Nuclear Magnetic Resonance (NMR) is a phenomenon in which certain nuclei in the presence of a magnetic field and radiofrequency (RF) radiation emit a certain amount of signal at a frequency equal to that of the RF radiation. Proton Magnetic Resonance Spectroscopy (1H-MRS) is an NMR technique capable of measuring the chemical composition, often referred to as metabolites, of the human body non-invasively and in vivo. It is commonly used as a research tool in the investigation of neurological disorders such as multiple sclerosis, brain tumors, stroke, clinical depression, and schizophrenia. Accurate quantification of the metabolites of interest requires a reference standard of known and fixed concentration. Brain tissue water has been previously reported to have a fairly constant and known concentration, and so has been suggested to be a suitable reference concentration in absolute quantitative 1H-MRS of the human brain. In practice, however, it is challenging to measure the actual tissue water concentration; hence, some studies choose to use estimates of tissue water concentration from the literature. These literature values are usually averages from a healthy study group. There are however indications that brain tissue water content could vary widely in certain disease conditions such as in brain tumors and inflammation. In such situations, absolute metabolite quantification using the literature estimates of tissue water content will be inaccurate while the measurement of cerebral water content using the available techniques will be impractical for the patients due to scanning time considerations. It is therefore necessary to develop a technique that can be used to quantify both the reference water and metabolite concentrations, simultaneously without subject tolerance issues. The main objective of this thesis was to investigate the response of water NMR signal from human brain tissue under various measurement conditions using the single-voxel 1H-MRS technique. As part of the investigation, the thesis also focused on the development of methods for the absolute quantification of cerebral water and metabolite concentrations. A standard 1H-MRS water-suppressed acquisition on the General Electric (GE) MR scanner acquires some unsuppressed-water spectra at the beginning of the PRESS pulse sequence. Using the Spectroscopy Analysis by GE (SAGE) software package (version 7), this thesis developed methods to optimise the unsuppressed-water and suppressed-water signals from which, respectively, cerebral water and metabolite concentrations were estimated. The unsuppressed-water signal response characteristics were investigated in experiments at 3 T that involved: 1) variation of the MRS voxel position over a three-dimensional RF field within an eight-channel head coil; 2) measurement of the relaxation times of brain tissue water using standard saturation recovery and multi spin-echo MRS techniques; 3) measurement of brain tissue water content in peripheral inflammation; and 4) estimation of the BOLD effect on the water spectral peak. The stability of the MR scanner used for all the investigations was assessed. Over the project period, the worst precision measurements of the scanner (for both water and metabolite signals) were observed to be about 12 % and 26 % in serial phantom and human studies, respectively. The MRI/MRS scanner was therefore found to measure water and metabolite signals with good precision, both in vivo and in vitro. By recording the water NMR signal responses at various locations within the phased-array head coil, RF sensitivity profile (voxel position-dependent) equations of the head coil were obtained. The coordinates of any in vivo voxel could be substituted into an appropriate profile equation to estimate an unsuppressed-water signal area that could be used as a reference signal to quantify brain tissue water content. This novel technique of quantifying cerebral water content is superior to the previous techniques of performing multi-echo unsuppressed-water signal acquisitions. The method does not require extra unsuppressed-water acquisitions, or corrections for variations in the sensitivity of the eight-channel head coil as both the in vivo and reference signals are acquired from the same voxel position. Brain tissue water content was subsequently quantified accurately using the newly developed method of referencing. In frontal brain voxels, the average water content, WC of grey matter, GM was found to be higher than that of white matter, WM (GM/WM WC ± SE = 46.37 ± 2.58/42.86 ± 2.46 mol/kg; p = 0.02); parietal voxels also showed a similar comparison (GM/WM WC ± SE = 37.23 ± 1.70/34.14 ± 2.02 mol/kg; p = 0.03). These findings were consistent with previous reports of cerebral water content. For regions of mixed proportions of grey and white matter tissues, the average water contents of each tissue type considered separately (by voxel segmentation) and together were found to compare with literature estimates. Using data from five voxel positions, average brain tissue water content was observed to be uniformly distributed across the human brain by one-way ANOVA (p = 0.60), and did not vary significantly with gender (p > 0.05) and age (p > 0.05). For the first time, cerebral water content was observed in this thesis to remain fairly constant in psoriatic arthritis, a peripheral inflammatory condition (one-way ANOVA, p = 0.63). Among five brain metabolites quantified in the psoriasis patients, only the mean concentration of creatine, Cr was found to be significantly lower in the frontal grey matter voxels of the patients, PsA compared to healthy controls, HC at baseline (PsA/HC ± SE = 6.34 ± 0.38/7.78 ± 0.38 mM/kg; p = 0.01) and post-TNF-alpha blockade medication (PsA/HC ± SE = 6.69 ± 0.25/7.78 ± 0.38 mM/kg; p = 0.03). None of the metabolite concentrations, including Cr (p = 0.27), changed significantly with medication. The condition of PsA was not observed to affect the mood of the patients, as indicated by their BDI scores. The significant finding of Cr concentration alteration in psoriatic arthritis thus suggests that Cr may not be a reliable denominator in studies of psoriasis that express the metabolite levels as ratios. The T1 and T2 relaxation times of water and the metabolites were measured in the prefrontal grey matter (T1/T2 ± SE = 1574 ± 61/147 ± 6 ms) and bilateral Hippocampi (T1/T2 ± SE; left = 1475 ± 68/178 ± 83 ms, right = 1389 ± 58/273 ± 98 ms). The relaxation time estimates for the metabolites were in agreement with literature values; relaxation times for water however were measured for the first time in those regions and at 3 T. The measured relaxation times were used to correct the water and metabolite signals for relaxation effects during their absolute quantification, and could as well serve the same purpose in future studies. There is increasing interest in the BOLD response of cerebral metabolites and water during tasks. This thesis thus also assessed changes in brain tissue metabolite and water contents while a subject experienced a visual stimulus. In the presence of the visual stimulus, the BOLD effects on the metabolite and water spectral peaks were found to be comparable, as has been observed in previous studies. For the first time, this thesis further investigated the impact of temporal resolution (determined by NEX) on the amount of the BOLD signal acquired from cerebral water and metabolites. In a single visual activation paradigm, the BOLD effect resulted in increased water peak area which differed significantly between NEX values of 2 and 8 (p < 0.01); this observation also was true for NAA and Glu. The findings thus suggest that temporal resolution of the MRS data could result in significant differences in the results of functional MRS studies.

Even though spinal cord research has expanded enormously during the past decades, we still lack a precise understanding of how spinal interneuron networks perfectly integrate sensory feedback with motor control, and how these neuron circuits give rise to specific functions. The present study thus has three basic aims: (1) to investigate propriospinal interneurons connecting rostral and caudal lumbar spinal cord in the rat; (2) to investigate input properties of identified spinal interneurons interposed in different pathways; (3) to investigate cholinergic terminals in the ventral horn of adult rat and cat. To realize the first aim, the B-subunit of cholera toxin (CTb) was injected into the motor nuclei at the L1 or L3 segmental level to retrogradely label propriospinal interneurons in the L5 segment of rat spinal cord. These cells had a clear distribution pattern which showed that they were located mainly in ipsilateral dorsal horn and contralateral lamina VIII. A series triple-labelling experiments revealed that about 1/4 of the CTb-positive cells were immunoreactive for calbindin and/or calretinin. It was also found that a small population of CTb labelled cells were cholinergic and were observed mainly in three locations: lamina X, the medial part of intermediate zone and lamina VIII. In addition, injection of CTb also anterogradely labelled axon terminals, which arose from the commissural interneurons (CINs) within the site of injection, crossed the midline and aroborized in the contralateral lateral motor nuclei of the L5 segment. The neurotransmitter systems in labelled axon terminals of CINs were investigated by using antibodies raised against specific transmitter-related proteins. The results showed that approximately 3/4 terminals were excitatory and among those excitatory terminals about 3/4 forming contacts with motoneurons. To achieve the second aim, 21 interneurons located in the intermediate zone and lamina VIII from 7 adult cats were characterised electrophysiologically and labelled intracellularly with Neurobiotin. Seventeen of these cells were activated monosynaptically from primary muscle afferents but the remaining four cells received monosynaptic inputs from the medial longitudinal fasciculus (MLF). Quantitative analysis revealed that cells in the first group received many contacts from excitatory terminals that were immunoreactive for the vesicular glutamate transporter 1 (VGLUT1) but those cells from the second group received few contacts of this type and were predominantly contacted by terminals immunoreactive for vesicular glutamate transporter 2 (VGLUT2). This result was as predicted because VGLUT1 is found principally in the terminals of myelinated primary afferent axons whereas VGLUT2 is located in the terminals of interneurons in the spinal cord. Interneurons in the first group were then characterised as excitatory and inhibitory on the basis of the transmitter content contained within their axon terminals. Although there was a greater density of VGLUT1 contacts on excitatory rather than inhibitory cells, the difference was not statistically significant. GABAergic terminals formed close appositions with VGLUT1 contacts on both excitatory and inhibitory cells. These appositions were likely to be axoaxonic synapses which mediate presynaptic inhibition. In addition, the densities of VGLUT1 and VGLUT2 contacts on 30 dorsal horn CINs and 60 lamina VIII CINs that were retrogradely labelled with CTb from 3 adult rats were compared. The results showed that VGLUT2 terminals formed the majority of excitatory inputs to both dorsal horn and lamina VIII CINs but dorsal horn CINs received a significantly greater density of VGLUT1/2 inputs than lamina VIII CINs. In order to achieve the third aim, i.e. whether glutamate is a cotransmitter at motoneuron axon collateral terminals in the ventral horn, a series of anatomical experiments were performed on axon collaterals obtained from motoneurons from an adult cat and retrogradely labelled by CTb in adult rats. There was no evidence to support the presence of vesicular glutamate transporters in motoneuron axon terminals of either species. In addition, there was no obvious relationship between motoneuron terminals and R2 subunit of the AMPA receptor (GluR2). However, a population of cholinergic terminals in lamina VII, which did not originate from motoneurons, was found to be immunoreactive for VGLUT2 and formed appositions with GluR2 subunits. These terminals were smaller than motoneuron terminals and, unlike them, formed no relationship with Renshaw cells. The evidence suggests that glutamate does not act as a cotransmitter with acetylcholine at central synapses of motoneurons in the adult cat and rat. However, glutamate is present in a population of cholinergic terminals which probably originate from interneurons where its action is via an AMPA receptor. In conclusion, the present studies add to the understanding of the organization of neuronal networks involved in sensorimotor integration. Propriospinal interneurons located within the lumbar segments have extensive intra-segmental projections to motor nuclei. First order interneurons interposed in reflex pathways and descending pathways receive a significantly different pattern of inputs. A similar proportion of monosynaptic excitatory input from primary afferents has been found in both excitatory and inhibitory interneurons and these two types of cells are subject to presynaptic inhibitory control of this input.

Background: Hyperglycaemia is common in acute stroke and is associated with a poor outcome. Underlying aetiology and mechanism of action is poorly understood. Management remains uncertain. Methods: We undertook a randomised placebo controlled trial to assess the effect of GKI (Glucose-Potassium-Insulin) versus placebo on lesion volume progression and cerebral lactate levels using magnetic resonance imaging (MRI) and spectroscopy (MRS). An observational study of the capillary blood glucose within 48 hours of stroke onset was performed to define the temporal profile of glucose, with a subset followed prospectively to determine the prevalence of abnormal glucose metabolism in patients with stress hyperglycaemia. The association between insular cortex involvement and hyperglycaemia was determined by analysing MRI data sets from two randomised trials. Stroke unit practice for the management of glucose was assessed in a review of the stroke unit trialists’ collaboration data set. Results: • GKI infusion failed to attenuate infarct growth in patients with moderate hyperglycaemia within 24 hours of acute ischaemic stroke. A trend towards attenuation of increased lactate concentration was evident in the GKI treatment arm. Exploratory analyses raised the possibility that GKI may be harmful in patients with persistent arterial occlusion. • Over the 48hour monitoring period 75% of patients developed Hyperglycaemia. Stroke severity was not predictive of admission hyperglycaemia whereas glycosylated haemoglobin was (OR 2.97; 95%CI 1.84-4.78; p<0.001). 50% of patients screened were found to have abnormal glucose metabolism at follow-up. • Insular cortex involvment on MRI was not predictive of admission hyperglycaemia. • Testing for blood glucose concentration in stroke units was infrequent. Of the minority of units that had a protocol in place, the threshold for intervention with insulin was >10mmol/l. Conclusion: We found no evidence that GKI infusion attenuated infarct growth in patients with mild hyperglycaemia following acute ischaemic stroke. In post-hoc analysis the possibility that GKI infusion may be harmful in patients with total occlusion suggests an effect dependent on recanalisation status. A non-significant trend towards attenuation of increased lactate concentration was evident. Stroke severity was not found to be a predictor of post stroke hyperglycaemia. Underlying dysglycaemia was common in non-diabetic patients manifesting hyperglycaemia within 48hours of stroke ictus. Screening of high risk patients with oral glucose tolerance testing is justified and provides a potential opportunity for secondary prevention. Insular cortex involvement did not independently predict hyperglycaemia in acute stroke. Current management of hyperglycaemia is guided by consensus guidelines with little evidence base. Stroke unit practice varies with little change across stroke units over the years.

In the UK, stroke is the third most common cause of death after heart disease and cancer. Importantly, most strokes are not fatal meaning stroke is the leading cause of adult disability, with one third of survivors still functionally dependent after one year. Post stroke hyperglycaemia (PSH) predicts poor outcome independent of age, stroke type or severity and occurs in over 60% of patients without a diagnosis of diabetes and in more than 90% of diabetic patients. Around 56% of hyperglycaemic acute stroke patients are subsequently diagnosed with insulin resistance, manifesting as impaired glucose tolerance, impaired fasting glucose or the “metabolic syndrome.” Clinical guidelines recommend routine blood glucose monitoring, and intervention with insulin for hyperglycaemia. However, there is currently no clinical evidence of benefit from insulin use, and recent findings raise concerns about the safety of insulin use in predominantly non-diabetic acute stroke populations. Hyperglycaemia in animal models of cerebral ischaemia is associated with increased infarct size. However, a recent systematic review highlighted uncertainties about the relevance of many previously published preclinical studies to the typical clinical picture of PSH with respect to whether clinically relevant elevations in blood glucose exacerbate ischaemic brain damage in models other than those of type I diabetes, or whether responses are different in animals with and without features of metabolic syndrome. The aim of this thesis was to investigate the differential effects of clinically relevant hyperglycaemia on the acute evolution of damage in animals with and without features of the metabolic syndrome and to investigate the potential mechanistic role of oxidative stress in glucose mediated ischaemic damage. I hypothesised that hyperglycaemia would exacerbate acute lesion evolution and final infarct volume in animals with and without features of the metabolic syndrome and that oxidative stress mechanisms would be involved. Developing suitable animal models for investigating post stroke hyperglycaemia and metabolic syndrome The first aim of this thesis was to develop a series of animal models for the investigation of clinically relevant PSH, metabolic syndrome in the presence and absence of PSH, and an appropriate focal cerebral ischaemia model. A previously described model of metabolic syndrome using the spontaneously hypertensive stroke-prone rat (SHRSP) fed on a 60% fructose diet for two weeks was set up in the laboratory and glucose tolerance, adiposity, plasma insulin, plasma triglycerides, cholesterol were measured and compared to the SHRSP reference strain, the Wistar-Kyoto (WKY). Fructose fed-SHRSP rats exhibited glucose intolerance, hypertriglyceridaemia, hyperinsulinaemia, increase adiposity and reduced HDL cholesterol compared to the WKY controls. This suitably modelled the features of the metabolic syndrome. Clinically relevant levels of PSH were achieved using a bolus intraperitoneal injection of 15% glucose (10ml/kg) 10 minutes prior to permanent middle cerebral artery occlusion (MCAO). Two methods of MCAO were compared; intraluminal filament (ILF) model and a distal diathermy MCAO model. The ILF method produced expansive lesions incorporating the majority of the ipsilateral hemisphere whereas the distal diathermy occlusion produced smaller cortical infarcts which were shown to produce a large volume of penumbra. The diathermy model was selected for all future experiments. Investigating the effects of hyperglycaemia on acute lesion growth after focal cerebral ischaemia in rats with and without components of the metabolic syndrome The second aim of this thesis was to determine the effects of hyperglycaemia on acute ischaemic lesion evolution and infarct volume in rats with and without features of the metabolic syndrome. Hyperglycaemia was induced in WKY or fructose-fed SHRSP rats 10 minutes prior to permanent MCAO. Magnetic resonance imaging (MRI) was used to quantify the expansion of the lesion using ADC maps calculated from diffusion weighted imaging (DWI) carried out over the first 4 hours after MCAO. Acute hyperglycaemia, at clinically relevant levels, exacerbated early ischaemic damage in both normal and metabolic syndrome rats. Hyperglycaemia worsened infarct volume at 24h in normal but not metabolic syndrome rats. These data suggest that management of hyperglycaemia may be most beneficial in the absence of an underlying dysglycaemia. Using a marker of oxidative stress to probe the tissue sections suggested that perhaps hyperglycaemic effects were mediated through oxidative stress mechanisms. Investigation of oxidative stress mechanisms in hyperglycaemia-dependant ischaemic damage. Based on the potential involvement of oxidative stress mechanisms in hyperglycaemia-associated ischaemic damage this was investigated in a proof of concept study using pre-treatment with a SOD/catalase mimetic, EUK-134. The rationale for this study was based on the involvement of increased superoxide production in chronic hyperglycaemia and diabetic complications. Sprague-Dawley rats received the same dose of glucose previously used 10 minutes prior to MCAO by distal diathermy. EUK-134 (2.5mg/kg) or vehicle (saline) was administered 20 minutes prior to MCAO. Infarct volume, calculated from RARE T2-weighted MR images and neurological scoring was measured 24 hours post MCAO. Arterial superoxide levels were measured along with the lipid peroxidation marker malondialdehyde in cerebral tissue. Hyperglycaemia increased infarct volume although no effect of high blood glucose on tissue lipid peroxidation was observed. EUK-134 failed to reduce infarct volume. EUK-134 reduced lipid peroxidation in ipsilateral cortex of normoglycaemic rats but not in hyperglycaemic rats. Thus, hyperglycaemia associated damage was not reduced by attenuating oxidative stress. Generating a threshold for quantitative T2 maps Using the data generated in the previous Chapter a study was carried out to determine if improvements could be made in the analysis of MRI data in pre-clinical rat studies. RARE T2-weighted images, where T2 relaxation time denotes contrast, have been used for infarct quantification 24 hours post MCAO. Generating a lesion volume from these images requires manual tracing of the visually defined infarct from the images. A degree of error is associated with this methodology either as inter-investigator reproducibility or in repeats conducted by the same investigator. The RARE T2 images are not quantifiable as arbitrary values denote contrast and this prevents direct comparison of T2 relaxation times for a particular structure between subjects. Using a MSME T2 sequence, quantitative T2 maps can be generated which calculate absolute T2 relaxation times for each voxel. Using quantitative maps an attempt was made to establish a threshold to discriminate between normal and ischaemically damaged tissue in animals that had undergone MCAO using the distal diathermy model. This threshold aimed to minimise inter- or intra-investigator error. A T2 threshold of abnormality for the quantitative T2 maps was established from the RARE T2 weighted images. Tissue that had a T2 relaxation time of 76 ms or more was deemed to be infarct according to the derived threshold. The suitability of the threshold was then assessed in a separate cohort and was found to produce similar infarct volumes to the manual delineation of RARE T2-weighted images. The 76ms threshold was also applied to animals that had undergone MCAO using the intraluminal filament model. However, the calculated threshold was not suitable for the intraluminal filament model due to interferences from cerebrospinal fluid signal. A more selective method for determining infarct volume in animals subjected to intraluminal filament MCAO needs to be established.

Background:The Addenbrooke’s Cognitive Examination-Revised (ACE-R) is a widely used screening tool for Dementia. Although it is recommended for use in detecting cognitive impairment in people who misuse alcohol (Scottish Government 2007), the ACE-R has not been validated with this population. This study compared the performance of a group of people who misuse alcohol on the ACE-R with published normative data. The study examines whether deficits in ACE-R performance are associated with previous experience of a withdrawal from alcohol, duration of alcohol use and units consumed per week. Methods:Data from 77 attendees at the Alcohol Liaison Service in NHS Ayrshire and Arran who had completed the ACE-R was extracted from an existing database and included in the study. The ALS group ACE-R total and domain scores were compared to those of the original validation control group used by Mioshi et al (2006). Using independent t–tests, differences in overall ACE-R performance and domain performance were examined. Independent t-tests were also used to determine the impact of previous withdrawal on ACE-R scores. Correlation analyses and multiple regression were used to examine relationships between aspects of drinking history (previous withdrawal, duration of use and units consumed per week) and ACE-R outcome. Results:Total ACE-R scores, memory and fluency domain scores were significantly lower in the ALS group compared to normative data (p<0.001) It was not possible compare attention, language and visuospatial domain scores between groups as parametric assumptions were not met and only mean control group data was available. Attendees with a history of alcohol withdrawal had significantly poorer scores on the domain of attention compared to those who had not (p=0.009). They appeared to have lower overall ACE-R scores although this differnce was not significant (p=0.128). This analysis was underpowered. Longer duration of alcohol drinking was associated with lower verbal fluency (r=-0.362), lower memory (r=-0.239) and lower visuospatial (rs=-0.234) domain scores. Units consumed weekly were not significantly associated with any ACE-R domain score or total score. Longer duration of alcohol use and previous withdrawal experience together accounted for 10% of the variance in ACE-R total scores (p=0.02). Conclusion:It is likely that most people who chronically and hazardously misuse alcohol will experience persisting cognitive impairment. The ACE-R appears to be a good measure for the assessment such difficulties in this population. This study suggests that it is not possible to accurately judge the severity of cognitive impairment in people who drink hazardously on the basis of duration of alcohol use and previous withdrawal experience alone. The study has methodological limitations and more rigorous research examining the use of the ACE-R with this population is necessary.

Insomnia disorder is characterised by difficulties with initiating and/or maintaining sleep. Similar to most psychiatric and mental health conditions, insomnia is defined according to subjective complaint, and achieves disorder ‘status’ when associated daytime functioning impairment is present. Yet, ironically, it is these two cornerstones of insomnia disorder, combined, that have achieved relatively minimal attention in the literature. That is, perhaps surprisingly, the subjective experience, and impact of insomnia, at least from the patient (‘expert’) perspective, has been under-researched. Night-time symptoms and sleep parameters have typically been the target of both treatment and non-treatment (clinical, epidemiological, mechanistic) research. In this thesis, a multi-method, multi-level approach is adopted to better understand the daytime experience of those with chronically disturbed sleep. First, a brief overview (chapter one) of insomnia is provided to familiarise the reader with the ‘problem of insomnia’. A narrative review (chapter two) then sets the scene in relation to the assessment and measurement of health-related quality of life (HRQoL) and daytime functioning. This work reveals several inadequacies and limitations of existing work, and outlines a prospective research agenda. Chapter three describes the first ever phenomenological study carried out in primary insomnia patients. Here, two qualitative methodologies, focus groups and audio-diaries, are combined to help better understand the proximal and distal impairments attributed to chronic sleep disturbance. Chapter four builds on this work by describing the creation of two new clinical scales, developed to quantify, in both valid and novel ways, the impact of poor sleep on aspects of daytime functioning and insomnia-related quality of life. Chapter five combines the aforementioned qualitative and questionnaire approaches to explore the experience of an effective behavioural intervention for insomnia, sleep restriction therapy (SRT). The application of these refined methods provided insight into the effects of SRT on both sleep and daytime functioning, but also permitted exploration of treatment-related issues - such as adherence, side-effects, and mechanisms of action - that have otherwise been difficult to probe using traditional quantitative methodologies. Chapter six tackles the issue of objective daytime impairment, typically assessed using computerised reaction time tasks. Through ‘mining’ an existing brain and behavioural database, and applying an algorithm to select poor and normal sleepers, it was possible to investigate cognitive functioning at two broad stages of processing – event-related potentials generated from the scalp-recorded electroencephalogram (EEG), and performance output using neuropsychological testing. The results provide some interesting hypotheses concerning possible cognition-arousal and -effort interactions. Importantly, as a by-product of this work, a methodological template for the future standardized assessment of brain and behavioural function in insomnia is considered. Finally, chapter seven synthesises the results of each preceding experimental chapter, with particular emphasis on how this work will advance research, measurement and understanding of insomnia-related functioning. Immediate clinical implications and relevance to other areas of insomnia research are also briefly considered.

Abstract Clinical trials are at the centre of advances in our understanding of stroke and its optimal treatment. In this thesis the uses and properties of outcome assessment scales for stroke trials are described, with particular attention given to the modified Rankin Scale (mRS). Through comprehensive literature review I will show that mRS is the most frequently used functional outcome scale in clinical trials but efficacy of the scale is potentially limited by inter-observer variability. Using a “mock” clinical trial design I demonstrate that inter-observer mRS variability in contemporary practice is moderate (k=0.57). Adding these data to systematic review of published data, confirms an overall moderate inter-observer variability across ten trials (k=0.46). Differing strategies to improve mRS reliability will then be described. I will outline development of a bespoke training package, international training scores across 2942 raters again confirms suboptimal reliability (k=0.67). A pilot trial using endpoint committee review of video recorded interviews demonstrates feasibility of this approach. Attempts to improve reliability by deriving mRS from data recorded in patients’ hospital records are not successful (k=0.34). In the final chapters I present a novel methodology for describing stroke outcomes – “home-time”. This measure shows good agreement with mRS, except at extremes of disability. Finally to put mRS in a historical context, the career of John Rankin and the development of his eponymous scale is recounted.

The post infectious paralytic autoimmune disease, Guillain-Barré syndrome (GBS), has been associated with the generation of cross-reactive auto-antibodies after Campylobacter jejuni infection. These auto-antibodies interact with both the ganglioside mimicking C. jejuni lipo-oligosaccharides (LOS) and endogenous gangliosides. This study sought to investigate novel interactions of the ganglioside mimicking LOS with immune system ganglioside specific receptors. In addition, studies investigated if such receptor recognition affects antigen trafficking or the immunostimulatory potency of the LOS, which could participate in auto-antibody generation. Results presented in this thesis demonstrate for the first time that certain members of the siglec receptor family are capable of recognising LOS from a GBS associated strain of C. jejuni. This interaction did not definitively result in enhanced, or altered, potency of ganglioside mimicking LOS in stimulating immune cells. Interestingly, ganglioside mimicry was shown to enhance phagocytosis of C. jejuni, however, in vivo differences in bacterial trafficking were not observed.

There has been much controversy as to whether people with Autism Spectrum Disorders (ASDs) have a specific impairment in processing biological motion, with some studies suggesting there is an impairment (Blake, et. al. 2003; Klin et. al. 2003, Klin & Jones, 2008, Klin et. al. 2009) and others finding that people with ASDs show intact abilities to detect biological motion and categorise actions, but are impaired in emotion categorisation (Moore et. al. 1997; Hubert et. al. 2007, Parron et. al. 2008). Recent studies have found that although behavioural measures of biological motion processing show no differences, adults with ASDs show different patterns of brain activation to controls in response to intact point-light displays (PLDs), with the STS, MT+ and ITG regions showing reduced activity in this population (Herrington et. al. 2007; Parron et. al. 2009). The current thesis aimed to clarify the nature of these difficulties and to try to elucidate the brain regions used to process configural information from PLDs using novel techniques and stimuli. The first set of experiments were designed to behaviourally test people with ASDs ability to detect biological motion in noise, to categorise actions and to categorise affect from PLDs. Despite finding differences in the two groups in detection of biological motion and affect categorisation in pilot experiments, there were no significant differences between the groups in the main experiments. However, the ASD group showed slightly poorer performance at detecting biological motion and significantly more variability in the action categorisation tasks, suggesting that there may have been an underlying difference between the two groups. Furthermore, an analysis of the pattern of errors tentatively suggested that the ASD group may be using different strategies to categorise affect than controls, particularly for negative affects. We then devised a novel technique for manipulating the amount of configural information available in a PLD without the need to add different degrees of background noise and used this technique to assess the contribution of configural cues in a direction discrimination task behaviourally and neurally. The results confirmed that in typically developed individuals configural cues significantly improved the participants’ ability to correctly determine the direction of locomotion of a point light walker. Furthermore, the fMRI task found that regions of the inferotemporal, parietal and frontal regions were sensitive to the amount of configural information present in the displays that corresponded to increases in individual participants’ behavioural performance. Lastly, we used the same technique, though with a more powerful fMRI design, to assess the behavioural and neural differences between people with ASDs and controls in response to displays containing different degrees of configural information. We found that both groups were comparable in their ability to discriminate the direction of locomotion from PLDs. However, the brain regions used to process this information were found to be substantially different. In displays in which the configural information enabled participants to accurately judge the direction of locomotion, the control group utilised a similar group of regions as found in the previous experiment. The ASD group showed a pattern of activation suggesting that they predominantly used regions in the temporal and occipital cortex, and more specifically a region in the fusiform gyrus. The results of Granger Causality Mapping analysis, which allows for the mapping of directional to and from seeded regions, confirmed that whereas the control group utilised a network of regions starting from the ITG and connecting to parietal and occipital regions, the ASD group seemed to utilise two separate networks, processing form information in the fusiform gyrus and motion information separately in middle-temporal regions. The results are discussed in terms of a potential dysfunction of the ITG region in early childhood and two different models of biological motion processing that have been proposed in the recent literature. In TD individuals the model of Giese & Poggio (2003) may be more applicable, in that it proposes the integration of static form cues with motion signals in areas such as the STS. However, a dysfunctional ITG or dysfunctional connections from the ITG to more dorsal regions would disrupt the integration of form and motion processing and force the brain to place additional processing demands on form processing regions in the fusiform gyrus. This would be more in line with the model proposed by Lange and Lappe (2006) in which information can be derived from biological motion in noise without recourse to the actual motion information, through a process of temporal analysis of static postures. Both systems though, may be intact in TD individuals and may share processing requirements depending on the task. Furthermore, it is hypothesised that a dysfunctional ITG may force the brain to place additional demands on regions in the fusiform gyrus and this neural rewiring may be the cause of the developmental delay seen in processing biological motion in people with ASDs (Annaz et. al. 2009). Future studies should examine the roles of the ITG and fusiform area in more detail, both in TD people and in people with ASDs, and determine the specific nature of these neural differences and there behavioural implications for both groups.

Injury to the peripheral nervous system (PNS) stimulates a finely regulated regenerative response that generally leads to some recovery of function. In contrast, the response to injury in the adult mammalian central nervous system (CNS) is abortive and adult CNS neurons do not normally regenerate. We used a microarray approach to identify putative regeneration-associated changes in gene expression in the L4 dorsal root ganglion (DRG) in rat models of PNS and CNS injury. Our models included crush injury to both branches of the bifurcating axon of sensory neurons with cell bodies in the DRG (DRGNs). Injury to the peripheral branch at the level of the spinal nerve (SN) results in axonal regeneration and reinnervation. Crush injury of the central branch in the dorsal root (DR) results in active regeneration up to the point of CNS entry at the DR entry zone (DREZ) and subsequent arrest of further growth, while transection injury within the CNS at the level of the dorsal columns (DC) results in abortive and unsuccessful regeneration attempts. These DRGN injury models therefore allowed us to compare the gene expression programmes elicited during active, arrested and abortive regeneration. Following a pilot microarray experiment to optimize experimental parameters and tract tracing and electrophysiological experiments to confirm time points for harvest of DRGs after DR and SN injury, respectively, male Sprague-Dawley rats underwent an L4 SN crush, an L4 DR crush or a bilateral DC transection at the L3/L4 spinal segment boundary. L4 DRGs were collected at 2 weeks (active regeneration) and 6 weeks (arrested regeneration) after DR crush. DRGs were harvested at 6 weeks after SN crush and 2 weeks after DC transection. DRGs harvested from naïve rats served as a control group. Microarray analysis (Affymetrix Rat genome 230 2.0 array) identified several hundred genes showing differential expression (5% FDR) in comparisons of regenerating with non-regenerating conditions. Selected genes were chosen for validation by qRT-PCR. These genes could represent putative regeneration-associated genes and may suggest novel therapeutic interventions to encourage regeneration of the spinal cord following injury. Additionally, we have identified genes upregulated in the DR active regeneration state relative to DR arrested state, which have relevance to root avulsion injury and may provide insight into the mechanisms that prevent regeneration of DR axons through the DREZ to re-enter the spinal cord. We also present evidence that a transcriptional programme consistent with regeneration is mounted within the DRG following DC transection. This lends support to the idea that CNS neurons have intrinsic regenerative capability and that manipulations of the CNS environment may be sufficient to permit regeneration of CNS axons.