Imaging of Demyelination, Repair and Remyelination in Multiple Sclerosis

Multiple Sclerosis (MS) is characterised pathologically by both inflammatory demyelination and neurodegenerative neuroaxonal loss, occurring in varying degrees in the white matter (WM) and in the grey matter (GM). Studies of MS commonly use imaging surrogates of inflammation (e.g. MRI lesions) and neurodegeneration (e.g. atrophy) as outcome measures to assess potential neuroprotective effects. As trials of potentially remyelinating agents become more important in the spectrum of MS research, imaging outcomes sensitive to myelin, such are magnetisation transfer ratio (MTR), are required to adequately assess any such agents. With the above in mind, for this thesis, I performed 4 studies: 1. MTR and atrophy localisation in the GM using voxel-based morphometry - MRI measures of GM MTR and volume were used to assess the regional localisation of reduced MTR (in part reflecting demyelination) and atrophy (in part reflecting neuro-axonal loss) in 98 patients with MS, as well as 29 controls. Subgroups of MS patients were compared with controls, adjusting for age and gender. Overall, whilst some regionally consistent reductions in MTR and atrophy were seen in GM, this study found that these mostly do not co-localise. The differing location and extent of regional MTR and volumetric abnormalities in MS subgroups argues against a single mechanism for demyelination and neuronal loss in the GM of MS patients. 2. MRI substudy of Dronabinol (Δ⁹-THC) vs placebo – 273 patients with secondary progressive MS (SPMS) received either Dronabinol or placebo (in a ratio of 2:1), with the aim of assessing the potential neuroprotective effects of Dronobinol. T2-weighed (T2w) and T1-weighted (T1w) lesions, and percentage brain volume change (PBVC) were assessed over 3 years. Over the course of the entire study, the occurrence of new or enlarging T2w or T1w lesions, or PBVC was not affected by Dronabinol. 3. Individual lesion area MTR analysis of autologous mesenchymal stem cells (AMSC) in patients with SPMS – A proof-of-concept individual lesion area MTR analysis pathway was developed and used post-hoc on 10 patients with SPMS and optic nerve disease from the MSCIMS study, which investigated the potential reparative effects of AMSC. For T2w lesion areas, a significant difference in rate of change of MTR was noted after infusion; this was not seen with T1w lesion areas. 4. Individual lesion MTR analysis in a crossover study of AMSC in patients with active MS – the proof-of-concept work above was refined for use in STREAMS, a crossover study of AMSC. 12 patients with active MS received either AMSC or placebo for 24 weeks, and then crossover to the other arm for a further 24 weeks. MTR was measured at week 0, 12, 24, 26, and 48 in both old and newly appearing lesions. There was not noted to be any significant effect of AMSC on the MTR of either old or newly appearing lesions

Assessment and optimisation of MRI measures of atrophy as potential markers of disease progression in multiple sclerosis

There is a need for sensitive measures of disease progression in multiple sclerosis (MS) to monitor treatment effects and understand disease evolution. MRI measures of brain atrophy have been proposed for this purpose. This thesis investigates a number of measurement techniques to assess their relative ability to monitor disease progression in clinically isolated syndromes (CIS) and early relapsing remitting MS (RRMS). Presented, is work demonstrating that measurement techniques and MR acquisitions can be optimised to give small but significant improvements in measurement sensitivity and precision, which provided greater statistical power. Direct comparison of numerous techniques demonstrated significant differences between them. Atrophy measurements from SIENA and the BBSI (registration-based techniques) were significantly more precise than segmentation and subtraction of brain volumes, although larger percentage losses were observed in grey matter fraction. Ventricular enlargement (VE) gave similar statistical power and these techniques were robust and reliable; scan-rescan measurement error was <0.01% of brain volume for BBSI and SIENA and <0.04ml for VE. Annual atrophy rates (using SIENA) were -0.78% in RRMS and -0.52% in CIS patients who progressed to MS, which were significantly greater than the rate observed in controls (-0.07%). Sample size calculations for future trials of disease-modifying treatments in RRMS, using brain atrophy as an outcome measure, are described. For SIENA, the BBSI and VE respectively, an estimated 123, 157 and 140 patients per treatment arm respectively would be required to show a 30% slowing of atrophy rate over two years. In CIS subjects brain atrophy rate was a significant prognostic factor, independent of T2 MRI lesions at baseline, for development of MS by five year follow-up. It was also the most significant MR predictor of disability in RRMS subjects. Cognitive assessment of RRMS patients at five year follow-up is described, and brain atrophy rate was a significant predictor of overall cognitive performance, and more specifically, of performance in tests of memory. The work in this thesis has identified methods for sensitively measuring progressive brain atrophy in MS. It has shown that brain atrophy changes in early MS are related to early clinical evolution, providing complementary information to clinical assessment that could be utilised to monitor disease progression

Computational modelling of imaging markers to support the diagnosis and monitoring of multiple sclerosis

Multiple sclerosis is a leading cause of neurological disability in young adults which affects more than 2.5 million people worldwide. An important substrate of disability accrual is the loss of neurons and connections between them (neurodegeneration) which can be captured by serial brain imaging, especially in the cerebral grey matter. In this thesis in four separate subprojects, I aimed to assess the strength of imaging-derived grey matter volume as a biomarker in the diagnosis, predicting the evolution of multiple sclerosis, and developing a staging system to stratify patients. In total, I retrospectively studied 1701 subjects, of whom 1548 had longitudinal brain imaging data. I used advanced computational models to investigate cross-sectional and longitudinal datasets. In the cross-sectional study, I demonstrated that grey matter volumes could distinguish multiple sclerosis from another demyelinating disorder (neuromyelitis optica) with an accuracy of 74%. In longitudinal studies, I showed that over time the deep grey matter nuclei had the fastest rate of volume loss (up to 1.66% annual loss) across the brain regions in multiple sclerosis. The volume of the deep grey matter was the strongest predictor of disability progression. I found that multiple sclerosis affects different brain areas with a specific temporal order (or sequence) that starts with the deep grey matter nuclei, posterior cingulate cortex, precuneus, and cerebellum. Finally, with multivariate mechanistic and causal modelling, I showed that brain volume loss causes disability and cognitive worsening which can be delayed with a potential neuroprotective treatment (simvastatin). This work provides conclusive evidence that grey matter volume loss affects some brain regions more severely, can predict future disability progression, can be used as an outcome measure in phase II clinical trials, and causes clinical and cognitive worsening. This thesis also provides a subject staging system based on which patients can be scored during multiple sclerosis

Imaging and therapy in multiple sclerosis

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.Grand Charity of the Freemason

Improving the clinico-radiological association in neurological diseases

Despite the key role of magnetic resonance imaging (MRI) in the diagnosis and monitoring of multiple sclerosis (MS) and cerebral small vessel disease (SVD), the association between clinical and radiological disease manifestations is often only moderate, limiting the use of MRI-derived markers in the clinical routine or as endpoints in clinical trials. In the projects conducted as part of this thesis, we addressed this clinico-radiological gap using two different approaches. Lesion-symptom association: In two voxel-based lesion-symptom mapping studies, we aimed at strengthening lesion-symptom associations by identifying strategic lesion locations. Lesion mapping was performed in two large cohorts: a dataset of 2348 relapsing-remitting MS patients, and a population-based cohort of 1017 elderly subjects. T2-weighted lesion masks were anatomically aligned and a voxel-based statistical approach to relate lesion location to different clinical rating scales was implemented. In the MS lesion mapping, significant associations between white matter (WM) lesion location and several clinical scores were found in periventricular areas. Such lesion clusters appear to be associated with impairment of different physical and cognitive abilities, probably because they affect commissural and long projection fibers. In the SVD lesion mapping, the same WM fibers and the caudate nucleus were identified to significantly relate to the subjects’ cerebrovascular risk profiles, while no other locations were found to be associated with cognitive impairment. Atrophy-symptom association: With the construction of an anatomical physical phantom, we aimed at addressing reliability and robustness of atrophy-symptom associations through the provision of a “ground truth” for atrophy quantification. The built phantom prototype is composed of agar gels doped with MRI and computed tomography (CT) contrast agents, which realistically mimic T1 relaxation times of WM and grey matter (GM) and showing distinguishable attenuation coefficients using CT. Moreover, due to the design of anatomically simulated molds, both WM and GM are characterized by shapes comparable to the human counterpart. In a proof-of-principle study, the designed phantom was used to validate automatic brain tissue quantification by two popular software tools, where “ground truth” volumes were derived from high-resolution CT scans. In general, results from the same software yielded reliable and robust results across scans, while results across software were highly variable reaching volume differences of up to 8%

Magnetic resonance imaging and histology correlation in the neocortex in temporal lobe epilepsy.

OBJECTIVE: To investigate the histopathological correlates of quantitative relaxometry and diffusion tensor imaging (DTI) and to determine their efficacy in epileptogenic lesion detection for preoperative evaluation of focal epilepsy. METHODS: We correlated quantitative relaxometry and DTI with histological features of neuronal density and morphology in 55 regions of the temporal lobe neocortex, selected from 13 patients who underwent epilepsy surgery. We made use of a validated nonrigid image registration protocol to obtain accurate correspondences between in vivo magnetic resonance imaging and histology images. RESULTS: We found T1 to be a predictor of neuronal density in the neocortical gray matter (GM) using linear mixed effects models with random effects for subjects. Fractional anisotropy (FA) was a predictor of neuronal density of large-caliber neurons only (pyramidal cells, layers 3 and 5). Comparing multivariate to univariate mixed effects models with nested variables demonstrated that employing T1 and FA together provided a significantly better fit than T1 or FA alone in predicting density of large-caliber neurons. Correlations with clinical variables revealed significant positive correlations between neuronal density and age (rs  = 0.726, pfwe  = 0.021). This study is the first to relate in vivo T1 and FA values to the proportion of neurons in GM. INTERPRETATION: Our results suggest that quantitative T1 mapping and DTI may have a role in preoperative evaluation of focal epilepsy and can be extended to identify GM pathology in a variety of neurological disorders

Serum Neurofilament Light Association With Progression in Natalizumab-Treated Patients With Relapsing-Remitting Multiple Sclerosis

OBJECTIVE: The objective of this study was to investigate the potential of serum neurofilament light (NfL) to reflect or predict progression mostly independent of acute inflammatory disease activity in patients with relapsing remitting multiple sclerosis (RRMS) treated with natalizumab. METHODS: Patients were selected from a prospective observational cohort study initiated in 2006 at the VU University Medical Center Amsterdam, The Netherlands, including patients with RRMS treated with natalizumab. Selection criteria included an age of 18 years or older and a minimum follow-up of 3 years from natalizumab initiation. Clinical and MRI assessments were performedon a yearly basis, and serum NfL was measured at 5 time-points during the follow-up, including on the day of natalizumab initiation (baseline), 3 months, 1 year and 2 years after natalizumab initiation, and on last follow-up visit. Using general linear regression models, we compared the longitudinal dynamics of NfL between patients with and without confirmed EDSS progression between year 1 visit and last follow-up, and between individuals with and without EDSS+ progression, a composite endpoint including the EDSS, 9 hole peg test and timed 25 foot-walk. RESULTS: Eighty-nine natalizumab-treated patients with RRMS were included. Median follow-up time was 5.2 years (IQR 4.3-6.7, range 3.0-11.0) after natalizumab initiation, mean age at time of natalizumab initiation was 36.9 (SD: 8.5), and median disease duration was 7.4 years (IQR 3.8-12.1). Between year 1 and the last follow-up, 28/89 (31.5%) individuals showed confirmed EDSS progression. Data for the EDSS+ endpoint was available for 73 out of the 89 patients and 35/73 (47.9%) showed confirmed EDSS+ progression.We observed a significant reduction in NfL levels 3 months after natalizumab initiation, which reached its nadir of close to 50% of baseline levels 1 year after treatment initiation. We found no difference in the longitudinal dynamics of NfL in progressors versus non-progressors. NfL levels at baseline and 1 year after natalizumab initiation did not predict progression at last follow-up. DISCUSSION: In our cohort of natalizumab-treated patients with RRMS, NfL fails to capture or predict progression that occurs largely independently of clinical or radiological signs of acute focal inflammatory disease activity. Additional biomarkers may thus be needed to monitor progression in these patients. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that serum NfL levels are not associated with disease progression in natalizumab-treated patients with RRMS

RimNet: A deep 3D multimodal MRI architecture for paramagnetic rim lesion assessment in multiple sclerosis.

In multiple sclerosis (MS), the presence of a paramagnetic rim at the edge of non-gadolinium-enhancing lesions indicates perilesional chronic inflammation. Patients featuring a higher paramagnetic rim lesion burden tend to have more aggressive disease. The objective of this study was to develop and evaluate a convolutional neural network (CNN) architecture (RimNet) for automated detection of paramagnetic rim lesions in MS employing multiple magnetic resonance (MR) imaging contrasts. Imaging data were acquired at 3 Tesla on three different scanners from two different centers, totaling 124 MS patients, and studied retrospectively. Paramagnetic rim lesion detection was independently assessed by two expert raters on T2*-phase images, yielding 462 rim-positive (rim+) and 4857 rim-negative (rim-) lesions. RimNet was designed using 3D patches centered on candidate lesions in 3D-EPI phase and 3D FLAIR as input to two network branches. The interconnection of branches at both the first network blocks and the last fully connected layers favors the extraction of low and high-level multimodal features, respectively. RimNet's performance was quantitatively evaluated against experts' evaluation from both lesion-wise and patient-wise perspectives. For the latter, patients were categorized based on a clinically relevant threshold of 4 rim+ lesions per patient. The individual prediction capabilities of the images were also explored and compared (DeLong test) by testing a CNN trained with one image as input (unimodal). The unimodal exploration showed the superior performance of 3D-EPI phase and 3D-EPI magnitude images in the rim+/- classification task (AUC = 0.913 and 0.901), compared to the 3D FLAIR (AUC = 0.855, Ps &lt; 0.0001). The proposed multimodal RimNet prototype clearly outperformed the best unimodal approach (AUC = 0.943, P &lt; 0.0001). The sensitivity and specificity achieved by RimNet (70.6% and 94.9%, respectively) are comparable to those of experts at the lesion level. In the patient-wise analysis, RimNet performed with an accuracy of 89.5% and a Dice coefficient (or F1 score) of 83.5%. The proposed prototype showed promising performance, supporting the usage of RimNet for speeding up and standardizing the paramagnetic rim lesions analysis in MS

Post mortem and in vivo study of multiple sclerosis pathogenesis

Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system. A number of pathological mechanisms could be responsible for acute demyelination and chronic tissue remodelling in MS, including inflammation, oxidative stress, microglia activation, and astrocyte infiltrates. In the present work, we aim to further explore the heterogeneity of MS pathogenesis on post mortem brains, and to evaluate the possibility to study MS pathogenesis by using magnetic resonance imaging (MRI) and peripheral blood biomarkers. In the first part of the study, we applied a data driven approach to classify MS patients in relation to the variety of pathological changes occurring in lesional and normal-appearing (NA) white matter (WM) and grey matter (GM), with subsequent clinical correlates. Tissue blocks from 16 MS brains were immunostained and quantified for neuro-axonal structures (NF200), myelin (SMI94), macrophages (CD68), B-lymphocytes (CD20), T-lymphocytes (CD3), cytotoxic T-lymphocytes (CD8), microglia (IBA1), astrocytes (GFAP), and mitochondrial damage. After semi-automatic registration of digitized histologic sections, regions-of-interest (ROIs) were manually defined in lesion and NA WM and GM. A latent class analysis was employed to characterize pathology subtypes in MS; different goodness of fit parameters (AIC, BIC, and G2 statistics) were used to identify the number of classes that better characterize the MS sub-populations. Profile 1 (active remodelling) was characterized by normal-appearing neuro-axonal structures and intact energetic metabolism, with high levels of macrophages/microglia and astrocytes. Profile 2 (mitochondrial dysfunction) was characterized by severely impaired mitochondrial function, along with demyelination and neuroaxonal loss, and ongoing inflammatory changes, mainly driven by cytotoxic T-cells (CD8+); patients in profile 2 presented with more severe symptoms at onset and faster disability accrual, when compared with other profiles. Profile 3 (inactive) was characterized by severe demyelination and axonal loss, with similarly reduced mitochondrial function, without any concomitant pathological process contributing to further tissue remodelling and/or damage. The possibility to classify each patient depending on his/her prevalent pathology profile support the concept of MS immunopathological homogeneity within the same patient and heterogeneity between different patients, and could be used to better profile MS patients and individualize their treatment. In the second part of the study, we explored post mortem pathology-MRI correlates and specifically focused on an advanced MRI technique (magnetization transfer ratio -MTR-), ideally detecting myelin content. MTR is widely used in MS observational studies and clinical trials, but its pathological correlates remain unclear. MTR maps were acquired at 3 Tesla from sixteen fixed MS brains and four healthy controls. 101 tissue blocks were immunostained and quantified, as previously described. After semi-automatic registration of digitized histologic sections and MTR maps, ROIs were manually defined. Associations between MTR and each stain were explored using linear mixed regression models (with cassettes nested within patients); differences in the associations between ROIs were explored using interaction terms. Lower MTR was associated with lower levels of NF200, SMI94, CD68, IBA1 and GFAP, with higher levels of CD8 and greater mitochondrial damage; MTR was more strongly associated with SMI94 in GM than WM. In a multivariate linear mixed regression model including all ROIs and brains, SMI94 was the best correlate of MTR. Myelin immunostain intensity is the strongest correlate of MTR, especially when measured in the GM. However, the additional histological correlates of MTR have to be kept in mind when interpreting the results of MTR clinical studies and designing experimental trials in MS. Finally, we evaluated the possibility to study (and to modify) MS pathology in vivo, by using biomarkers in the peripheral blood. Considering that oxidative stress is a driver of MS pathology, we evaluated the effect of coenzyme Q10 (CoQ10) on laboratory markers of oxidative stress and inflammation, and on MS clinical severity, and, then, calculated the sample size needed to detect significant variations to define most promising biomarkers. We included 60 relapsing-remitting MS patients treated with Interferon-Beta1a-44μg with CoQ10 for 3 months, and with Interferon-Beta1a-44μg alone for 3 more months (open-label cross-over design). At baseline, 3- and 6-month visits, we measured markers of scavenging activity, oxidative damage and inflammation in the peripheral blood, and collected data on disease severity. After 3 months, CoQ10 supplementation was associated with improved scavenging activity (as mediated by uric acid), reduced intracellular reactive oxygen species production, reduced oxidative DNA damage, and shift towards a more anti-inflammatory milieu in the peripheral blood (with higher IL-4 and IL-13, and lower Eotaxin, GM-CSF, HGF, IFN-γ, IL-1α, IL-2R, IL-9, IL-17F, MIP-1α, RANTES, TNF-α and VEGF). Also, CoQ10 supplementation was associated with lower expanded disability status scale, fatigue severity scale, Beck's depression inventory, and visual analogic scale for pain. For sample size estimates, we used adjusted-beta-coefficients of observed 3-month variation for each laboratory measure (and respective standard deviation); we assumed that the observed variation was the highest achievable treatment effect (100%), and we estimated sample size for conservative treatment effects (e.g., 70%), smaller than what observed. Setting 5% alpha-error and 80% power, low sample size requirements to detect 70% observed variation from a baseline pre-treatment timepoint to a 3-month follow-up were found for IL-3 (n=1), IL-5 (n=1), IL-7 (n=4), IL-2R (n=4), IL-13 (n=6), IL-6 (n=14), IL-8 (n=22), IL-4 (n=23), RANTES (n=25), TNF-α (n=26), IL-1β (n=27), and uric acid (n=29). CoQ10 supplementation improved scavenging activity, reduced oxidative damage, and induced a shift towards a more anti-inflammatory milieu, in the peripheral blood of relapsing-remitting MS patients treated with Interferon-Beta1a 44μg, along with clinical improvements. Peripheral biomarkers of oxidative stress and inflammation could be used in small proof-of-concept studies to quickly screen the mechanisms of action of new or already-existing medications for MS