional hypoxia during the period of remyelination resulted in a significant decrease in the number of remyelinated axons at 15 Dpl but not at 10 Dpl, compared to mice undergoing remyelination in normoxia (room air). Importantly, the observed decrease in the number of remyelinated axons was not due to a loss of axons. I also noticed a difference in thickness of the inner cytoplasmic tongue (ICT) than expected for a given myelin thickness. The ICT is the inner most uncompacted myelin layer facing the axon. The ICT is in direct connection to the oligodendrocyte soma and via myelin 'channels' which provide passage of metabolites to the underlying axons and this is the site of new myelin membrane deposition during myelin growth. I observed that the ICT was larger than expected for myelin thickness at 10 Dpl in remyelinated axons irrespective whether they were exposed to normoxia or hypoxia, followed by its recovery at 15 Dpl in normoxic mice, compared to non-demyelinated controls. At 15 Dpl, mice that remyelinated under additional hypoxic conditions showed a larger ICT for a given myelin thickness, compared to non-lesioned mice. This enlargement of the ICT may represent a metabolic compensatory mechanism during early remyelination in response to the lesion-associated hypoxia that may help sustain remyelination through provision of metabolites. However prolonged hypoxia leads to decompensation and together with an unfavourable lesion environment results in the loss of myelin and poor remyelination. I next assessed whether preconditioning of brain tissue to mild hypoxia before inducing a focal demyelinating lesion affects subsequent remyelination using the same model. Evidence from preconditioning paradigms in different organs, has shown that this confers resilience to further damage, aiding tissue repair. Lesion size was significantly reduced in mice preconditioned to hypoxia compared to controls, but there was no difference in the number of remyelinated axons at the two post-lesion timepoints. In conclusion, this work provides evidence that additional longer period of hypoxia during the period of remyelination may be detrimental to remyelination and that hypoxia preconditioning warrants future investigation to fully address its possible protective effect in reducing lesion size.

Myelin-oligodendrocyte glycoprotein associated disease (MOGAD) is a recently described inflammatory demyelinating disorder of the central nervous system. It has been shown to be distinct from both aquaporin-4 (AQP4) antibody (Ab) neuromyelitis optica spectrum disorder (NMOSD) and multiple sclerosis (MS). Spinal cord involvement is a hallmark feature of all three diseases and a central cause of disability, and so, is an important phenotype to study. This thesis consists of a retrospective clinical study and then a cross-sectional research MRI study that compares structural, quantitative and resting-state functional MRI scans across MOGAD, AQP4-NMOSD, MS and healthy volunteers (HV). The principal clinical research findings are: • MOGAD myelitis presents more frequently with short cord lesions, multiple cord lesions, conus lesions and involvement of either the brain or optic nerves at the time of the acute attack; these MRI lesions are likely to resolve at follow-up. • MOGAD myelitis results in significant sphincter dysfunction, despite good motor recovery. Conus lesions are associated the need for long term catheter dysfunction and the presence of a brainstem lesion during the acute attack may predict higher long term disability. The principal research MRI findings are: • AQP4-NMOSD myelitis shows the most severe, localised damage in areas of the cord involved during the acute episode. In MS, metrics show significant differences compared to HV but the relationship with lesion location is not as pronounced. In MOGAD, metrics are comparable to HV after recovery from myelitis but localised grey matter atrophy is noted in the affected area of the cord. • Patients with relapsing MOGAD show significantly more thoracic cord atrophy than monophasic MOGAD, even where the relapse occurred outside of the spinal cord. This may indicate that relapsing MOGAD denotes a more severe phenotype. • Non-conventional MRI metrics show clinical relevance: cervical cord cross-sectional area associates with disability outcomes and spinothalamic fractional anisotropy with pain scores, independent of disease type. • Distinct and robust functional networks exist in the spinal cord without a particular task. Damage in the spinal cord may lead to either a compensatory increase or a decrease in connectivity related to atrophy and cord lesions. The publications from this work, which form part of this thesis, have added to the growing body of research into the study of MOGAD. Additionally, advanced spinal cord imaging techniques were used, which add novelty to the research and its conclusions.

Theory of Mind (TOM) is an important ability in guiding social behaviour, with impairments in TOM being associated with psychosocial difficulties. Over recent years, individuals with multiple sclerosis (MS) have been consistently shown to have impaired TOM abilities, however, little is known about the psychosocial impact of these impairments in this population. This thesis has two sections. Initially, a systematic review was completed that assessed and compared the psychometric properties of the TOM measures which have been used with individuals with MS. This highlighted that seventeen different TOM measures have been used with individuals with MS. Through exploring the psychometric properties of these seventeen measures, it was revealed that no measure has been validated on an MS sample and more generally there was little information about the psychometric properties of these measures. Whilst recommendations are tentatively made on TOM measure selection for individuals with MS, the paper discusses the remaining challenges in measure selection, accuracy of measurement and the need for further research into these measures. Secondly, an empirical study explored the relationships between TOM abilities and psychosocial outcomes in individuals with MS. In total, 36 individuals participated, which involved completing neuropsychological tasks of TOM and executive functioning abilities, as well as self-report measures of their psychological, interpersonal and social abilities. Results indicated that TOM was not associated with psychological, interpersonal and several areas of social functioning, however, TOM performance was significantly related to social withdrawal, employment and quality of life. These findings are discussed and areas for future research identified.

Multiple sclerosis (MS) is a common, debilitating autoimmune disorder of the central nervous system (CNS). The most visible element of MS pathology is white matter (WM) lesions. However, extra-lesional abnormalities are recognised and appear most marked at the outer (subpial) brain surfaces at post-mortem. Magnetisation transfer ratio (MTR) is a non-conventional MRI sequence that correlates with myelin density and axonal count, and has recently uncovered reductions in the innermost (periventricular) layers and outermost (cortical) layers of patients with longstanding MS. These abnormalities are termed “outside-in” changes. The cause, extent, evolution and treatment-responsiveness of these outside-in gradients is unknown. Most patients present with relapsing-remitting (RR) MS for which numerous immunomodulatory disease-modifying therapies (DMTs) are licensed. However, after approximately 20 years most convert to secondary progressive (SP) MS where immunomodulatory therapies have little if any effect. Whether DMTs delay or prevent this transition remains unclear. During this PhD I examined whether outside-in MTR gradients occur in different disease stages, their clinical associations and predictive capabilities, and (for periventricular gradients) their response to a potent immunomodulatory DMT. In a separate project, I used real-world data to explore whether DMT use is associated with a lower risk of conversion to SPMS. Outside-in MTR gradients of tissue damage were seen in periventricular and cortical regions at all stages of relapse-onset MS and primary-progressive MS. The periventricular MTR gradient was reversed by peripheral immunomodulation and independently predicted subsequent relapse activity on and off DMTs. The underlying process(es) remain unknown but appear at least partially distinct from those underlying lesion formation; a CSF-mediated process – secondary to meningeal inflammation or primarily degenerative – might explain both periventricular and cortical gradients. Among patients with RRMS, initial treatment with fingolimod, natalizumab or alemtuzumab was associated with a lower risk of conversion to SPMS compared to initial treatment with glatiramer acetate or interferon beta over a median 5.8 years of follow-up. A lower risk of conversion was also associated with early (versus late) commencement of glatiramer acetate or interferon beta; and with early escalation from these therapies to fingolimod, natalizumab or alemtuzumab compared to late escalation. These findings, considered along with these therapies’ risks, may help inform decisions about DMT selection.

The store-operated calcium entry (SOCE) pathway is an important route for generating cytosolic Ca2+ signals that regulate a diverse array of biological processes. Abnormal SOCE seem to underlie several diseases that notably include allergy, inflammation, acute pancreatitis and cancer. Therefore, any modulator of this pathway is likely to have significant impact in cell biology under both normal and abnormal conditions. In this study, the FDA-approved drug library was screened for agents that share significant similarity in 3D shape and surface electrostatics with few, hitherto best-known inhibitors of SOCE. This has led to the identification of five drugs that showed dose- dependent inhibition of SOCE in cell-based assay, probably through blocking SOCE pathway extracellularly. Of these drugs, leflunomide and teriflunomide could suppress SOCE significantly at clinically-relevant doses and this provides for an additional mechanism towards the therapeutic utility of these drugs as immunosuppressants. Few more compounds from each of the 4 different pharmacological classes were further tested using cell-based methods to find agents with more potent SOCE-inhibitory potency. Three candidates with better potency namely trequinsin, roflumilast N-oxide and vidofludimus, were identified. Of these compounds, vidofludimus which is known to be an inhibitor of the dihydroorotate dehydrogenase (DHODH) enzyme, proved to be the most potent (IC50~1.4μM) SOCE inhibitor among all the drugs tested, inhibitor of SOCE as it could suppress SOCE significantly at nanomolar dose. Next, selected drugs identified with SOCE suppressive ability were evaluated against the proliferation of different lung cancer cells namely H520, H1752, and LK2. In agreement with previous studies with bona fide SOCE inhibitors, the chosen drugs significantly reduced proliferation of those lung cancer cells. Interestingly, the anti-proliferative activity of most of the selected drugs were observed at concentrations at which they could significantly inhibit SOCE identified in those lung cancer cells. Although the possible contribution of the inhibition of the known targets of these drugs towards their anti-proliferative activity could not be ruled out, it was nevertheless indicative of the fact that their SOCE-inhibitory property was definitely implicated. This, in agreement with existing literature, hinted towards the fact that SOCE can be targeted to achieve potential therapeutic benefit against lung cancer and other cancer cells of epithelial origins. Of the drugs identified at the early phase of the present work to possess SOCE suppressive ability, teriflunomide is approved for multiple sclerosis (MS) which is believed to be autoimmune origin. The pro-drug of teriflunomide is leflunomide which is approved for managing rheumatoid arthritis. Since oligodendrocytes progenitor cells (OPCs) play important role in MS due to their myelin forming ability upon differentiation, SOCE was characterized in OPCs using single cell Ca2+ imaging approach and effect of some SOCE inhibitory compounds were then evaluated for any role in OPC differentiation. Employing different biochemical and imaging methods, AnCoA4, a known SOCE blocker was found to promote differentiation of OPCs. This finding could help to identify new target to develop therapeutic for demyelinating diseases like MS.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) that is a leading non-traumatic cause of disability in young adults. The 18 kDa Translocator Protein (TSPO) is a mitochondrial protein and positron emission tomography (PET)-imaging target that is highly upregulated in MS brain lesions and may potentially represent both an inflammatory biomarker and a therapeutic target for the monitoring and treatment of MS. However, its pathological significance is not well understood. While experimental autoimmune encephalomyelitis (EAE) in the common marmoset is a well-established primate model of MS, TSPO expression in this model has not yet been studied. Moreover, the comparison of TSPO expression and PET radioligand signal in inflammatory conditions has been limited by the lack of suitable unfixed pathological samples for concurrent measurement of protein expression and radioligand-binding. In this thesis, I seek to characterize patterns of TSPO expression in fixed CNS tissues from EAE marmosets using multiplex immunofluorescence and directly compare TSPO protein expression measured by flow cytometry with radioligand-binding signal in the peripheral blood of healthy donors and MS patients. Through these studies, I have established that marmoset EAE recapitulates temporal and cellular patterns of TSPO expression seen in MS, rendering it a suitable model for the study of human disease that may give insight into the pathological significance of TSPO in immune dysregulation. Furthermore, I have found TSPO expression in the peripheral blood of MS patients to be significantly increased, suggesting TSPO to be a useful peripheral biomarker of CNS disease. However, I found that radioligand-binding by second-generation PET radioligand PBR28 is paradoxically decreased in the peripheral blood of MS patients, implying that the clinical utility of PBR28 may be limited, likely by conformational changes occurring in inflammation. Collectively, these results allow us to better interpret the pathological significance of TSPO expression and TSPO-radioligand binding in the CNS and peripheral blood in neuroinflammatory disease.

Autoimmune disease is characterised by the development of abnormal immune responses directed against antigenic components of the host i.e. self-antigen. Multiple sclerosis (MS) is a chronic autoimmune, inflammatory disease directed against an unknown antigen in the central nervous system. This leads to focal demyelinating lesions and axonal damage. MS is caused by a complex interplay between multiple genetic and environmental risk factors and its incidence is increasing in both developed and developing countries worldwide. Current disease-modifying therapies non-specifically suppress components of the inflam- mation in MS. They are effective but with significant adverse events. There is an unmet need for safe targeted immunotherapies and for treatments that actively promote remyelination. This thesis examines two potential novel therapeutic strategies in autoimmunity and MS. The first approach arose from the observation, by previous research at GSK, that there is a population of CD4+ lymphocytes with apparent constitutive pSTAT1 expression independent of any stimulus and refractory to JAK-1 inhibition. This pSTAT1hi population had been found to be enriched in patients with systemic lupus erythematosus. The starting hypothesis of this thesis was that these cells might be pathogenic in autoimmune disease. However, following deep FACS phenotyping of this lymphocyte subset, the constitutive pSTAT1hi population was demonstrated to be an artefact of non-specific antibody binding, resulting in termination of this project. The remainder of the thesis focuses on the immunological effects of bexarotene. This retinoid X receptor agonist is currently being tested in a phase II clinical trial for its ability to promote remyelination [“CCMR One” trial]. Here, we focused on the ability of bexarotene to modulate the human Th17/iTreg axis in vitro, owing to literature reporting that RXR ligands augment RAR agonists’ effect on murine Th17/iTreg cells. We show that bexarotene promotes the differentiation of human Foxp3+ iTregs from naive CD4+ T cells independent of RAR engagement, whilst concomitantly inhibiting the differentiation of Th17 cells and their secretion of IL-17a. Bexarotene was not found to alter the methylation status of the Treg-specific demethylated region, with iTregs remaining fully methylated within this study. Next, I examined the peripheral blood of patients receiving six months of oral bexarotene treatment within the CCMR One trial. There was no change in serum IL-17a, the frequency of CD25hi CD127 Foxp3+ Tregs, nor in the secretion of IL-17a from effector T cells. Mirroring the data in vitro, no change was observed in the TSDR methylation status of Tregs isolated from these patients. Taken together, these results demonstrate the ability for RXR-specific ligands to modulate the Th17/iTreg axis in favour of iTreg differentiation in vitro, and thus their potential in the treatment of those autoimmune diseases which are driven by a failure of self-tolerance and imbalance of Tregs and inflammatory Teffectors.

In Multiple Sclerosis (MS), the insulating membrane that ensheaths axons termed myelin, is lost by a process known as demyelination. Initial regeneration of myelin, termed remyelination, restores neuronal function. However, remyelination failure with disease leads to a loss of cognitive, sensory and motor functions. There are no currently approved therapies aimed at inducing remyelination, highlighting the need to investigate new targets that can promote this process. Astrocytes are the most abundant glial cell type in the central nervous system (CNS). They are known to support myelin formation in development and its maintenance during adulthood, yet their roles during remyelination are controversial and remain poorly understood. The work from this thesis explores the changes in astrocytes at the cellular and molecular level during remyelination. Using two different in vivo models of toxin induced demyelination, I have found that astrocytes during remyelination undergo a series of changes in numbers, reactivity (assessed by GFAP, vimentin, nestin and NFIA), and morphology, which indicate a temporal regulation of reactivity. Interestingly, I observed that astrocytes increase in reactivity at the time at which the myelin-forming cells, termed oligodendrocytes, differentiate to remyelinate axons. These findings suggest that astrocytes are important for regulating remyelination. To explore this, unbiased RNA-sequencing of astrocyte translating mRNA, isolated using translational ribosome affinity purification (TRAP), was used to identify changes in their gene expression. I found that astrocytes upregulate genes indicative of neuroprotective functions early after demyelination, characterised by the upregulation of the Nrf2 pathway. Subsequently, at the time of oligodendrocyte differentiation, astrocytes downregulate Nrf2 signalling and activate cholesterol biosynthesis, which is essential during myelination. Here, I hypothesised whether Nrf2 is an critical regulator of astrocyte functions that are important for remyelination. Hence, I used a transgenic mouse model in which Nrf2 is overexpressed in reactive astrocytes. The results suggested that upon sustained Nrf2 overexpression, cholesterol biosynthesis in astrocytes is impaired and consequently remyelination is poor. This study identifies a mechanism by which astrocytes regulate their functions after demyelination to control the timing of remyelination. These findings are of translation relevance, given that some MS therapies are thought to act via Nrf2 activation.

Multiple Sclerosis affects around 2 million people worldwide (Kantarci and Wingerchuk, 2006; Dutta and Trapp, 2011). The disease is typified by the destruction of the central nervous system neurons' myelin sheaths, caused by the individual's own immune system (Hauser and Oksenberg, 2006). This destruction results in the inflammation and chronic degeneration of the CNS, causing varying symptoms including pain, fatigue, cognitive impairment and paralysis (Costelloe et al., 2008). Not only is the life expectancy of individuals with MS 10 years below that of the age-matched general population (Ragonese et al., 2008), but life quality is often severely affected from the start of disease onset (typically around 30 years of age (Hauser and Oksenberg, 2006)). There are several treatments available to aid in the relief of specific symptoms; however, the treatment is lifelong which places a burden on healthcare services. Current research looks to expand the available knowledge on the causes of MS, to improve preventative measures (such as lifestyle changes to accommodate environmental conditions) and targeted treatments (for example, focusing on the product of an MS-associated gene variant). Of particular interest to MS research are the two population isolates of Orkney and Shetland, which together make up the Northern Isles of Scotland. Shetland has 295 MS cases per 100,000 individuals, while Orkney has the highest global prevalence of MS at 402 cases per 100,000 individuals (Visser et al., 2012). Orkney, at a lower latitude than Shetland, has a significantly higher prevalence than what would be expected. Multiple theories behind the excess of MS cases in Orkney have been investigated, including vitamin D deficiency and homozygosity: neither were found to cause the high prevalence of MS. It is possible that this excess prevalence may be explained through unique genetics. This thesis sought to better understand these high rates of MS, with the aim of passing this knowledge on to the island residents of Orkney and Shetland and to contribute the findings to the wider understanding of MS. Analyses were conducted using the ORCADES and VIKING datasets. ORCADES contained 2215 individuals from the Orkney islands, including 97 MS cases (some recruited because they were cases); VIKING contained 2015 individuals from the Shetland islands, including 15 cases. First, a heritability study was conducted using GCTA to determine the SNP heritability of MS in both Orkney and Shetland and how it compared to published estimates of heritability. The SNP heritability of MS in Orkney was estimated at 0.31 (95% CI 0.13, 0.49). An estimate of SNP heritability for MS in Shetland could not be determined due to low case numbers. Second, a genome-wide association study was conducted using a combined ORCADES/VIKING dataset containing 112 cases and 4223 controls. The aim of this study was to determine if unique common MS risk variants existed in the Northern Isles. Here, 89 SNPs were identified to suggestive significance, mostly within six key regions of the genome. Within the literature, only one of these (chromosome 6 SNP rs9268154) was associated with Multiple Sclerosis. Four of the five other regions had possible functions within the immune or nervous system. However, as these did not reach genome-wide significance it is likely these results were due to chance; further investigation is needed to clarify this. Finally, a polygenic risk score study looked at the contribution of common risk variants to MS. The 127 most strongly associated MS SNPs were used to calculate risk scores in mainland Scotland, Orkney and Shetland. These risk scores were compared between controls in all three populations to determine if the Northern Isles, by chance, had higher frequencies of common risk variants and if this contributed to the excess of cases. These common risk variants explained 3% of variance in MS risk, and had an AUC score of 0.69 (95% CI 0.65, 0.74). However, no difference existed between common risk variants in the three populations, aside from one variant: rs9271069, a tag SNP for HLA-DRB1*1501. This SNP was found to have a significantly higher frequency in Orkney (RAF = 0.23, p-value = 8 x 10-13) and Shetland (RAF = 0.21, p-value = 2.3 x 10-6) than mainland Scotland (RAF = 0.17). This SNP accounted for 6 cases (95% CI 3, 8) out of 150 observed excess cases per 100,000 individuals in Shetland and 9 cases (95% CI 8, 11) of the observed 257 excess cases per 100,000 individuals in Orkney. The question of why the Northern Isles have such high rates of MS remains open. This thesis explains a small proportion of this excess. It is hoped that the findings and discussions found here will encourage dialogue within the Northern Isles and bring awareness to the genetic, environmental and lifestyle factors that contribute to MS within the islands.

Aim As part of the broad strategy in Edinburgh and beyond to discover new treatments for progressive Multiple Sclerosis (MS), the aims for my PhD project were: (1) to address the lack of an in vitro phenotypic drug screening platform that is able to fully recapitulate myelin sheath formation, with the long-term goal being to enhance the discovery of pro remyelination therapies in progressive MS, and target the poor drug discovery rates in brain disorders which is partially due to poor disease modelling, and (2) the exploration of non linear optical imaging microscopy techniques that targets both the resolution and speed to study myelination. Background In multiple sclerosis, an inflammatory autoimmune process destroys the oligodendrocytes that provide neuronal support by forming multi-layered compact myelin sheaths around axons, leading to neurodegeneration. Although there are drugs available to suppress the inflammatory attack to limit the formation of demyelinated lesions, no treatments currently exist to promote the regeneration of these myelin sheaths once the damage has occurred. Cell based screens have formed an important part of the strategy used to discover such regenerative drugs. The majority of published cell-based phenotypic drug screens to target repair have focused on the differentiation of oligodendrocyte precursor cells into oligodendrocytes rather than their ability to form mature protective myelin sheaths. However, in many MS lesions, pre-myelinating oligodendrocytes are present, and studies on oligodendrocyte biology show that differentiation and myelination are regulated by distinct mechanisms. There is therefore a need for novel drug screens that target the later myelinating stages of oligodendrocyte development. Results Using a 3D microfibre system for in vitro myelin sheath formation (described by Bechler, Byrne and ffrench-Constant (2015)), I first asked whether compounds that had been v identified as increasing differentiation in conventional 2D culture systems enhanced myelination in the 3D cultures. While Benztropine and Clemastine showed an increase in the number of MBP+ (differentiated) oligodendrocytes in the 2D system, consistent with previous publications, no increase in myelin sheath formation was seen with any of the drugs in the 3D system, highlighting the potentially important differences between differentiation and myelin sheath formation for drug discovery. Next I developed a multiwell plate based assay to allow 3D myelination assays to be used for drug screening. Using electrospinning to produce PLA microfibres, I was able to develop and optimise a technique to insert and suspend the fibres across the bottom of a 96-well plate that can be incorporated into an automated pipeline for high-throughput drug screening. The 3D myelin sheaths could be imaged using the Leica SP8 confocal system with the MatrixScreener extension and the Opera Phenix high-content screening system. Finally, I addressed the problem of imaging myelin in such screens. CARS was shown to be able to preferentially detect myelin sheaths in fixed and live slices in regions and time-points of varying myelin densities. As the development of new myelin sheaths requires the formation of lipid and therefore the incorporation of hydrogen, the consumption of D2O (heavy water) with a 2H atom allowed the non-invasive labelling and detection of myelin sheaths using SRS. Future experiments will allow us to confirm whether this deuterium detection is preferential and/or specific to new myelin sheaths. Significance With only about 10% of drugs that enter Phase I trials successfully launching into clinics, there is an important need for more effective drug screens that better model disease-relevant processes and so reduce late-stage failures. The high-throughput-compatible 96-well plate with suspended PLA microfibres that combines the recent progresses in 3D cellular model systems with bioengineering and the recent advances in high content imaging systems may give us the opportunity to more accurately model these disease-relevant structures in vitro, and therefore improve drug discovery for regenerative therapies in multiple sclerosis and other myelin diseases. The research on Raman-based label-free imaging of myelin sheaths is not only applicable for imaging myelin sheaths in the context of drug validation, but could be important for live imaging of brain slices and detection of newly-formed myelin sheaths without the need for complex and expensive transgenic animals.

Diagnosis and monitoring progression of neurodegenerative diseases relies on the assessment of clinical symptoms such as cognitive impairment in Alzheimer`s disease (AD) and disability in Multiple Sclerosis (MS). Detection of molecular and morphological hallmarks in the brain or spinal cord is invasive, insensitive and expensive. It has been hypothesized that changes in the retina may mirror changes in the brain. In contrast to brain imaging, imaging the eye is quick, non-invasive, inexpensive and might offering a unique “window to the brain” To test this hypothesis eye imaging studies were set up of cohorts with different type of neurodegenerative conditions such as typical and atypical Alzheimer’s disease (AD) or Multiple Sclerosis (MS). In this thesis I report on results using optical coherence tomography (OCT) as “gold standard” in neuro ophthalmology, alongside with new eye imaging and testing modalities such as ultra-widefield laser scanning ophthalmoscopy imaging (UWFI), adaptive optics (AO) and widefield perimetry combined with recently developed image analysis methods and algorithms. In this thesis I present evidences that there are detectable changes on different eye imaging modalities that correlate with disease type and stage and outline how future studies will benefit from these results, studies that are already under way.

Multiple Sclerosis is an autoimmune disease mediated by activated lymphocytes entering the central nervous system. Treatments with the greatest efficacy either prevent the entry of activated lymphocytes, or deplete the lymphocyte population, before allowing lymphocyte reconstitution. IL-7Rα was identified by genetic studies in MS pathogenesis and is involved in the homeostasis and proliferation of lymphocytes. This thesis investigates the role of antagonism of IL-7Rα in the treatment of MS in humans and in vitro differences between genetically stratified subjects based on IL-7Rα genotype. It also explores the role for biomarkers during reconstitution of lymphocytes after Alemtuzumab treatment in MS, in which IL-7Rα plays a major role. Chapter 3 describes the prematurely aborted clinical trial of subjects with an IL-7Rα antagonist. This first-time-in-human trial demonstrated the drug was safe and well tolerated in this limited cohort of subjects. Chapter 4 investigated the differences between individuals based on IL-7Rα genotype with in vitro IL-7Rα antagonism and stimulation. It demonstrated greater activation through IL-7Rα in individuals with the protective genotype. Differences in negative feedback mechanisms of IL-7Rα were explored. Chapter 5 investigated the tolerability of palifermin, a keratinocyte growth factor, with alemtuzumab, which was well tolerated as part of a dose escalation sub-study of the CAMTHY trial. The main CAMTHY trial investigated if palifermin could cause increased thymic lymphopoiesis, offsetting the IL-7 driven homeostatic proliferation of lymphocytes and secondary autoimmunity associated with alemtuzumab. Chapter 6 investigated the use of CD4+ lymphocytes as a biomarker for relapses after Alemtuzumab treatment. This contradicted the findings of a previously published paper, using a much larger cohort of patients in Cambridge. This work underlines the importance of IL-7 in the pathogenesis and treatment of MS. It points towards the IL-7Rα pathway as a future avenue for biomarkers and novel treatments for MS.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS) which has a complex aetiology that involves the interplay of both genetic and environmental factors. Although more than 200 susceptibility loci have been identified through genome-wide association studies, our understanding of the molecular mechanisms underlying these associations remains limited. My predecessor in the laboratory established that the MS associated variants rs9282641 and rs4810485 respectively influence the expression of CD86 and CD40 in freshly collected (ex-vivo) B cells. In my research, I sought to extend this work by investigating the impact of these variants in the context of activation in an effort to refine understanding of the molecular mechanisms underlying the effects of these genotypes on the risk of developing MS. In the first phase of my work I processed venous blood samples from 108 healthy volunteers, who were recruited from the Cambridge BioResource (CBR) to enable a balanced representation of each possible genotype. I isolated peripheral blood mononuclear cells (PBMCs) from these samples and then activated these with CD40L-transfected L cells. In line with the ex vivo results, I found that the expression of CD86 and CD40 remained associated with the genotypes of the respective MS associated variants. I then went on to investigate the effects of these stimulated cells on co-cultured naïve T cells by studying a second cohort of 28 volunteers recruited in pairs from the CBR; the individuals in each pair carrying either the AA or the GG genotype at rs9282641. In these experiments I co-cultured naïve T cells with purified B cells that had been activated with CD40L-transfected L cells. In these 12-day co-cultures, the T cells from subjects homozygous for the risk allele (GG) at rs9282641 demonstrated significantly enhanced proliferation and displayed significantly higher IFN-γ and IL-17 production, and lower IL-10 production. In my PhD I have therefore shown that the altered expression of co-stimulatory molecules on B cells induced by MS risk variants results in enhanced proliferation of pro-inflammatory T cells.

Metabolism is a key process to the function and survival of all living organisms. The role of metabolism is key in many pathologies, and presents a prime opportunity to both understand and treat disease in new ways. In particular, lactate is a molecule which has been known and studied for many years by a broad spectrum of scientists from many research fields, with it appearing to have a number of roles in the routine operation and function of living organisms, as well as signaling pathological disturbance of normal physiology in many circumstances. This thesis aims to further the understanding of how measuring and quantifying both lactate and sodium with both NMR and spectral photometric techniques can be a powerful tool for the researcher in cell, pre-clinical, and clinical research. The research for my PhD has focused on basic biology, pre-clinical large animal imaging, clinical sodium imaging in Multiple Sclerosis, and a physiological first in man study of the healthy brain with hyperpolarised [1-13C] pyruvate.

Previous work in embryonic cell lines has implicated LSH in the deposition of DNA methylation, a key regulatory epigenetic modification during mammalian development. We have generated a conditionally reversible knock-out of Lsh (Lshoff) to investigate its role in vivo. Lshoff/off mice survive to adulthood but develop a tremor and ataxia phenotype. Transmission election microscopy of nervous tissue shows this phenotype is concurrent with central nervous system (CNS) hypomyelination. Lshoff/off animals also have a reduction in mature oligodendrocyte (mOL) cell numbers, but similar number of oligodendrocyte (OL) lineage cells in total, indicating the presence of an OL differentiation defect. Conversion to Lshon/on in OPCs leads to complete rescue of the phenotype, suggesting LSH expression in the OL lineage is necessary for terminal differentiation. OPC specific knock-out of Lsh however produces no phenotype. Sequencing of Lshoff/off adult optic nerve RNA reveals under-expression of genes associated with mOLs, the myelinating cells of the CNS, but no cognate upregulation of oligodendrocyte precursor cell (OPC) markers. Interrogation of DNA methylation in rescued OPCs however reveals scant to no rescue in the hypomethylation present in Lshoff/off OPCs. Taken together these results show an essential role for LSH in OL differentiation and that LSH dependent DNA methylation patterns are not required prior to differentiation to mOLs. We hypothesize that early life LSH expression instead establishes a DNA methylation independent chromatin state required for differentiation and this is maintained in the absence of LSH expression following OPC cell cycle exit.

Purpose Investigate changes in retinal structure and function caused by multiple sclerosis (MS) using established and novel modes of image acquisition and analysis, in order to identify candidate biomarkers of disease. Use longitudinal analysis to determine the rate at which changes to the retinal anatomy occur and whether this rate-of-change alters throughout the MS disease course. Method Within the Anne Rowling Regenerative Neurology Clinic (Royal Infirmary of Edinburgh), 72 MS participants (53 female, 19 male, age range 20-79, median age 44) and 80 healthy volunteers (HV) (61 female, 19 male, age range 23-73, median age 38) received spectral domain optical coherence tomography (SD-OCT) and ultra-wide field scanning laser ophthalmoscope (UWF-SLO) scans at baseline. Within the MS group, 16 (9 right eyes and 10 left eyes) had at least one clinically diagnosed episode of optic neuritis (ON). Follow up scans were taken at approximately 3, 6 and 12-month intervals over the course of two years. Visual acuity (VA) as an indicator of retinal function was also measured at each visit using Sloan letter charts at 100% and 2.5% illumination. Through proprietary software, automatic segmentation of SD-OCT images quantified changes to retinal nerve fibre layer (RNFL) thickness and macular volume. Novel image processing algorithms specifically adapted to semi-automatically segment the vasculature in UWF-SLO images (using multi-scale filtering and supervised classification) allowed for measurement of vessel diameter. Data from the 3 participant groups (MS (no ON), MS (ON) and HV) was first tested statistically for significant differences. Linear regression modelling with the inclusion of disease, sex, length-of-disease and age as predictors was then used to estimate the true effects of disease on the retinal anatomy without confounders. Results Baseline data showed that individuals with MS (no ON) have a reduced RNFL thickness in all peripapillary regions and a reduced macular volume when compared with HVs. The reduction in RNFL thickness was seen globally in both eyes (OS: -10μm, OD: -11μm, p<0.001) but was greatest in the inferior temporal region (OS: -15μm, OD: -13μm, p<0.01). In the MS (ON) group the reduction in RNFL thickness was even greater, again being largest in the inferior temporal region (OS: -29μm, OD: -24μm). Linear regression models confirmed the effect of disease on RNFL thickness and macular volume with significant coefficients in all regions but the inferior nasal. Follow-up analysis showed that the MS (no ON) group had a larger annual rate-of-thinning of the RNFL than HVs in the majority of regions, the largest difference being in the inferior nasal (OS: -1.32μm/year, OD: -1.06μm/year, p<0.05) and superior nasal (OS: - 1.11μm/year, OD: -1.13μm/year, p<0.05) regions. Retinal arteries were shown to have a significantly reduced diameter in only the inferior nasal quadrant, where the greatest reduction in vessel width was found (OS: -0.005mm, OD: - 0.007mm, p<0.05). For mean venous width, the inferior nasal quadrant of the right eye also showed a statistically significant difference between the two groups (OS: -0.005mm, OD: - 0.005mm). Linear regression models were more frequently significant for arterial rather than venous data, despite both vessel types having significant disease coefficients in the inferior nasal quadrant. Follow-up data was inconclusive on the effect that disease has on the annual rate-of-change of vessel thinning. Median baseline high contrast VA (OS: -6, OD: -3, p< 0.05) and low contrast VA (OS: -6, OD: -6, p<0.01) scores were significantly reduced in the MS (no ON) group when compared to HVs. In this study, MS seemed responsible for a 1 line drop in both high contrast and low contrast VA scores in the MS (no ON) group. A significant difference between the HV group and MS (ON) group was only found in the median low contrast VA score of the left eye (OS: -21, OD: -4, p< 0.05). As with vessel analysis, follow-up data showed no significant rates-of-change in VA score. Conclusions At baseline, the RNFL and blood vessels of the retina were significantly thinner in the MS (no ON) group when compared to HVs in particular regions. Consistent with previous reports in the literature, both RNFL and macular volume showed a significant decrease in individuals with MS (no ON). However, this is the first study to show that both arteries and veins in the retinal periphery also have a reduced width in the inferior nasal quadrant. Such changes in the retinal anatomy correlate with a drop in visual function as measured by VA. An increased annual rate of RNFL thinning was also seen in participants with MS (no ON) indicating that anatomical changes occur over time rather than acutely, however this rate-of-change is largest early in the disease course. A similar pattern was observed with the rate at which macular volume decreases in individuals with MS (no ON). Whether changes to the retinal blood vessels occur over time was not determinable within this study, due to the effect size being too small for the number of participants recruited. Future studies should focus on replicating findings pertaining to retinal blood vessels and determining when in the MS disease course these changes occur.

Myelin damage in central nervous system white matter disorders such as multiple sclerosis (MS) leads to axonal dysfunction/degeneration and clinical disability. Regeneration of myelin (termed remyelination) can occur and requires oligodendrocyte progenitor cells (OPCs) to differentiate into mature oligodendrocytes, which are then able to make contact with axons and ensheath them. However, this process fails in progressive MS. The lack of approved therapies aimed at promoting remyelination highlight the need to identify mechanisms driving this regenerative process to develop novel therapeutic strategies. Previous work in the lab identified the TGF-β superfamily member activin-A as being increased during remyelination in vivo and sufficient in stimulating activin receptor-driven OPC differentiation into mature oligodendrocytes in vitro. Here, these studies were followed up by undertaking a comprehensive assessment of the role of activin receptors and their ligands during remyelination. Using an ex vivo brain explant model of demyelination, the stimulation of activin receptors using activin-A was sufficient to enhance remyelination. Blocking activin receptors using an endogenous inhibitor (Inhibin) hindered remyelination, demonstrating the requirement of activin receptor signalling for this process. Surprisingly, blocking the binding of endogenous activin-A to activin receptors using follistatin did not impact remyelination, suggesting that other activin receptor ligands are involved in driving remyelination. As activin receptors may bind other ligands in the TGF-β superfamily, the expression and function of alternative ligands was investigated, and each was found to be important for remyelination (albeit with distinct timing/ effects). Both activin receptors and their ligands were expressed on microglia/macrophages in mouse and human disease tissue. Finally, analysis of activin receptor expression on oligodendrocytes in human tissue revealed potential functional differences between receptor subtypes. Together, these results demonstrate previously undefined roles of a subset of TGF-β superfamily members in regulating remyelination, and have implications for the development of novel approaches to enhancing remyelination in disease.

Failed regeneration of myelin in the central nervous system (CNS) contributes to axon loss/dysfunction in prevalent neurodegenerative disorders, for which there is an unmet need for effective therapies. Previous work from this lab has demonstrated that remyelination requires a transition in microglia activation from pro-inflammatory (inducible nitric oxide synthase (iNOS)+) to pro-regenerative (arginase-1 (Arg-1+)). However, the mechanisms underpinning microglia activation remain unknown, and the assumption that in situ tissue macrophages can transition in activation state has never been substantiated. Here, unexpectedly it is revealed that microglia activation during remyelination is driven by their death and subsequent repopulation. More specifically, pro-inflammatory microglia undergo controlled necrosis (necroptosis), followed by repopulation via residual microglia and CNS-resident Nestin+ cell differentiation into microglia. Blocking necroptosis prevented microglial death and maintained their pro-inflammatory (iNOS+) activation, which consequently hindered remyelination. In human brain tissue, necroptosing and repopulating microglia were only significantly increased in white matter lesions with a high potential for remyelination. These results overturn current assumptions that following injury, microglia simply switch their activation from an initial pro-inflammatory phenotype to become pro-regenerative. Impairment in microglia death and/or repopulation may thus underpin the chronic pro-inflammatory microglia activation associated with failed remyelination in human pathology, highlighting a novel therapeutic strategy in the treatment of chronic inflammatory and neurodegenerative diseases.

Background Multiple sclerosis (MS) is a highly variable disease of the central nervous system with inflammatory and neurodegenerative components, associated with both physical and cognitive disability. Abnormalities are visible on routine magnetic resonance imaging (MRI) of the brain, with 'white matter hyperintensities' (WMHs) representing sites of previous inflammation. Techniques for measuring WMHs have not been standardised, although manual outlining is conventionally taken to be the reference standard, despite its subjective element. WMHs have been found to only partly explain the degree of cognitive impairment, forming part of the 'clinico-radiological paradox'. Research interest has largely moved to advanced imaging techniques, one such technique being diffusion tensor imaging (DTI). Through sensitivity to water molecule movement, DTI reflects the integrity of white matter tracts and thus its measures may be relevant to both the inflammatory and degenerative disease components. Aims The work described in this thesis aims to improve our understanding of the true relationship between measures of white matter damage and cognitive impairment in people with MS, to determine the optimum measurement technique(s) for quantifying WMHs, including developing and testing a novel visual rating scale, and to assess whether information provided by DTI can strengthen the association of imaging and clinical findings. Methods A systematic review of the literature and meta-analysis relating WMHs to cognition was conducted, focussing on image analysis technique. Three separate methods for quantifying WMHs were then investigated. The reproducibility of manual outlining was assessed using scans available from 43 people with secondary progressive MS (SPMS). An automated software method was optimised for the same cohort, based on the results of the manual outlining. A novel semi-quantitative visual rating scale was developed, with validation using the same scans within a larger, more varied cohort. All available information regarding the participants studied was then used to construct a linear regression model predicting cognitive outcomes and determining the utility of the various imaging markers derived from conventional imaging techniques. A non-linear relationship for WMHs was also considered. White matter DTI metrics in the same smaller cohort of 43 people were then investigated, primarily considering tissue outwith WMHs, as well as that within major tracts and the novel diffusion marker `peak width of skeletonised mean diffusivity'. The additional explanatory power of DTI metrics within the linear models developed previously was then determined. Results High variability was found in the literature regarding imaging marker measurement and reporting of technique reproducibility. Manual outlining was found to be associated with considerable measurement error, dependent on observer and cohort factors. It was possible to optimise the automated software for a particular cohort, either for volumetric or spatial outputs. Visual rating of MS imaging features was found to be feasible and measures of WMH burden were closely related to fully quantitative measures. The overall association of WMHs to cognitive function was similar to that found in the published literature, with no additional association following addition of DTI metrics. A trend towards a greater effect of WMH volume at higher levels was found, consistent with a non-linear relationship between imaging metrics and cognitive phenotype. Conclusions Substantial heterogeneity in the reporting of the reproducibility of WMH measurement supports a move towards benchmarking against reference datasets. Poor reliability of the current reference standard, manual segmentation, should be recognised as a key limitation for the field. Rich information can be captured quickly using visual rating of imaging features. The close correlation of visual ratings of WMHs with quantitative measures may represent a practical alternative in the appropriate circumstances. Combining visual rating features provided additional explanatory power, supporting a multidimensional substrate for the cognitive phenotype. Finally, both automated and visual rating analyses support a non-linear relationship between disease burden and cognitive performance in MS.

Background: Relapsing-remitting multiple sclerosis (RRMS) is an autoimmune inflammatory disorder of the central nervous system, with significant morbidity and mortality. The lymphocyte depleting, anti-CD52 monoclonal antibody alemtuzumab is a highly effective treatment option in RRMS, though associated with high rates of secondary autoimmune disorders. As alemtuzumab is now in routine clinical use, following licensing in Europe and the US, understanding the effects of repeated treatment cycles and reducing the risk of secondary autoimmunity is timely and essential. In this thesis, I explore how repeated treatment impacts the extent of lymphodepletion. I also study the role of soluble CD52, recently described as suppressive via interaction with Siglec-10, in the mechanism of secondary autoimmunity, and determine the biomarker potential of pre-treatment cytokine levels. Findings: CD4 and CD8 lymphocytes are less effectively depleted with repeated treatment cycles. Lymphocyte surface CD52 density was found by flow cytometry to be significantly lower after alemtuzumab treatment, although CD52-negative clones are not seen. In a cytolysis assay, reduced CD52 density was shown to correlate with reduced susceptibility to alemtuzumab. In addition, activated proliferating T lymphocytes, as observed after treatment, downregulated CD52 expression in a gene expression assay and shed the antigen from cell surface as demonstrated by ELISA and flow cytometry. The development of immunoassays to detect and quantify anti-idiotype antibodies to alemtuzumab is presented. The occurrence of antibodies at retreatment did not reduce lymphocyte depletion. A regulatory role for soluble CD52 was not found in suppression assays, and the proportion of antigen-activated CD52hi T cells did not vary between healthy controls and RRMS individuals. Siglec-10 was not seen on cell surface of activated T cells by flow cytometry and gene expression; thus concluding that soluble CD52 does not play a role in post-alemtuzumab induced autoimmunity. Prior to treatment, the only serum cytokine found to distinguish individuals who develop autoimmunity from those who do not was IL-21 (higher in the autoimmune cohort), however currently commercially available IL-21 immunoassays have no utility as predictive biomarker tests.

Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases, in part by modulating adaptive and innate immune responses. Since metabolism governs the phenotype and function of immune cells, the aim of this thesis was to investigate whether NSCs have the ability to regulate the immunometabolic components underpinning neuroinflammation. Herein I have identified a new mechanism by which transplanted somatic and directly-induced NSCs counteract CNS-compartmentalised chronic inflammation in mice. NSC transplantation reduces the immunometabolite succinate in the cerebrospinal fluid, while decreasing the burden of mononuclear phagocyte (MP) infiltration and secondary CNS damage. Mechanistically, the anti-inflammatory activity of NSCs arises in response to succinate released by inflammatory MPs, which activates succinate receptor 1 (SUCNR1)/GPR91 on NSCs, thus initiating prostaglandin E2 secretion and extracellular succinate scavenging. This work uncovers a succinate-SUCNR1 axis in NSCs that clarifies how stem cells respond to inflammatory metabolic signals to inhibit the activation of pro-inflammatory MPs in the chronically inflamed brain.

This thesis looks at risk factors for multiple sclerosis (MS), a chronic, degenerative autoimmune disease which is usually diagnosed between the ages of 20 and 50 years. It is estimated to affect over 100,000 people in the UK. The research setting was Orkney and Shetland, two archipelagos situated north of mainland Scotland, and both of which have very high MS prevalence as do other countries at high latitudes. I examine genetic and environmental risk factors in Orkney and Shetland using multiple methods over four studies. I also review the vitamin D and UV exposure literatures as these are risk factors pertinent to MS in Orkney and Shetland. After devoting three chapters to introducing the purpose of the thesis, MS, and Orkney and Shetland, in the fourth chapter, I aim to establish whether the birthplace of cases show any spatial, temporal, or spatiotemporal clustering. Evidence of these kinds of clustering may indicate that there are environmental risk factors present in some areas or that were present over particular periods, which raise risk of developing MS. Although I find statistically significant temporal, spatial, and spatiotemporal clustering in Orkney, and a spatial cluster in Shetland, for multiple reasons these results need to be interpreted with caution. I conclude that the clusters are very likely to be artefacts. Furthermore, there are multiple possible alternative explanations for such clusters that could not be explored by the available data. Chapter 5 examines the heritability of MS in Orkney and Shetland to estimate the proportion of phenotypic variance attributable to additive genetic effects. I also look at the birthplaces of ancestors of cases and controls to see if any locations contribute a greater amount of ancestral DNA to the gene pool of modern MS cases, which I term ‘genetic clustering’. In Orkney I obtained a heritability estimate of 0.36 (95% CI -0.26, 0.98); in Shetland this estimate was 0.20 (95% CI -1.88, 2.28). These modest estimates are consistent with the heritability literature. The genetic clustering analyses highlight two Orkney registration districts, Kirkwall and Westray, which earlier studies identified as areas of MS clustering. I also identify three Shetland registration districts, however these locations had not shown any evidence of clustering in earlier studies. Again, I advise caution in interpreting results, particularly as all the error bars across registration districts overlap. Chapter 6 presents a scoping review to map the literature and identify evidence of an association between vitamin D and UV exposure with MS. In methodically searching the literature, I identify a large and heterogeneous evidence base comprising multiple observational, intervention, and genetic studies. Overall, many studies support an association between vitamin D deficiency and MS. There is also evidence for an association between UV exposure and MS, although UV exposure is considerably less explored than vitamin D. I finally identify gaps in the literature and make suggestions for future research. In Chapter 7 I aim to compare vitamin D levels in Orkney and mainland Scotland, and establish the determinants of vitamin D status in Orkney. I firstly compare mean vitamin D and prevalence of deficiency in cross-sectional data from studies in Orkney and mainland Scotland. I secondly use multivariable regression to identify factors associated with vitamin D levels in Orkney. I find that mean (standard deviation) vitamin D is significantly higher in Orkney compared to mainland Scotland (35.3 (18.0) and 31.7 (21.2), respectively), and prevalence of severe deficiency is lower in Orkney (6.6% to 16.2% p = 1.1 x 10-15). Factors associated with higher vitamin D in Orkney include older age, farming occupations and foreign holidays. I conclude that although mean vitamin D levels are higher in Orkney compared to mainland Scotland, there is substantial variation within the Orkney population which may influence MS risk. Chapter 8 examines the correlates and determinants of UVB exposure in Shetland. I firstly construct correlation matrices to visualise how 1) personal characteristics such as sex, occupation, and skin type, 2) physical activity, and 3) body weight and fat, correlate with UVB exposure. I then use multivariable regression to identify factors associated with UVB exposure in Shetland. I run two multivariable models. The first includes the full sample size where activity data were measured by questionnaires. The second includes both questionnaire physical activity data and step-count data from pedometers, however as only a subset of participants had been supplied with pedometers, this analysis comprises a smaller sample size. I find that the amount of skin exposed was most strongly correlated with UVB exposure. Step count and activity minutes were also moderately positively correlated, and indoor occupations moderately negatively correlated, with UVB exposure. The regression analysis using the full sample with questionnaire activity data found that factors associated with greater UVB exposure were age and ambient UVB, while working indoors was significantly associated with lower UVB exposure. The model including the pedometer data found that found that age, total steps, and the amount of ambient UVB were significantly associated with greater UVB exposure. I conclude that atmospheric conditions, working outdoors and older age are important factors in UVB exposure in Shetland. It remains to be seen how UVB exposure translates to vitamin D levels in Shetland. I found evidence for environmental and genetic risk factors for MS in Orkney and Shetland. The two environmental risk factors, vitamin D deficiency and reduced UV exposure, are more likely to affect the younger population who are still within their lifetime risk of developing MS.

Introduction: The objective of this thesis was to examine aspects of emotion regulation in Multiple Sclerosis (MS). A systematic review was carried out of studies investigating the prevalence of alexithymia in individuals with MS, and its relationship with anxiety and depression. An empirical study was conducted to explore whether there were differences in emotion regulation abilities in individuals with different variants of MS, compared to healthy individuals. Methods: Twelve journal articles were identified via systematic search utilising predefined criteria. Seventy nine individuals with relapsing-remitting type of MS, and 38 individuals with chronic progressive type of MS were recruited to the empirical study. Participants filled in self-report questionnaires measuring difficulties in emotion regulation, illness severity, illness representations and quality of life. Also, 55 healthy volunteers took part in the current study. Results: The estimate of prevalence of alexithymia in MS was approximately twice as high as in general population. Positive relationships between alexithymia and/or anxiety and depressive symptoms were found. Those with MS reported difficulties accepting emotional distress which, to some extent, were predicted by strong illness identity and negative emotional responses to having MS. These findings were independently of illness severity, and type of MS. Perceptions of negative consequences of MS were the only partial mediator of the relationship between illness severity and quality of life. Conclusions: Larger and more representative samples are needed to clarify the impact of alexithymia on the clinical presentations of patients with MS. Psychological interventions targeting non-acceptance of emotional distress and negative illness perceptions are warranted to support those with MS.

Purpose: The systematic review explored whether self-management interventions improve patient activation in long-term conditions, and if any improvements are greater than the amount of change experienced by patients in usual care or active control conditions. It also aimed to determine if positive effects on activation are maintained at follow-up. The empirical study sought to explore relationships between patient activation, psychological factors (depression and valued living), perceived clinician empathy, perceived symptom severity, self-management and demographic variables. It also examined whether depression, valued living and perceived clinician empathy are unique predictors of activation, and if activation is a unique predictor of self-management for MS, when relevant confounding variables are controlled for. Methods: For the systematic review, a comprehensive search of multiple electronic databases was conducted to identify intervention research reporting on patient activation outcomes, as measured by the short-form Patient Activation Measure (PAM-13), in people with long-term conditions. For the empirical study, a cross-sectional survey of 118 people with MS explored patient activation, MS symptom severity, valued living, depression, perceived clinician empathy, self-management for MS and demographic factors. Correlation and hierarchical regression analyses were employed to explore relationships between variables. Results: Twenty-five studies were eligible for inclusion in the systematic review, reporting a wide range of long-term conditions. Twenty-one studies (10 RCTs; 1 non-randomised study; and 10 uncontrolled studies) found an improvement in patient activation at post-intervention. Nine studies reported a significantly greater improvement in activation in self-management conditions compared with usual care or an active control at post-intervention. In six out of eight studies, gains in patient activation were maintained in the intervention group at follow-up. However, in four of these six studies, patient activation in the control group also improved over time. Findings from the empirical study suggested that only valued living was a significant predictor of patient activation after controlling for demographic variables and MS symptom severity. Neither depression nor perceived clinician empathy significantly predicted activation. After controlling for valued living, depression and perceived clinician empathy, patient activation independently predicted 5.5% of variance in self-management for MS. Both activation and perceived clinician empathy were significant predictors of self-management for MS. Conclusions: Self-management interventions improve patient activation in long-term conditions compared with usual care or active control. Patient activation gains appear to be maintained longer-term; however, the impact of self-management interventions on activation is unclear due to increases in activation in control groups over time. Valued living is associated with patient activation in MS, while patient activation and perceived clinician empathy are associated with MS self-management. Self-management interventions targeting valued living and the patient-clinician relationship may be effective for addressing low levels of activation in some patients with MS.

In the brain, axons are wrapped by myelin sheaths which ensure fast saltatory conduction of impulses and provide metabolic support. In multiple sclerosis (MS), the myelin sheaths are lost which leaves the axon denuded. This not only results in slower conduction of action potentials, but if prolonged, can also lead to axon death due to the loss of metabolic support. This neurodegeneration is the main cause of permanent disability in multiple sclerosis patients. The axon death and disability which stem from it could be prevented by restoring the myelin wrap before axon damage has occurred. This remyelination process is carried out by oligodendrocyte precursor cells which are present throughout life. To remyelinate, OPCs migrate to the area of damage and differentiate into myelinating oligodendrocytes which ensheathe axons with new myelin. In multiple sclerosis, this process occurs but is insufficient to overcome the damage. Therefore, central to the therapeutic efforts in multiple sclerosis is the aim to improve endogenous remyelination. Enhancing recruitment of oligodendrocyte precursor cells (OPCs) to the areas of damage is a clinically unexplored target. To investigate the therapeutic potential of OPC recruitment modulators, I have looked at 2 different targets involved in migration NDST1/HS and Sema3A/NP1. The first target, heparan sulfate (HS) is a proteoglycan which is important to OPC migration, investigated by Pascale Durbec's group in France. In a demyelinating mouse model, its key synthesising enzyme, NDST1, is upregulated by oligodendroglia in a belt around the lesion to aid OPC recruitment. Loss of NDST1 in oligodendrocytes was found to impair remyelination and reduce OPC migration in mice. In collaboration with them, I investigated the relevance of this molecule in post-mortem MS human tissue. I found that in human as well as mouse, NDST1 was primarily expressed by oligodendroglia. The protein level and the proportion of oligodendroglia expressing NDST1 was increased in MS compared to control indicating NDST1 upregulation as a disease response in human. We also found that low numbers of NDST1+ oligodendroglia correlate with bigger sizes of lesions and chronic lesion types that fail to repair, highlighting its importance in repair. Moreover, high numbers of NDST1+ cells in a patient correlated with increased remyelination potential. This indicates that in human, intra-patient variation in NDST1 level may explain differences in potential for endogenous repair. Secondly, I looked at Sema3A, a chemorepulsive molecule which is upregulated in demyelinated injury rodent models aswell as multiple sclerosis lesions, particularly in OPC-depopulated chronic active lesions. Research has consistently found that the level of Sema3A negatively correlates to remyelination because Sema3A hinders OPC migration. This has highlighted Sema3A as a potential target to improve OPC recruitment in MS however the size and shape of the molecule make it hard to design therapeutics against it. Therefore, I looked at its druggable receptor, Neuropilin 1 (NP1), to see whether inhibition of NP1 had the same positive effect on OPC recruitment and remyelination as lowering the level of Sema3A. NP1 is a tyrosine kinase receptor for both Sema3A and vascular endothelial growth factor (VEGF) and is found in many cell types. To check if NP1 inhibition is beneficial, I assessed remyelination in a mouse where the Sema3A binding site of NP1 has been mutated to prevent Sema3A binding and exerting its effect. This is a proxy for a (currently unavailable) ideal NP1 inhibitor of the Sema3A site only. Contrary to my expectations, OPC recruitment and remyelination in the mutant mice were not improved. However, the NP1 mutation resulted in an altered immune response. To exclude the possibility that no improvement in the OPC recruitment and remyelination of those mice was seen because it was negated by the altered immune response, I explored a cell specific mutant mouse in which NP1 was deleted in oligodendroglia only. In this mutant as well, I did not see improvement of OPC recruitment and remyelination. I therefore propose that Neuropilin 1 is not imperative for Sema3As action in remyelination and is not suitable as a therapeutic target in multiple sclerosis. Loss of the whole NP1, but not loss of the Sema3A site also resulted in biggermyelinated and unmyelinated axons as well as a different myelin thickness post remyelination. This showed that VEGF and the VEGF site on NP1 in oligodendroglia have a previously unknown but important role in determining axon size and myelin thickness which should be further investigated. To further elucidate those results in a simple system, I looked at how Sema3A, NP1-Sema3A inhibitors, VEGF and NP1-VEGF inhibitor affect OPC behaviour. I confirmed Sema3As chemorepulsive effect but also showed that at different concentrations it can improve proliferation and survival of OPCs. Inhibiting the Sema3A site and the VEGF site of NP1 by specific blocking antibodies also affects OPC proliferation and maturation. This suggested that NP1s ligands are involved in more than just OPC migration. In summary, this work supports the relevance of the mouse findings that NDST1 is upregulated in demyelination and important for repair for human illustrating that it might be a suitable therapeutic target to investigate. However, despite the importance of Sema3A in MS models, its only reported receptor, NP1, is not essential for Sema3As action. Therefore, it is an unsuitable therapeutic target. The fact that NP1 is an inappropriate drug target for MS is further demonstrated by the involvement of its ligands in multiple OPC behaviours both in positive and negative aspects.

Axonal degeneration is the major cause of disability in progressive multiple sclerosis (MS). It has been shown that in MS and relevant disease models, demyelinated axons harbor an increased number of mitochondria, which is reflected in bigger stationary sites of mitochondria, increased mitochondrial activity and increased transport speed of mitochondria. This axonal response of mitochondria to demyelination (ARMD) is protective, as there is an increase in energy demand due to the redistribution of sodium channels along the axon following demyelination. However, it remains to be determined how this ARMD is mounted and how mitochondrial dynamics are involved. By using in vivo and in vitro systems we are determined to elucidate the transport and fusion dynamics of the ARMD and where these additional mitochondria come from. Using a cerebellar slice culture system with lysolecithin induced demyelination, we show that the increase in mitochondrial occupancy of the axon already occurs at 24 hours after demyelination and plateaus around 3 to 4 days after demyelination. At 24 hours, there was a steep increase in the mitochondrial numbers inside the axon, which is then followed by an increase in mitochondrial size over the following days. All parameters decrease again over the following days, but remain elevated compared to baseline even 12 days after demyelination. To determine the source of these additional mitochondria and to assess the fusion dynamics within the axon, we used a lentivirus expressing a mitochondrial targeted photoconvertible dye (mEOS2) to label mitochondria in Purkinje cells. The mitochondria that are labelled green in the Purkinje cell axons are then photoconverted to red by illuminating the initial part of the axon with a 405-nm laser and imaged over the following 20 minutes to determine the transport and fusion dynamics. This showed an increased number of mitochondria moving from the cell body into the axon, as well as an increase in retrograde transport of mitochondria in the demyelinated compared to the myelinated axons. Furthermore the size of newly transported mitochondria and their speed was increased in the anterograde direction. Furthermore, the fusion rate of newly transported mitochondria with stationary converted mitochondria was increased in the demyelinated axons compared to myelinated control. These changes can also be observed in unmyelinated axons, as well as axons of cerebellar slices of the dysmyelinating shiverer mutant with or without lysolecithin treatment. The manipulation of mitochondrial dynamics after demyelination with the fission inhibitor mdivi-1 and the ATPase inhibitor oligomycin both showed an increasing or decreasing effect on the mitochondrial parameters after demyelination respectively. The effect on the axonal health after demyelination was detrimental with both of these treatments. Increasing mitochondrial biogenesis with pioglitazone increased axonal mitochondrial parameters, as well as ameliorated axonal damage after demyelination with lysolecithin. As the neuronal cell bodies in MS harbour mitochondrial DNA deletions, which affects their physiology, including energy production efficiency, another aim of this thesis was to model this deficiency in vitro. As it was not possible to model these mitochondrial defects in vitro within the experiments of this thesis, the characterization of a mitochondrial mutant in vivo model was done as a contribution to a greater set of experiments performed by other members of the Mahad lab.

Cerebellar signs and cognitive dysfunction often occur in parallel in Relapsing-Remitting Multiple Sclerosis (RR-MS). Motor planning is thought to be one area of cognition susceptible to damage but also a surrogate of cerebellar integrity. Therefore, the objective was to investigate the longitudinal relation of cognition to cerebellar function in RR-MS and how changes relate to motor planning and function. A total of 11 RR-MS patients with cerebellar symptomatology (RR-MSc: 5 males, x̅ age: 41), 17 RR-MS patients without cerebellar symptomatology (RR-MSnc: 2 males, x̅ age: 40.94) and 9 matched control participants (HC: 2 males, x̅ age: 37.78) completed the Brief International Cognitive Assessment for MS (BICAMS), the Nine-hole Peg Test (NHPT) and the Grooved Pegboard Test (GPT) at baseline and at 12 months. The GPT - NHPT difference was used to compute a Motor Planning index (MPI), the NHPT serving as a control for sensorimotor impairment. Mixed ANOVA revealed a consistent significant group separation on all cognitive and motor tests other than visual memory that was maintained over the follow-up period. There was a significant effect of time for the visual and verbal memory suggestive of learning. There were no significant interactions between group and time. Post hoc tests showed the RR-MSc group was significantly outperformed in tests of cognition, motor function and motor planning by both RR-MSc and HC. There was a significant correlation between measures of information processing speed and MPI. RR-MSc were characterised by greater impairment on tests of cognition, motor function and motor planning than the RR-MSc. These differences were maintained over a year. The association of the MPI and SDMT indicates information processing speed may act as a moderator of motor planning. The next step is to validate the MPI with an MRI study exploring how it relates to cerebellar lesion load and atrophy.

Multiple Sclerosis (MS) is a chronic, demyelinating disease which affects neurons in the central nervous system. Symptoms of MS include difficulties with mobility, gait, bowel and bladder dysfunction, fatigue and cognition. People with MS (PwMS) are often diagnosed in their 30s and 40s and up to 80% become unemployed within 10 years of being diagnosed. This results in a negative impact on quality of life. Levels of physical disability do not fully account for the impact of the disease on employment outcomes. In fact, approximately 45% of people with low levels of physical disability are unemployed. It is likely that the impact of 'invisible' symptoms of MS, such as cognition, can explain this discrepancy. Evidence suggests that cognition is also a mediating factor between physical disabilities and unemployment. Since unemployment can have a detrimental impact to quality of life, it is important to ensure that people with MS remain employed for as long as they desire.

Objectives: The World Health Organisation has predicted that by 2020, brain injury will be one of the leading causes of disability in the world (Hyder et al, 2007). Psychological difficulties are common in this population, with up to 60% of individuals experiencing mental health difficulties (Acquired Bain injury Outreach Service, 2012). Therefore, with the rapid rise in referrals, services are under increasing pressure to provide innovative ways of offering effective and cost-efficient care. This research aimed to evaluate a novel transdiagnostic Acceptance and Commitment Therapy group approach for supporting individuals adjusting to life following the diagnosis of a neurological condition. Methods: A mixed-methods waiting-list control design was used and carried out across two sites of Hertfordshire Neurological Outpatients Service. The outcomes of the group were assessed using four outcome measures evaluating acceptance, self-identified difficulties, low mood and anxiety, as well as a semi-structured interview to identify mediators of change. Results: Ten participants from the intervention group completed, equating to a 76.9% completion rate. The results indicated that those in the intervention group made significant improvements across all measures of acceptance, self-identified difficulties and psychological distress. Those in the waiting list groups did not experience any change in these domains. The qualitative feedback from participants was also highly positive. Participants reported the usefulness of the ACT strategies, in addition to valuing being in a group with others with a range of difficulties. Participants reported greater awareness and acceptance, as well as increased activity and improved mood. Conclusions: The ACT group is a potentially effective and cost-efficient method of supporting individuals with adjustment following diagnosis of a neurological condition. Despite these promising findings it is important to acknowledge the limitations, such as the small sample size and research design. Further research would be beneficial in order to evaluate the intervention using more rigorous methods.

Background: Pain is frequently reported by people with multiple sclerosis (MS). It has been associated with decreased quality of life, psychiatric morbidity, interference with day to day activities, and frequent healthcare attendance. It has been reported by people with multiple sclerosis to be one of their most important symptoms, and available treatments are limited in their effectiveness. Despite this, our understanding of the epidemiology and mechanisms of pain in people with MS are limited. Our understanding of the interactions of central nervous system mechanisms and pain states overall is growing. However, the application of this knowledge to MS is incomplete. Previous studies have shown that the descending pain modulatory system (DPMS) is an endogenous network of cortical and subcortical brain structures which act to limit, or accentuate, an individual’s perception of pain, via descending brainstem pathways. Associated clinical measures include depression, anxiety, and cognitive flexibility. Our understanding of the function or dysfunction of this system in MS is limited. We do not know if the MS disease process may adversely affect the structure or function of the DPMS. Hypothesis: In people with neuropathic limb pain in relapsing remitting MS (RRMS), compared to people with RRMS who do not have pain, there will be disruption of the endogenous descending pain modulatory system. This will manifest as impaired descending inhibition of pain. Aims and Methods Establishing the background using systematic reviews: The first aim of this thesis was to establish the prevalence, natural history and associations of pain (and pain syndromes) occurring in people with MS. The second aim was to explore existing knowledge of how the MS disease process may contribute to pain states, using a systematic review of neuroimaging studies. Prospective clinical study: A case-control study of 47 people with RRMS was then carried out. 31 of these had neuropathic pain in the limbs, and 16 did not have pain. Using targeted assessments, function of the descending pain modulatory system was assessed in the following ways: First: Detailed clinical, behavioural and neuropsychological assessment, focussing on cognitive, behavioural and affective features known to be closely related to the DPMS. Second: MRI imaging of brain structure, focussing on the volume and location of MS lesions, as well as the volume of key grey-matter structures involved in the DPMS. Third: Resting state functional MRI imaging of the brain, focussing on functional connectivity between the rostral anterior cingulate cortex and two other key DPMS structures (dorsolateral prefrontal cortex, and periaqueductal gray). Results: Systematic reviews: Meta-analysis of existing prospective studies confirmed that pain is very common in MS, affecting about 63% of people with MS on average (95%CI between 55 and 70%). Many different types of pain contribute to this overall estimate. No significant associations with disease course or stage emerged. Several neuroimaging studies have assessed people with MS-associated pain using MRI. These studies were often small, and with associated methodological issues. It is likely that location of MS lesions is implicated in aetiology of pain syndromes in some cases, though our overall knowledge is limited. Prospective study: In a prospective study, people with and without pain were matched for age and gender. Furthermore, groups were balanced for a range of other variables. The pain group more frequently received gabapentinoid medications. The presence of pain was significantly associated with increased scores for depression, fatigue and catastrophising, as well as with specific impairments at neuropsychological assessment, including cognitive flexibility. Many of these impairments are directly relevant to existing models of the DPMS. Overall volume of MS lesions was not different in people with pain, though lesions were more likely to occur in the brainstem. Some alterations of grey-matter volumes in people with pain which mirrored studies of pain disorders outside MS were found, but these did not involve structures key to the DPMS. Affected structures included trigeminothalamic nucleus (relative volume increase in pain group), posterior cingulate cortex and parahippocampal gyrus (volume decrease in pain group). Functional connectivity of the rostral anterior cingulate cortex to the periaqueductal grey matter, a key structure in the descending modulation of pain, was stronger in the group without pain. Conversely, functional connectivity to the dorsolateral prefrontal cortex, repeatedly implicated in the DPMS and thought to be involved in cognitive evaluation and flexibility, was stronger in the pain group. MS lesion volume appeared to account for some of this difference in a multivariate analysis. Limitations: Key limitations of this work include cross-sectional design, small sample size, and number of statistical comparisons carried out. Conclusions: Systematic reviews examined the prevalence, natural history and associations of pain in MS, as well as examining existing neuroimaging studies which investigated how the MS disease process could contribute to pain states. A prospective study found evidence of both emotional/affective and cognitive dysfunctions relevant to the hypothesis of dysfunction in the DPMS. Higher likelihood of MS lesions in the brainstem could be relevant to DPMS function. Separately, there were structural grey-matter volume alterations reflecting those found in many pain studies outside MS. Importantly, however, these did not affect key DPMS structures. Resting state functional MRI however demonstrated altered connectivity of core DPMS structures, which may be partly mediated by MS lesion volume. Functional connectivity findings could be consistent with the hypothesis of impaired descending pain inhibition, in people with relapsing remitting MS affected by neuropathic limb pain.

Multiple Sclerosis (MS) patients are faced with complex risk-benefit profiles of disease-modifying drugs (DMDs). It is important that MS patients are able to understand the risks and benefits of DMDs in order to make an informed treatment decision. The goal of the present thesis was to identify the best methods of presenting treatment risks and benefits to improve MS patients' understanding and reduce conflict in their treatment decisions. First, systematic reviews and surveys were conducted to inform the need to improve MS patients' understanding of treatments. Subsequently, three experiments were designed to assess the effect of different presentation methods on MS patients' understanding of hypothetical treatments. Experiment 1 found that treatments presented using non-graphical formats had an effect on patients' understanding (p < .001), with numerical frequencies improving understanding the greatest. Understanding of treatments was also affected by presentation using graphical formats (p < .001), and was greatest for bar charts and line graphs. Experiment 2 found that presenting treatments using ratios (p < .001) and frames (p < .05) also affected patients' understanding. Experiment 3 showed that the methods to communicate differences in the risks and benefits of clinical trial groups further had an effect on patients' understanding of treatments (p < .001). The BRIMMS protocol was developed by integrating the best presentation methods from Experiments 1-3 and was compared with standard consultation using a randomised controlled trial with crossover design. The BRIMMS protocol improved understanding (p < .001) and reduced decisional conflict (p < .001) in comparison to standard consultations. In conclusion, some methods to present treatments can improve MS patients' understanding of treatment risks and benefits. The BRIMMS protocol could potentially be implemented into current clinical practice to improve MS patients' understanding and engagement in the decision-making process.

Cerebellar symptoms are associated with poor prognosis in MS. A small number of studies have reported a specific cognitive profile associated with cerebellar damage, including reduced information processing speed, impairments in visuospatial functions and verbal fluency deficits. The cerebellum is thought to encode models of sensory information, providing mental representations of the world used to plan motor actions. Motor planning may be a key factor in the disability that cerebellar involvement imposes. This study explores how the cognitive profile of those with MS with cerebellar symptoms (RR-MSc) relates to motor planning and motor function, and how this differs from those with MS without cerebellar symptoms (RR-MSnc) and healthy controls (HC). Participants were assigned to three groups: HC (n = 21), RR-MSnc (n = 21) and RR-MSc (n = 14) using a validated self-report cerebellar symptom questionnaire (NARCOMS-TACS). Participants completed a cognitive test battery: BICAMS (CVLT-II, SDMT, BVMT-R), WLG, PASAT3 and TOPF. For assessment of motor functioning and motor planning, participants completed the 9-hole peg test (9HPT) as a sensorimotor control, and grooved pegboard test (GPT) which has greater motor planning demands. Subtraction of 9HPT from GPT was used to compute a motor planning index (MPI). One-way ANCOVAs demonstrated significant differences between groups on all cognitive tests other than WLG. Post-hoc tests showed significant differences between RR-MSc and HC on all tests except WLG, and RR-MSc and RR-MSnc on SDMT, CVLT-II and PASAT3. On 9HPT, GPT and MPI, HC performed significantly better than both MS groups, and the RR-MSnc performed significantly better than RR-MSc. There were significant correlations between neuropsychological variables (other than WLG) and MPI. RR-MSc showed greater impairment than RR-MSnc on cognitive tests and tasks requiring motor functioning and motor planning. RR-MSc demonstrated a specific cognitive profile associated with more significant and widespread impairments. Clinical implications are discussed.

The subject of this thesis is the use of Magnetic Resonance (MR) Imaging to quantify biometric MR indices in the Multiple Sclerosis (MS) fixed post-mortem central nervous system (CNS) tissue. Evaluating these indices in fixed tissue allows for the use of histology to verify the findings of MRI. However, it must first be discovered if the indices can be evaluated in fixed post-mortem spinal cord tissue. There is very little literature in this specific area, though some in the fixed brain, the results of which have been assumed to be equivalent in the spinal cord without proof. Therefore, the methodology must first be verified before the consideration of any index as useful and translatable to in-vivo spinal cord. This thesis concentrates on the evaluation of MR relaxometry methods using the indices T1 and T2 by themselves and to evaluate the myelin content of fixed post-mortem CNS tissue. The Carr-Purcell-Meiboom-Gill (CPMG) and Multicomponent Driven Equilibrium Single Pulse Observation of T1 & T2 (mcDESPOT) sequences are used to calculate T1, T2 and the Myelin Water Fraction (MWF) which is believed to be proportional to myelin content in the CNS. This is performed at 3T in a clinical scanner and at 7T in a small animal and wholebody scanner. The methods are first evaluated for use in fixed post-mortem CNS tissue. The two myelin measurement methods are then compared to histological staining if appropriate and where available to verify that the results obtained are proportional to myelin content. The T1 and T2 values in fixed tissue were found to be shortened in fixed tissue, T2 values were so short as to be at the limits of measurement by a clinical scanner, and values converged in white and grey matter, and therefore contrast was found to be limited between these tissues. Proton density images provided the most contrast between tissues. However, even with shortened T2 values, the CPMG sequence was able to identify the myelin water component in fixed tissue. The mcDESPOT algorithm struggled to separate the myelin water component due to clinical scanner limitations and the shortened, converging T1 and T2 values. However, the mcDESPOT algorithm was successful in discerning the myelin water component in the high signal situation of a small bore 7T peclinical scanner. An evaluation was then made of the usefulness of these indices for translation into clinical imaging. The CPMG sequence was found to be proportional to myelin content under all conditions, and therefore useful for disease monitoring in demyelinating diseases. The mcDESPOT sequence, was found to be proportional to myelin in some conditions, and is likely to be useful for monitoring myelination, though the sequence could not be fully validated in this thesis.

Basic science and early clinical trial evidence suggest the safe diuretic drug amiloride, may exert a neuroprotective effect in multiple sclerosis (MS) through blockade of the acid sensing ion channel. Neuroprotective treatments are a key unmet need in multiple sclerosis. Optic neuritis (ON) is a discrete CNS inflammatory event leading to neuro-axonal injury in the optic nerve and retina. The optic nerve is part of the visual system, one of the most functionally and structurally eloquent systems in the central nervous system, which affords a number of unique modalities to assess neurodegeneration and neuroprotection. The visual system can be classified into two parts, the anterior and posterior visual systems, which are defined by the lateral geniculate nucleus, where the two components synapse. The extent of neurodegeneration following ON in the anterior visual system can be imaged in vivo through scanning laser polarimetry (GDx) and optical coherence tomography (OCT). The posterior visual system can be imaged by quantitative and functional magnetic resonance imaging (MRI) of the brain, giving insights into white matter structural integrity and cortical plasticity over time. Combining these modalities in a longitudinal study, allows assessment of the impact of neurodegeneration in the anterior visual system on neurodegeneration downstream in the posterior visual system and on changes in functional connectivity over time in the visual cortex. Furthermore, in the clinical trial setting the neuroprotective effect of any intervention both on direct anterior neurodegeneration and downstream processes can be assessed. The functional relevance of changes in all of these biomarkers can be tested through a number of visual measures, including low contrast visual acuity. In MS, the contribution of transsynaptic neurodegeneration to the global neuronal loss experienced by patients is an area of incomplete understanding. In addition, the role of the visual cortex, through neuroplasticity, in aiding visual recovery from optic neuritis, is unclear. To address these issues, this thesis reports the results of the first clinical trial of amiloride in ON, and shows that despite the pre- and early clinical evidence of neuroprotection of amiloride, no neuroprotective benefit was found. It goes on to explore reasons for this lack of effect including the finding of early retinal neurodegeneration in ON, and the need for early recruitment windows in the future. From there, it makes a detailed assessment of the longitudinal changes in retinal OCT for 12 months following ON, including a novel finding of the temporal evolution of inner nuclear layer swelling, previously reported only cross-sectionally. Next, for the first time macular retinal neurodegeneration is shown to influence diffusion tensor MRI derived measures of white matter integrity in the optic radiations, indicating transsynaptic neurodegeneration. Finally, longitudinal changes in resting state functional connectivity following ON are found in the visual system for the first time. The interaction between this cortical functional, retinal neurodegeneration and visual recovery is probed.