Anatomy and Neuroscience

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). The immune system plays a significant role in the pathophysiology of the disease and research into the mechanisms that may be potentially contributing to the disease is essential to help advance knowledge in the field and ultimately seek better outcomes for MS patients. A key set of cells that are believed to play a role in MS immunopathology are mononuclear cells which consist of B-cells, T-cells, monocytes and their derivatives. The aims of this thesis was to assess the effects of 1) genetic susceptibility and 2) immunosuppressant treatment, on mononuclear cell phenotype in MS. The first aim involved interrogating a key co-stimulatory molecule, cluster of differentiation 40 (CD40), expressed on mononuclear cells which is known to play a fundamental role in the regulation of the humoral immune response. CD40 is a risk gene for the development of MS and I sought to assess the effects of the single nucleotide polymorphism (SNP) rs1883832 on the phenotype of CD40-expressing mononuclear cells. Utilising a range of bimolecular methods and samples taken from MS individuals and healthy matched-controls (HC), I showed that SNP rs1883832, particularly the ‘risk’ genotype (homozygous TT) was correlated with reduced soluble CD40 (sCD40) levels in HC, but not MS patients. I did not observe any phenotype-dependent differences in sCD40 levels between MS (in quiescent state) and during active demyelination compared to HC. Upon assessment of key cytokines produced by B-cell and monocytes, I observed elevated gene expression of the pro-inflammatory cytokine interleukin-1 beta (IL-1β) in the MS population linked to the CD40 risk TT-genotype. In the second component, I showed that the immunosuppressant drug Cladribine, is highly toxic to mononuclear cells, and that it down-regulates the expression of the key phagocytic marker MERTK at the protein level (preliminary data, study is ongoing). In summary, the data presented in this thesis suggests that the CD40 risk gene and the immunosuppressant Cladribine play roles in altering the phenotype in mononuclear cells. The understanding from this body of work provides some insights into how genetic susceptibility can drive changes in mononuclear cell phenotype which may impact on the modulation of co-stimulatory mechanisms in humoral immunity. Moreover, the findings from the Cladribine study may assist the field in the discovery of alternative mechanisms of the drug on mononuclear cells and therefore inform a refined approach to treatment of MS.

Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) which affects white matter (WM), grey matter (GM) and normal appearing brain tissue (NABT). In 85-90% of MS patients, the first manifestation of the disease is an acute, remitting clinically isolated syndrome (CIS). One of the most frequent symptoms of CIS is optic neuritis (ON). The risk of conversion to MS in isolated optic neuritis (ION) patients with abnormal baseline magnetic resonance imaging (MRI) is about 40% one year post onset. The chance of conversion to MS significantly increases based on the number of lesions on baseline MRI. Therefore, ION patients at risk of MS provide an excellent opportunity to study the events occurring at the early stages of the disease. Pathological studies and MRI provide good diagnostic and prognostic information about MS. MRI techniques as in-vivo non-invasive tools have helped to better understand the substrate, pathophysiology and mechanisms underlying the development and evolution of focal lesions and diffuse damage in MS. There is a strong correlation between pathological changes in GM and NABT and future clinical disability of MS patients. Therefore, advanced MRI techniques such as diffusion tensor imaging (DTI) have been widely applied to discover injuries in both GM and NABT with more specificity and sensitivity. Early diagnosis of MS is essential for initiation of early treatment leading to better prognosis and fewer disabilities in future. In this thesis, the main aim was to investigate longitudinal microstructural changes in NABT in the visual pathways and beyond in ON patients at risk of MS using different modalities of MRI including volumetric and diffusion imaging techniques. Twenty three patients (17f/6m; mean ± SD age = 34.6 ± 8.6 yrs) and eleven healthy age matched controls (8f/3m; mean ± SD age = 36.4 ± 4.9 yrs) were recruited for the studies of this thesis. Patients were scanned around two weeks after their first presentation of ON and then at 1, 3, 6 and 12 months, and controls were scanned with one month separation. Following each scan session; multifocal visual evoked potentials (mfVEP) and optical coherence tomography (OCT) were acquired by a neuro-ophthalmologist. After completing the MRI safety questionnaire, all participants were scanned at the Royal Children’s MRI Centre, Melbourne, Australia on a 3T Siemens MRI scanner with a 12 channel receiver head coil. Each MR session consisted of whole brain and optic nerve T1-weighted images, whole brain and optic nerve T2 fluid-attenuated inversion recovery (FLAIR) images, and one DTI study. The baseline MR session also included gadolinium enhanced MRI. For the purpose of this thesis, whole brain T1-weighted, T2-FLAIR, and DTI images were used. In the first study, in order to investigate longitudinal microstructural changes in NABT in the visual pathway following ON, we chose the body of optic radiations (BORs) as the regions of interest (ROIs). BORs are highly indicative of tightly packed and collinear axon bundles, and diffusion findings in these regions in comparison to the other areas of the optic radiations are more reliable. More than half of the participants (52.2%) converted to MS after 12 months which made our participants an ideal sample of patients in the very early stages of MS. By using DTI, we demonstrated longitudinal microstructural changes in NABORs in ION patients during one year follow up. Most of the longitudinal diffusion parameter alterations were observed in the right NABOR. Microstructural changes were found to commence at a very early stage of MS, nearly one month following ON. We also showed that in ION patients there was a progressive longitudinal decrease in retinal nerve fiber layer (RNFL) thickness, and that earlier axonal loss in the optic nerve correlated with later diffusion changes in NABORs. Furthermore, there was a correlation between left visual hemifield amplitude asymmetry and FA value in the right NABOR a year following ON. The results of this study confirmed previous imaging findings that DTI technique allows to explore subtle WM degeneration and to investigate integrity of neuronal bundles in the brain. Furthermore, our results provided evidence that the involvement of NABORs following ON can be the result of the trans-synaptic damages following ON. In the second study, we chose visual cortices (VCs) as the ROIs to investigate atrophy in normal appearing grey matter (NAGM) in the visual pathway following ON during one year in ION patients at risk of MS. Furthermore, to investigate microstructural changes in beyond the secondary order neurons in the visual pathways, we focused on the connection between primary visual cortices (V1s) and the splenium of the corpus callosum (CC), called cortico-splenial (CS) tracts. CS tracts were defined using probabilistic tractography. By using volumetric assessment, we demonstrated longitudinal decrease in the volume of the right normal appearing primary visual cortex (NAV1). Atrophy in the right NAV1 was observed to commence at the very early stage, nearly one month following ON. We also revealed a correlation between volume of the right NAV1 and left visual hemifield amplitude in the affected eye at 12 months. By using DTI, we showed that there is a progressive microstructural change in bilateral CS tracts. The commencement of the microstructural changes in the CS tracts was observed to be at the very early stage of MS. We also found a correlation between early axonal loss in the optic nerve and later microstructural changes in the CS tracts. The results of this study supported the potential role of MRI as a biomarker in early diagnosis of the disease. Moreover, this study provided further evidence of the involvement of NABT in the visual pathway and beyond following ON due to trans-synaptic damage. In our first two studies we provided evidence supporting the role of trans-synaptic damage in microstructural changes and atrophy in NABT in the visual pathway and beyond following ON. It would therefore be interesting to investigate whether only areas related to the visual pathways are affected in patients with ON, or are there other areas in the brain show microstructural damages? Early recognition of the involvement of the WM or GM and the possible mechanisms of the involvement in ION patients at risk of MS is very important because early diagnosis helps with decisions regarding appropriate treatments and options. Furthermore, early treatment can lead to better prognosis and fewer disabilities. In order to address these questions, we undertook an exploratory study to investigate other WM or GM regions of the brain which have microstructural changes or atrophy following ON. We used tract-based spatial statistics (TBSS) method to investigate areas of the whole brain WM with significant longitudinal diffusion changes during one year follow up in ION patients at risk of MS. The voxel-based morphometry (VBM) was used to investigate longitudinal decrease in the volume of GM areas in whole brain. We found progressive microstructural changes during one year in different WM areas of the brain in ION patients. The regions include ORs (mainly right), all parts of the CC; splenium, body and genu, as well as the left and right thalamus. Moreover, we demonstrated progressive atrophy in the GM areas of the brain including VCs, frontal lobe, and primary motor and somatosensory cortex during one year follow up in ION patients. The results of this study provided evidence for diffuse whole brain longitudinal WM microstructural changes and brain atrophy in ION patients at risk of MS during one year follow up. Pathophysiology underlying the diffuse involvement of the brain WM and GM in ION patients at risk of MS is still unclear. In summary, the results of our studies support DTI as a suitable technique for exploring subtle WM degeneration. Moreover, the findings provide evidence of trans–synaptic neuronal damage as the mechanism involved in the early progressive changes of the NABT in the visual pathways and beyond following ON at the early stage of MS even before the definite diagnosis of MS. Therefore, the results support the potential role of multi-modal MRI as a biomarker in the evaluation of disease evolution and progression, as well as response to treatment. The third study provided evidence for whole brain diffuse longitudinal WM microstructural changes and brain atrophy in ION patients at risk of MS during one year follow up. However, pathophysiology and mechanisms underlying the diffuse involvement of the brain WM and GM in ION patients at risk of MS require further investigations. Our findings suggest that ION patients with abnormal baseline MRI should be considered as an important group of patients with higher risk of conversion to MS and not just treated as patients with simple inflammatory disease. It has been shown that early treatment leads to less disability in future. Therefore, ION patients with abnormal MRI need to be closely monitored for early commencement of MS treatment. We hope that the findings of this thesis will lead to the identification of new outcome measures to monitor the evolution of MS and treatment response and, ultimately to improving disease management in individual patients.

Anatomy and Neuroscience - Theses

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