Prevalence of Grey Matter Pathology in Early Multiple Sclerosis Assessed by Magnetization Transfer Ratio Imaging

The aim of the study was to assess the prevalence, the distribution and the impact on disability of grey matter (GM) pathology in early multiple sclerosis. Eighty-eight patients with a clinically isolated syndrome with a high risk developing multiple sclerosis were included in the study. Forty-four healthy controls constituted the normative population. An optimized statistical mapping analysis was performed to compare each subject's GM Magnetization Transfer Ratio (MTR) imaging maps with those of the whole group of controls. The statistical threshold of significant GM MTR decrease was determined as the maximum p value (p<0.05 FDR) for which no significant cluster survived when comparing each control to the whole control population. Using this threshold, 51% of patients showed GM abnormalities compared to controls. Locally, 37% of patients presented abnormalities inside the limbic cortex, 34% in the temporal cortex, 32% in the deep grey matter, 30% in the cerebellum, 30% in the frontal cortex, 26% in the occipital cortex and 19% in the parietal cortex. Stepwise regression analysis evidenced significant association (p = 0.002) between EDSS and both GM pathology (p = 0.028) and T2 white matter lesions load (p = 0.019). In the present study, we evidenced that individual analysis of GM MTR map allowed demonstrating that GM pathology is highly heterogeneous across patients at the early stage of MS and partly underlies irreversible disability

Regional patterns of grey matter atrophy and magnetisation transfer ratio abnormalities in multiple sclerosis clinical subgroups: A voxel-based analysis study.

In multiple sclerosis (MS), demyelination and neuro-axonal loss occur in the brain grey matter (GM). We used magnetic resonance imaging (MRI) measures of GM magnetisation transfer ratio (MTR) and volume to assess the regional localisation of reduced MTR (reflecting demyelination) and atrophy (reflecting neuro-axonal loss) in relapsing-remitting MS (RRMS), secondary progressive MS (SPMS) and primary progressive MS (PPMS)

A novel approach with "skeletonised MTR" measures tract-specific microstructural changes in early primary-progressive MS

We combined tract‐based spatial statistics (TBSS) and magnetization transfer (MT) imaging to assess white matter (WM) tract‐specific short‐term changes in early primary‐progressive multiple sclerosis (PPMS) and their relationships with clinical progression. Twenty‐one PPMS patients within 5 years from onset underwent MT and diffusion tensor imaging (DTI) at baseline and after 12 months. Patients' disability was assessed. DTI data were processed to compute fractional anisotropy (FA) and to generate a common WM “skeleton,” which represents the tracts that are “common” to all subjects using TBSS. The MT ratio (MTR) was computed from MT data and co‐registered with the DTI. The skeletonization procedure derived for FA was applied to each subject's MTR image to obtain a “skeletonised” MTR map for every subject. Permutation tests were used to assess (i) changes in FA, principal diffusivities, and MTR over the follow‐up, and (ii) associations between changes in imaging parameters and changes in disability. Patients showed significant decreases in MTR over one year in the corpus callosum (CC), bilateral corticospinal tract (CST), thalamic radiations, and superior and inferior longitudinal fasciculi. These changes were located both within lesions and the normal‐appearing WM. No significant longitudinal change in skeletonised FA was found, but radial diffusivity (RD) significantly increased in several regions, including the CST bilaterally and the right inferior longitudinal fasciculus. MTR decreases, RD increases, and axial diffusivity decreases in the CC and CST correlated with a deterioration in the upper limb function. We detected tract‐specific multimodal imaging changes that reflect the accrual of microstructural damage and possibly contribute to clinical impairment in PPMS. We propose a novel methodology that can be extended to other diseases to map cross‐subject and tract‐specific changes in MTR

White and grey matter damage in primary-progressive MS: the chicken or the egg?

Objective: The temporal relationship between white matter (WM) and gray matter (GM) damage in vivo in early primary progressive multiple sclerosis (PPMS) was investigated testing 2 hypotheses: (1) WM tract abnormalities predict subsequent changes in the connected cortex (“primary WM damage model”); and (2) cortical abnormalities predict later changes in connected WM tracts (“primary GM damage model”). Methods: Forty-seven patients with early PPMS and 18 healthy controls had conventional and magnetization transfer imaging at baseline; a subgroup of 35 patients repeated the protocol after 2 years. Masks of the corticospinal tracts, genu of the corpus callosum and optic radiations, and of connected cortical regions, were used for extracting the mean magnetization transfer ratio (MTR). Multiple regressions within each of 5 tract-cortex pairs were performed, adjusting for the dependent variable's baseline MTR; tract lesion load and MTR, spinal cord area, age, and sex were examined for potential confounding. Results: The baseline MTR of most regions was lower in patients than in healthy controls. The tract-cortex pair relationships in the primary WM damage model were significant for the bilateral motor pair and right visual pair, while those in the primary GM damage model were only significant for the right motor pair. Lower lesion MTR at baseline was associated with lower MTR in the same tract normal-appearing WM at 2 years in 3 tracts. Conclusion: These results are consistent with the hypothesis that in early PPMS, cortical damage is for the most part a sequela of normal-appearing WM pathology, which, in turn, is predicted by abnormalities within WM lesions

Clinical correlates of grey matter pathology in multiple sclerosis

Traditionally, multiple sclerosis has been viewed as a disease predominantly affecting white matter. However, this view has lately been subject to numerous changes, as new evidence of anatomical and histological changes as well as of molecular targets within the grey matter has arisen. This advance was driven mainly by novel imaging techniques, however, these have not yet been implemented in routine clinical practice. The changes in the grey matter are related to physical and cognitive disability seen in individuals with multiple sclerosis. Furthermore, damage to several grey matter structures can be associated with impairment of specific functions. Therefore, we conclude that grey matter damage - global and regional - has the potential to become a marker of disease activity, complementary to the currently used magnetic resonance markers (global brain atrophy and T2 hyperintense lesions). Furthermore, it may improve the prediction of the future disease course and response to therapy in individual patients and may also become a reliable additional surrogate marker of treatment effect

The Contribution of Various MRI Parameters to Clinical and Cognitive Disability in Multiple Sclerosis

Next to the disseminated clinical symptoms, cognitive dysfunctions are common features of multiple sclerosis (MS). Over the recent years several different MRI measures became available representing the various features of the pathology, but the contribution to various clinical and cognitive functions is not yet fully understood. In this multiparametric MRI study we set out to identify the set of parameters that best predict the clinical and cognitive disability in MS. High resolution T1 weighted structural and high angular resolution diffusion MRI images were measured in 53 patients with relapsing remitting MS and 53 healthy controls. Clinical disability was inflicted by EDSS and cognitive functions were evaluated with the BICAMS tests. The contribution of lesion load, partial brain, white matter, gray matter and subcortical volumes as well as the diffusion parameters in the area of the lesions and the normal appearing white matter were examined by model free, partial least square (PLS) approach. Significance of the predictors was tested with Variable Importance in the Projection (VIP) score and 1 was used for threshold of significance. The PLS analysis indicated that the axial diffusivity of the NAWM contributed the most to the clinical disability (VIP score: 1.979). For the visuo-spatial working memory the most critical contributor was the size of the bilateral hippocampi (VIP scores: 1.183 and 1.2 left and right respectively). For the verbal memory the best predictors were the size of the right hippocampus (VIP score: 1.972), lesion load (VIP score: 1.274) and the partial brain volume (VIP score: 1.119). In case of the information processing speed the most significant contribution was from the diffusion parameters (fractional anisotropy, mean and radial diffusivity, VIP scores: 1.615, 1.321 respectively) of the normal appearing white matter. Our results indicate that various MRI measurable factors of MS pathology contribute differently to clinical and cognitive disability. These results point out the importance of the volumetry of the subcortical structures and the diffusion measures of the white matter in understanding the disability progression

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

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