Cognitive impairment and fatigue are prevalent and impactful symptoms of multiple sclerosis (MS). Effective markers are required by clinical studies to accurately test the efficacy of treatments for these symptoms. Graph analysis of brain networks based on magnetic resonance imaging (MRI) data can feasibly provide useful candidate markers of cognitive impairment and fatigability in MS which may be more objective, reliable and specific than existing markers. My original contribution to knowledge is therefore an exposition of the following hypothesis: “summary graph-theoretic descriptors of brain network organisation are good candidate markers of cognition or fatigue in MS”. To achieve this, network metrics were assessed based on three main criteria: reliability (“are the measurements the same across time and settings?”), validity (“do they measure what they are supposed to measure?”) and responsiveness (“are they altered when a change in cognitive state is induced?”). The applicability of the graph-theoretic approach was first established with a spatial meta-analysis of tract integrity and its relevance to cognition and disability. Reliability over time in healthy subjects was assessed by systematic review and reliability between different scanners and between MS and control groups was assessed in two longitudinal datasets by measuring intra-class correlation (ICC) of graph metrics. The validity criterion was assessed in an analysis of covariance and linear regression of cognitive and fatigue measures with brain network metrics in people with MS. Finally, an exploration of network dynamics during a sustained attention task with a sliding-window approach was performed to test the immediate responsiveness of the measures to alterations in cognitive state. Spatial meta-analysis of white matter tract degradation was performed using the Signed Differential Mapping method. Statistical maps were gathered from the original authors of studies which performed voxelwise correlations between fractional anisotropy (a measure of white matter integrity based on diffusion tensor imaging data) and measures of either cognitive impairment or physical disability. The combined sample included 495 people with MS and 253 controls from 12 studies. MS diagnosis was significantly associated with widespread lower tract fractional anisotropy. Distributions of voxels with significantly lower fractional anisotropy in relation to cognition and physical disability were only minimally overlapping. The number of and effect sizes for significant clusters in the cognition comparison were greater than those for the physical disability comparison, suggesting a greater relevance of cerebral white matter damage to cognition. The main results remained significant when using a stringent p-value threshold of 0.00001 to control for false positives. The next analysis was a systematic review of the reproducibility of graph metrics over time in healthy people. Online databases were searched for articles reporting ICCs for graph metrics based on imaging data and information was recorded on the sample size, acquisition method, inter-scan interval and reported ICCs. Twenty-six articles were included, with a combined sample size of 676. Overall, reproducibility over time was rated as “good”, but heterogeneity of methods precluded in-depth quantitative analysis. A qualitative synthesis of results highlighted the main methodologic factors affecting reproducibility, which included: ICC type, retest interval, fibre tracking algorithm, graph metric type, image processing strategy, region of interest size, graph threshold and acquisition method. Reliability of brain network metrics between scanners was tested using a travelling-subjects dataset in which 5 subjects each underwent a resting-state functional MRI scan at 10 sites. Graph metrics were calculated for each scan and then tested for ICC across sites. Reproducibility was “poor” for most metrics (characteristic path length ICC=0.23, global efficiency ICC=0.18, modularity ICC=0.24) and “fair” for two (clustering coefficient ICC=0.43, small-worldness ICC=0.42). There was limited evidence that some subjects tended to produce less reliable results and that magnets with higher field strengths did not produce more reliable results. The main implication is that multi-site studies using graph analysis of brain MRI data should investigate inter-site reproducibility beforehand. To investigate the validity of graph metrics as markers of cognitive impairment and fatigue, MRI and neurocognitive data were first gathered from 37 people with MS and 23 matched controls. The sample was characterised in detail and comprised a range of cognitive abilities. Data quality was investigated and the small-world structure of the data was confirmed by comparison to random and lattice graphs. Analysis of covariance controlling for age, sex and education showed significant group differences for all but one graph metric. Linear regression models predicted the main measures of cognitive impairment in the MS group, but not in the control group. Measures of fatigue were not well-explained by graph metrics. The direction of the relationships indicated that greater levels of cognitive impairment were related to increased network clustering and modularity, longer average path lengths, lower small-worldness, lower levels of education, old age and sleep disturbance. Finally, responsiveness of graph metrics was investigated in an analysis of functional network dynamics during performance of a sustained attention task. A “sliding-window” approach was taken, in which network metrics were calculated for 84 100-second windows at increments along the fMRI timeseries. Reaction times in the task showed a learning effect for both groups, but were consistently slower for the MS group. Plots of graph metrics over time showed differing responses to the task and to the transition between task and rest periods between groups. The small-worldness and clustering coefficient metrics were correlated with reaction times for both MS (small-worldness: r=0.623, < 0.001; clustering coefficient: r=0.554, p= < 0.001) and control (small-worldness: r=0.586, < 0.001; clustering coefficient: r=0.627, p= < 0.001) groups, but the characteristic path length metric was not (MS: r=-0.154, p=0.313; control: r=0.343, p=0.021). Disconnection of cortical areas by degradation of white matter is a viable explanation for cognitive symptoms in MS. There is some evidence that increased network segregation and decreased network integration may explain cognitive symptomatology. Graph theoretic summary brain network metrics do have potential for use as complimentary information to existing markers of cognitive impairment in clinical studies.

Background: Approximately 152,000 people have a stroke in the UK every year, a quarter are working age and only 40% return to work. Vocational rehabilitation (VR) provision is patchy in the UK and has not been evaluated for the stroke population. Aim: This study aimed to develop, deliver and evaluate stroke specific VR in a feasibility randomised controlled trial (RCT) in one English county. Method: A qualitative interview study with key stakeholders sought to explore barriers to and unmet needs for support for stroke survivors intending to return to work. The findings, two case studies and an expert panel informed the development of a stroke specific VR intervention. Its potential effectiveness in influencing occupational status at 12 months post baseline was measured in a feasibility RCT. Intervention content was analysed and the stroke survivors and employers who received it were interviewed about its usefulness and acceptability. Results: 18 key stakeholders identified barriers to VR in existing service design and delivery. Stakeholders identified the need for individualised, responsive, timely and flexible intervention including support for family members and employers. 46 people, with mainly minor and moderate strokes, were recruited to the feasibility trial and 23 randomised to stroke specific VR. Delivery and compliance with intervention was feasible. Only one participant withdrew. Follow-up was feasible at three, six and 12 months post baseline as indicated by an overall response rate of 73.9%. Twice as many participants returned to work in the intervention group. Data collection on income and benefit status was problematic due to missing data. Secondary measures included quality of life, function, mood and participation. A proforma was successfully used to record and measure intervention content, which showed that stroke specific VR is an individually tailored complex intervention involving cross sector working. 12 stroke survivors and six employers interviewed following the trial, valued this flexible, individualised intervention which positively influenced return to work experiences and outcomes. Discussion: Stroke severity influenced participation and a different model may be needed for those with severe stroke and those unable to return to an existing job. Employer contact was not always possible or desired by the stroke survivor. Funding, targeting and implementing this type of intervention requires further consideration. Conclusion: Early intervention can potentially influence job retention rates in people with mild and moderate stroke but a larger trial is needed to demonstrate effect.

1. The materials consisted of 20 Salmo salar (salmon parr), 12 Rana teroporaria (frog), 12 Lacerta viridis (Common green lizard), 45 Gallus domesticus (domestic fowl), and 20 Mus norvegious albinus (common white rat). 2. The brains, having been fixed by injection with 10% neutral formol-saline, were carefully dissected out. Some measurements and photographs were made, and the specimens were embedded in paraffin outing down the time of dehydration in the higher grades of alcohol to a minimum and avoiding cedar-wood oil as a clearing agent for reasons which have been given in the text. Three series of serial sections, out at 15?, were prepared from each brain embedded, and were respectively, (a) impregnated with silver by the Bodian Method, (b) stained with, carbol fuohsin and (c) stained with iron haematoxylin. One hundred and two such series were prepared. 3. The following conclusions were reached: (a) The size of the optic lobes, as observed from the surface and relative to the size of the cerebrum, was found to diminish as the vertebrate series of brains was ascended. Estimations of the volume of the optic lobes were, however, not made. (b) It was verified that the superior and the inferior colliculi of the mammals are homologous respectively with the optic tectum and the torus semi-circularis composing the sub mammalian optic lobe. Further, it was found that structurally the optic lobes (in: eluding the ventricles) of the pieces, amphibia and reptilia, and the tectum of the mammalia could be arranged in this order as a series. The optic lobes of the aves were, however, divergent, in that they could not be placed in the series as an intermediate stage between the reptilian and mammalian forms. (c) Throughout the vertebrate series the optic tectum including the superior colliculus of mammals presents a lamination of its constituent cells and fibres. The layers of the representative brains were numbered from without inwards and were compared and correlated with each other using Huber and Crosby's classification of the layers in the reptilian optic tectum as a standard. The lamination of the optic tectum at first increases as the vertebrate series is ascended; it reaches its peak development in the lizard and the bird, and undergoes a reduction again in the mammals. (d) Homologies in the structure of the torus semi-circular is of the lower vertebrates, the nucleus mesencephalicus lateralis pars dorsalis of the aves and the inferior colliculus of the Mammalia were noted. (e) The morphology of the torus longitudinalis, conspicuous in the teleostean fish, was examined. The torus is formed by the fusion in the median plane of two separate processes, from the dorso-medial part of the optic tectum all the layers of which take part in its formation.

The aim of this project was to determine the neurotrophic factor survival requirements of PNS neurons during development and to clarify the role of certain receptors in mediating responsiveness. Members of the neurotrophin family of neurotrophic factors (NGF, BDNF, NT3 and NT4/5) and neurotrophic cytokines (CNTF, LIF, OSM, IL-6 and CT-1) were studied. The activity of a recently identified neurotrophin, NT4/5, was investigated in vitro. In cultures of mouse neurons, mammalian NT4/5 promoted the survival of the same kinds of neurons as BDNF and was as potent as BDNF, which is consistent with the action of both neurotrophins on the same receptor, TrkB. However, both mammalian NT-4/5 and the Xenopus homologue were less potent than mammalian BDNF on chicken embryo neurons, which is consistent with the lower evolutionary conservation of NT4/5. Interestingly, mammalian NT4/5 exhibited differences in potency on certain populations of chicken neurons that responded equally well to BDNF, and this may reflect differences in TrkB receptors in these different populations of neurons. To clarify the role of the common neurotrophin receptor in modulating the response of neurons to NGF, I then compared the actions of NGF with a mutated NGF protein that binds to TrkA, but does not bind to p75. At subsaturating concentrations, the NGF mutant was less effective than NGF in promoting the survival of embryonic sensory neurons and postnatal sympathetic neurons but was equally effective as NGF in promoting the survival of embryonic sympathetic neurons, indicating that binding of NGF to p75 enhances the sensitivity of NGF-dependent neurons to NGF at certain stages of development. To investigate if neurotrophic cytokines act on developing sensory neurons, I studied their effects in vitro. Whereas trigeminal neurons were responded to cytokines in the late fetal period, nodose neurons were supported by these factors throughout embryonic development. These findings indicate that different populations of PNS neurons display different patterns of responsiveness to neurotrophic cytokines during development.

BDNF binds to two transmembrane receptors: trkB, which is a tyrosine kinase essential for signalling, and p75, which is a common neurotrophin receptor whose role is contoversial. To determine the relationship between BDNF synthesis, BDNF receptor expression, and neuronal responsiveness, the expression of BDNF, trkB, and p75 mRNAs were studied for different populations of sensory neurons whose axons reach their targets and become dependent on BDNF for survival at different times. BDNF mRNA was expressed in the peripheral and central targets of these neurons prior to the arrival of sensory axons. The onset of BDNF responsiveness was preceded by the expression of first p75 mRNA then trkB mRNA, and neurons that start responding to BDNF early were the first to express trkB mRNA. BDNF upregulated trkB mRNA expression just shortly before the onset of BDNF dependence. BDNF is expressed not only in sensory neuron targets but in some of these neurons themselves. To determine whether BDNF is synthesized in NGF-dependent or BDNF-dependent neurons, BDNF mRNA expression was studied in purified populations of cranial sensory neurons that depend on either NGF or BDNF for survival. During the period of neuronal death, BDNF mRNA expression was highest in NGF-dependent cutaneous sensory neurons, lower in BDNF-dependent cutaneous sensory neurons, and undetectable in BDNF-dependent proprioceptive neurons. In coculture, NGF-dependent neurons promoted the survival of BDNF-dependent neurons by the production and release of BDNF, implying a paracrine role for BDNF during the period of naturally occurring neuronal death. To determine if the level of p75 expression in sensory neurons is related to the particular neurotrophin they require for survival, p75 mRNA levels were measured in purified populations of cranial sensory neurons. No clear relationship between the level of p75 mRNA expression and neuron type was observed. Studies of the regulation of p75 mRNA expression in sympathetic neuroblasts revealed that retinoic acid increased and membrane depolarization using KCl decreased the levels of p75 mRNA.

Convulsive status epilepticus (CSE) is an emergency condition associated with mortality and morbidity. It is commonly treated with antiepileptic drugs (AEDs), but these may cause serious adverse events and even death in children. Research on their effectiveness for CSE, and related adverse events in children remains limited. The primary aim of this research was thus to evaluate the effectiveness and safety of AEDs in treating acute tonic-clonic seizure including convulsive status epilepticus (CSE). Two systematic reviews and meta-analyses were conducted to address these aims. The first evaluated the effectiveness of AEDs in children with acute tonic-clonic seizures including (CSE). The second evaluated the safety of AEDs in this population. The systematic review of AED effectiveness identified 20 studies published between 1946 and April 2015. It showed that buccal midazolam was more effective than rectal diazepam for treating acute tonic-clonic seizures including CSE in children, and was associated with a lower recurrence rate. Lorazepam and diazepam were equally effective in terminating seizures, but for lorazepam, intravenous administration was more effective than the buccal, sublingual or intranasal routes. Intravenous valproate appeared to be more effective than intravenous phenytoin and phenobarbital; however, the difference was not significant. The systematic review of AED safety for children with acute tonic-clonic seizures identified 25 studies, published between 1946 and April 2015. These studies were predominantly randomised controlled trials and of these 19 studies reported more than one adverse event, while 6 reported none. A total of 203 adverse events were documented, most commonly respiratory depression (101 children), mainly after treatment with diazepam (46 children). The rates of respiratory depression with buccal midazolam and rectal diazepam were similar (3.0% and 3.3%, respectively). Compared to intravenous diazepam, intravenous lorazepam was associated with less respiratory depression. No child suffered respiratory depression associated with intravenous valproate treatment, compared to one child with intravenous phenobarbital. When looking at all adverse events, intravenous valproate was significantly safer than intravenous phenobarbital. Respiratory depression was not noted in children who received intravenous levetiracetam; however, all levetiracetam studies identified in this review were cohort and non-comparative. In conclusion, in the treatment of acute tonic-clonic seizures (including CSE), buccal was the best administration route for children admitted to the emergency department. Intravenous lorazepam treatment was associated with less respiratory depression than intravenous diazepam. Where IV access was practicable, intravenous lorazepam was the drug of choice. More randomised control trials are needed to evaluate the effectiveness and safety of AEDs as a second-line treatment.

Multiple Sclerosis (MS) has long been considered as White matter (WM) disease. The last decade, the significance of cortical lesions (CL) and their contribution to MS pathology has been intensely investigated. They have been shown to play a major role in physical and cognitive impairment in MS patients. CL detection has proven to be challenging, mainly due to poor contrast between cortical lesion and surrounding normal grey matter (GM) tissue. Various magnetic resonance imaging (MRI) sequences have been used to improve cortical lesion detection in MS patients. In recent years, Double inversion recovery (DIR), Phase sensitive inversion recovery (PSIR) and 7 Tesla T2* have been found to improve CL detection. Magnetization Transfer Imaging (MTI) has the advantage over conventional imaging as it reflects tissue myelin content. In this thesis, I present our studies using MTI at 7 Tesla to study cortical pathology in MS. 1) For a pilot study aiming to validate the use of magnetization transfer ratio (MTR) to detect cortical lesions, We examined the sensitivity of MTR to detect cortical lesions in comparison with 3 T DIR, 7 T PSIR, and 7 T T2* in 18 MS patients and 9 healthy controls. 2) A further 42 patients (11 clinically isolated syndrome (CIS), 11 relapsing remitting MS (RRMS), 10 primary progressive MS (PPMS), and 10 secondary progressive MS (SPMS)) and 8 healthy controls were scanned at baseline, 23 of these patients had a follow up scan at 12 months. MTR at 7 Tesla has increased sensitivity to detect cortical lesions compared to 3T DIR, 7T PSIR and 7T T2*. CL myelin content as measured by the mean MTR lesional values were the lowest in SPMS patients in comparison with the rest of MS phenotypes. CL mean MTR values, more than volume was associated with the degree of physical and cognitive disability in MS patients. When MTR was studied in a longitudinal study, we have seen more changes in average MTR of cortical lesions in SPMS and CIS patients compared to RRMS and PPMS patients.

G protein coupled receptor agonists can be described by two parameters; affinity (ability to bind a receptor) and efficacy (ability to activate a receptor once bound). While affinity is now an accessible parameter through binding studies, agonist efficacies have historically been difficult to determine. This is due to the significant system dependence of agonist responses and is particularly true of traditional maximal response and potency measurements. The Black and Leff operational model determines agonist affinity and efficacy estimations directly from functional data. Its measure of efficacy (τ) offers improvement over maximal response determinations but remains a system dependent parameter. More recently, the Slack and Hall operational model has been described. This model uses receptor constitutive activity to estimate the system contribution to agonist responses to derive system independent measures of intrinsic agonist efficacy. This thesis explores the use of operational models in the analysis of agonist functional responses at the dopamine D2 receptor. This receptor is an important therapeutic target in the treatment of schizophrenia and Parkinson’s disease, and has well documented receptor constitutive activity. Maximal responses and potencies in two representative functional assays, CRE-SPAP reporter gene and receptor internalisation, were determined for a panel of ligands at the D2L receptor. In the receptor internalisation assay, agonist maximal responses and potencies were decreased, consistent with an assay of reduced receptor reserve. Applying the Black and Leff operational model to the responses of four representative agonists, in the absence and presence of the irreversible antagonist phenoxybenzamine, yielded affinity and efficacy estimations consistent with the known pharmacology of the compounds. However, experiments did not provide evidence for the receptor constitutive activity necessary to apply the Slack and Hall operational model. As such, site-directed mutagenesis was performed to generate a constitutively active D2L receptor. T6.34R mutation increased agonist potencies and relative maximal responses consistent with a receptor more likely to adopt the active state; however, potencies of the ergo-derived compounds were selectively decreased. Mutagenesis of three key binding site serine residues examined the mode of agonist binding in both wild type (WT) and T6.34R backgrounds. Each serine mutation had differential effects on the responses of structurally distinct agonists, suggesting agonists engage specific serine residues; while S5.42 and S5.46 were required for catechol agonist responses, S5.46 was essential for the responses of the ergo-derived compounds, bromocriptine and dihydroergocristine. It was unknown whether these specific modes of binding would generate functionally similar active conformations. Generally, the differences between the effects on CRE-SPAP reporter gene and receptor internalisation assay responses were sufficiently explained by differences in receptor reserve. However, there were differential effects of serine mutations on WT and T6.34R D2L receptors, particularly the opposing effect of S5.42A on quinpirole potencies in the CRE-SPAP reporter gene assay. This suggests that WT and T6.34R D2L receptors adopt different conformations. Finally, we have described a method to determine agonist affinity and efficacy from [35S]-GTPγS binding assays using operational models, which does not rely on irreversible antagonist treatments. Buffer Na+ substitution revealed consistent constitutive activation of the D2S receptor, but not of the D2L or D2L T6.34R receptors. Agonist responses at the D2S receptor, in the presence and absence of buffer Na+, were analysed with the Black and Leff and Slack and Hall operational models respectively, generating agonist affinity and efficacy estimations. A novel operational model, written specifically for the analysis of [35S]-GTPγS binding assays, was also examined. This model provided similar affinity derivations to the Black and Leff operational model but provided a τ value that may have reduced system dependency. Although there may be compromises in the associated experimental conditions and which receptors can be investigated, the Slack and Hall operational model can be applied to constitutively active systems to provide system independent measures of agonist intrinsic efficacy.

Introduction Patients with symptomatic carotid artery disease have increased risk of cognitive impairment. Multiple mechanisms of cognitive impairment have been proposed. However, the contribution of each mechanism to cognitive impairment and the interrelation between these mechanisms remain unclear. Lesional and non-lesional damage of brain tissues may lead to cognitive impairment in patients with symptomatic carotid artery disease. The research presented aims to use whole brain statistical brain mapping and quantitative neuroimaging techniques to understand vascular cognitive impairment (VCI) in a group of patients with symptomatic carotid artery disease. Methods Studies presented in this thesis were based on available multimodal MRI and cognitive performance data from a study in patients with symptomatic carotid artery disease. Totally 108 subjects had FLAIR and diffusion MRI scans and 62 of them had resting-state fMRI scans in a 3T MRI scanner. All participants (n=108) underwent cognitive assessments using Addrenbrooke’s cognitive examination-revised (ACE-R). Voxel-based lesion symptom mapping (VLSM) technique was used to identify the contribution of ischaemic lesion presence to cognitive performance. Tract-based spatial statistics (TBSS) approach was used to investigate the association between microscopic damage of white matter and cognitive performance. Independent component analysis (ICA) and seed-based analysis approaches were used to investigate functional disconnection of large-scale cognitive networks and its contribution on cognitive impairment. The association between cognitive performance and ischaemic lesions volumes and microscopic damage of brain tissues were also assessed. Results The presence of chronic subcortical ischaemic lesions located in strategic white matter tracts including anterior thalamic radiation, forceps minor and forceps major contributed to global cognitive impairment in patients with symptomatic carotid artery disease. Ischaemic lesion volumes and microscopic damage of brain tissues including strategic brain areas, strategic white matter tracts, normal appearing white matter and white matter tract skeleton were related to cognitive impairment. Functional disconnection of large-scale cognitive networks including default mode network (DMN), dorsal attention network (DAN), left fronto-parietal network and salience network was found in patients with probable vascular mild cognitive impairment (VaMCI). Functional disconnection beyond the large-scale cognitive networks, showing as decreased functional connectivity between bilateral hippocampi, bilateral dorsal lateral prefrontal cortex (DLPFC), anterior cingulate cortex (ACC) and posterior cingulate cortex (PCC) with the rest of the brain, was also found in patients with probable VaMCI. Also, the thesis presented preliminary results of the role of lesional and non-lesion brain damage in fluency performance in patients with symptomatic carotid artery disease. Conclusions This thesis provides further evidence for the role and to the nature of disconnection in VCI and proposes several disconnection related imaging biomarkers.

This thesis focuses on the use of the Magnetic Resonance Imaging (MRI) white matter lesion (WML) central vein as a biomarker for multiple sclerosis (MS). MS remains a clinical diagnosis, with reliance on MRI to support the diagnosis. Misinterpretation of the MRI can lead to misdiagnoses of diseases that mimic MS. With the increase in disease modifying treatments, accurate and timely diagnosis is needed now more than ever. Using T2* weighted imaging at 3 Tesla (T) MRI, I explored different aspects of WML central veins in patients with relapsing-remitting (RRMS), primary progressive MS (PPMS), and ischaemic small vessel disease (SVD) including: (1) the effect of using different T2* weighted sequences; (2) how T2* and susceptibility weighted imaging (SWI) and fused imaging techniques such as fluid attenuated inversion recovery (FLAIR)-SWI affected the proportion of WML central veins and; (3) determining if WML central veins were as prevalent in patients with PPMS. Further objectives included: (4) attempting to determine if vascular risk factors altered the proportion of WML central veins in patients with MS and; (5) using statistical modelling to calculate a simple diagnostic rule using WML central veins to differentiate MS from SVD. The proportion of WMLs with central veins differed significantly between patients with MS and SVD. Variations of the T2* sequence altered the proportion of WMLs with central veins, but the difference between MS and SVD remained statistically significant. T2* and SWI allowed a higher proportion of WMLs with central veins to be detected, with T2* being just as accurate as FLAIR-SWI in allowing the diagnosis of MS or SVD. Patients with PPMS and RRMS have a similarly high proportion of WMLs with central veins. High sensitivity and specificity for the diagnosis of MS versus SVD can be achieved by identifying a subset of WMLs with central veins. WML central veins could be used as an MRI biomarker using T2* imaging at 3T to differentiate cases of diagnostic uncertainty with RRMS, PPMS and SVD. Application of this imaging technique to patients with diagnostic uncertainty in prospective studies needs to be studied along with refining a clinical diagnostic rule.