Concurrent axon and myelin destruction differentiates X-linked adrenoleukodystrophy from multiple sclerosis

Cerebral disease manifestation occurs in about two thirds of males with X-linked adrenoleukodystrophy (CALD) and is fatally progressive if left untreated. Early histopathologic studies categorized CALD as an inflammatory demyelinating disease, which led to repeated comparisons to multiple sclerosis (MS). The aim of this study was to revisit the relationship between axonal damage and myelin loss in CALD. We applied novel immunohistochemical tools to investigate axonal damage, myelin loss and myelin repair in autopsy brain tissue of eight CALD and 25 MS patients. We found extensive and severe acute axonal damage in CALD already in prelesional areas defined by microglia loss and relative myelin preservation. In contrast to MS, we did not observe selective phagocytosis of myelin, but a concomitant decay of the entire axon-myelin unit in all CALD lesion stages. Using a novel marker protein for actively remyelinating oligodendrocytes, breast carcinoma-amplified sequence (BCAS) 1, we show that repair pathways are activated in oligodendrocytes in CALD. Regenerating cells, however, were affected by the ongoing disease process. We provide evidence that—in contrast to MS—selective myelin phagocytosis is not characteristic of CALD. On the contrary, our data indicate that acute axonal injury and permanent axonal loss are thus far underestimated features of the disease that must come into focus in our search for biomarkers and novel therapeutic approaches

Correlation of Diffusion and Metabolic Alterations in Different Clinical Forms of Multiple Sclerosis

Diffusion tensor imaging (DTI) and MR spectroscopic imaging (MRSI) provide greater sensitivity than conventional MRI to detect diffuse alterations in normal appearing white matter (NAWM) of Multiple Sclerosis (MS) patients with different clinical forms. Therefore, the goal of this study is to combine DTI and MRSI measurements to analyze the relation between diffusion and metabolic markers, T2-weighted lesion load (T2-LL) and the patients clinical status. The sensitivity and specificity of both methods were then compared in terms of MS clinical forms differentiation. MR examination was performed on 71 MS patients (27 relapsing remitting (RR), 26 secondary progressive (SP) and 18 primary progressive (PP)) and 24 control subjects. DTI and MRSI measurements were obtained from two identical regions of interest selected in left and right centrum semioval (CSO) WM. DTI metrics and metabolic contents were significantly altered in MS patients with the exception of N-acetyl-aspartate (NAA) and NAA/Choline (Cho) ratio in RR patients. Significant correlations were observed between diffusion and metabolic measures to various degrees in every MS patients group. Most DTI metrics were significantly correlated with the T2-LL while only NAA/Cr ratio was correlated in RR patients. A comparison analysis of MR methods efficiency demonstrated a better sensitivity/specificity of DTI over MRSI. Nevertheless, NAA/Cr ratio could distinguish all MS and SP patients groups from controls, while NAA/Cho ratio differentiated PP patients from controls. This study demonstrated that diffusivity changes related to microstructural alterations were correlated with metabolic changes and provided a better sensitivity to detect early changes, particularly in RR patients who are more subject to inflammatory processes. In contrast, the better specificity of metabolic ratios to detect axonal damage and demyelination may provide a better index for identification of PP patients

Understanding progression in primary progressive multiple sclerosis: a longitudinal clinical and magnetic resonance imaging study

The work in this thesis applies magnetization transfer imaging (MTI) and conventional MRI measures (brain volume, T2 lesion load and enhancing lesions) to investigate the mechanisms underlying progression in primary progressive multiple sclerosis (PPMS), and identifies MR markers to predict and monitor progression. First, we demonstrated that MTI was sensitive to change in the normal appearing brain tissues over one year, and that clinical progression over this period was predicted by baseline normal appearing white matter (NAWM) MT ratio (MTR). However, our second study showed that over three years, grey matter MTR became a better predictor of progression than any other MRI measure. Grey matter MTR and T2 lesion load changes reflected concurrent progression during this study. To localize the baseline grey matter injury more precisely, we developed a voxelbased technique to identify areas of grey matter MTR reduction and volume loss in patients compared with controls. The regions of grey matter MTR reduction identified correlated with clinical function in anatomically related systems. Finally, because our studies showed that lesion load influenced progression, we used contrast enhanced T1-weighted imaging to examine active focal inflammation. We found that while lesion activity declined over five years, levels of activity at the start of the study could influence mobility five years later. The work presented in this thesis suggests that grey matter damage has a predilection for certain brain regions and is an important determinant of progression in early PPMS. In the white matter, changes in lesion volume and activity continue to influence progression, but NAWM injury may have a declining role. MTR is a sensitive and responsive tool for predicting, monitoring, and localizing clinically relevant brain injury in early PPMS

T1/T2-weighted ratio in multiple sclerosis: A longitudinal study with clinical associations

Clinically isolated syndrome; Magnetic resonance imaging; Multiple sclerosisSíndrome clínicamente aislado; Imágenes por resonancia magnética; Esclerosis múltipleSíndrome clínicament aïllat; Imatges per ressonància magnètica; Esclerosi múltipleBackground T1w/T2-w ratio has been proposed as a clinically feasible MRI biomarker to assess tissue integrity in multiple sclerosis. However, no data is available in the earliest stages of the disease and longitudinal studies analysing clinical associations are scarce. Objective To describe longitudinal changes in T1-w/T2-w in patients with clinically isolated syndrome (CIS) and multiple sclerosis, and to investigate their clinical associations. Methods T1-w/T2-w images were generated and the mean value obtained in the corresponding lesion, normal-appearing grey (NAGM) and white matter (NAWM) masks. By co-registering baseline to follow-up MRI, evolved lesions were assessed; and by placing the mask of new lesions to the baseline study, the pre-lesional tissue integrity was measured. Results We included 171 CIS patients and 22 established multiple sclerosis patients. In CIS, evolved lesions showed significant T1-w/T2-w increases compared to baseline (+7.6%, P < 0.001). T1-w/T2-w values in new lesions were lower than in pre-lesional tissue (-28.2%, P < 0.001), and pre-lesional tissue was already lower than baseline NAWM (-7.8%, P < 0.001). In CIS at baseline, higher NAGM T1-w/T2-w was associated with multiple sclerosis diagnosis, and longitudinal decreases in NAGM and NAWM T1-w/T2-w were associated with disease activity. In established multiple sclerosis, T1-w/T2-w was inversely correlated with clinical disability and disease duration. Conclusion A decrease in T1-w/T2-w ratio precedes lesion formation. In CIS, higher T1-w/T2-w was associated with multiple sclerosis diagnosis. In established multiple sclerosis, lower T1-w/T2-w values were associated with clinical disability. The possible differential impact of chronic inflammation, iron deposition and demyelination should be considered to interpret these findings.This project was developed as a part of Mateus Boaventura ECTRIMS Clinical Training Fellowship Programme 2018–2019. This study was partially supported by the Project PI18/00823, from the Fondo de Investigación Sanitaria (FIS), Instituto de Salud Carlos III

A defect of sphingolipid metabolism modifies the properties of normal appearing white matter in multiple sclerosis

Maintaining the appropriate complement and content of lipids in cellular membranes is critical for normal neural function. Accumulating evidence suggests that even subtle perturbations in the lipid content of neurons and myelin can disrupt their function and may contribute to myelin and axonal degradation. In this study, we determined the composition and quantified the content of lipids and sterols in normal appearing white matter (NAWM) and normal appearing grey matter (NAGM) from control and multiple sclerosis brain tissues by electrospray ionization tandem mass spectrometry. Our results suggest that in active-multiple sclerosis, there is a shift in the lipid composition of NAWM and NAGM to a higher phospholipid and lower sphingolipid content. We found that this disturbance in lipid composition was reduced in NAGM but not in NAWM of inactive-multiple sclerosis. The pattern of disturbance in lipid composition suggests a metabolic defect that causes sphingolipids to be shuttled to phospholipid production. Modelling the biophysical consequence of this change in lipid composition of NAWM indicated an increase in the repulsive force between opposing bilayers that could explain decompaction and disruption of myelin structure

Longitudinal microstructural MRI markers of demyelination and neurodegeneration in early relapsing-remitting multiple sclerosis:Magnetisation transfer, water diffusion and g-ratio

INTRODUCTION: Quantitative microstructural MRI, such as myelin-sensitive magnetisation transfer ratio (MTR) or saturation (MTsat), axon-sensitive water diffusion Neurite Orientation Dispersion and Density Imaging (NODDI), and the aggregate g-ratio, may provide more specific markers of white matter integrity than conventional MRI for early patient stratification in relapsing-remitting multiple sclerosis (RRMS). The aim of this study was to determine the sensitivity of such markers to longitudinal pathological change within cerebral white matter lesions (WML) and normal-appearing white matter (NAWM) in recently diagnosed RRMS. METHODS: Seventy-nine people with recently diagnosed RRMS, from the FutureMS longitudinal cohort, were recruited to an extended MRI protocol at baseline and one year later. Twelve healthy volunteers received the same MRI protocol, repeated within two weeks. Ethics approval and written informed consent were obtained. 3T MRI included magnetisation transfer, and multi-shell diffusion-weighted imaging. NAWM and whole brain were segmented from 3D T1-weighted MPRAGE, and WML from T2-weighted FLAIR. MTR, MTsat, NODDI isotropic (ISOVF) and intracellular (ICVF) volume fractions, and g-ratio (calculated from MTsat and NODDI data) were measured within WML and NAWM. Brain parenchymal fraction (BPF) was also calculated. Longitudinal change in BPF and microstructural metrics was assessed with paired t-tests (α = 0.05) and linear mixed models, adjusted for confounding factors with False Discovery Rate (FDR) correction for multiple comparisons. Longitudinal changes were compared with test-retest Bland-Altman limits of agreement from healthy control white matter. The influence of longitudinal change on g-ratio was explored through post-hoc analysis in silico by computing g-ratio with realistic simulated MTsat and NODDI values. RESULTS: In NAWM, g-ratio and ICVF increased, and MTsat decreased over one year (adjusted mean difference = 0.007, 0.005, and −0.057 respectively, all FDR-corrected p < 0.05). There was no significant change in MTR, ISOVF, or BPF. In WML, MTsat, NODDI ICVF and ISOVF increased over time (adjusted mean difference = 0.083, 0.024 and 0.016, respectively, all FDR-corrected p < 0.05). Group-level longitudinal changes exceeded test-retest limits of agreement for NODDI ISOVF and ICVF in WML only. In silico analysis showed g-ratio may increase due to a decrease in MTsat or ISOVF, or an increase in ICVF. DISCUSSION: G-ratio and MTsat changes in NAWM over one year may indicate subtle myelin loss in early RRMS, which were not apparent with BPF or NAWM MTR. Increases in NAWM and WML NODDI ICVF were not anticipated, and raise the possibility of axonal swelling or morphological change. Increases in WML MTsat may reflect myelin repair. Changes in NODDI ISOVF are more likely to reflect alterations in water content. Competing MTsat and ICVF changes may account for the absence of g-ratio change in WML. Longitudinal changes in microstructural measures are significant at a group level, however detection in individual patients in early RRMS is limited by technique reproducibility. CONCLUSION: MTsat and g-ratio are more sensitive than MTR to early pathological changes in RRMS, but complex dependence of g-ratio on NODDI parameters limit the interpretation of aggregate measures in isolation. Improvements in technique reproducibility and validation of MRI biophysical models across a range of pathological tissue states are needed

The relation between inflammation and neurodegeneration in multiple sclerosis brains

Some recent studies suggest that in progressive multiple sclerosis, neurodegeneration may occur independently from inflammation. The aim of our study was to analyse the interdependence of inflammation, neurodegeneration and disease progression in various multiple sclerosis stages in relation to lesional activity and clinical course, with a particular focus on progressive multiple sclerosis. The study is based on detailed quantification of different inflammatory cells in relation to axonal injury in 67 multiple sclerosis autopsies from different disease stages and 28 controls without neurological disease or brain lesions. We found that pronounced inflammation in the brain is not only present in acute and relapsing multiple sclerosis but also in the secondary and primary progressive disease. T- and B-cell infiltrates correlated with the activity of demyelinating lesions, while plasma cell infiltrates were most pronounced in patients with secondary progressive multiple sclerosis (SPMS) and primary progressive multiple sclerosis (PPMS) and even persisted, when T- and B-cell infiltrates declined to levels seen in age matched controls. A highly significant association between inflammation and axonal injury was seen in the global multiple sclerosis population as well as in progressive multiple sclerosis alone. In older patients (median 76 years) with long-disease duration (median 372 months), inflammatory infiltrates declined to levels similar to those found in age-matched controls and the extent of axonal injury, too, was comparable with that in age-matched controls. Ongoing neurodegeneration in these patients, which exceeded the extent found in normal controls, could be attributed to confounding pathologies such as Alzheimer's or vascular disease. Our study suggests a close association between inflammation and neurodegeneration in all lesions and disease stages of multiple sclerosis. It further indicates that the disease processes of multiple sclerosis may die out in aged patients with long-standing disease

The investigation of early MRI in diagnosis and prognosis in patients presenting with a clinically isolated syndrome characteristic of demyelination

This thesis explores the use of early MRI in prognosis and diagnosis in patients presenting with a clinically isolated syndrome (CIS) characteristic of demyelination. This has been investigated in a cohort recruited within 3 months of CIS onset between 1995 and 2004 and followed up clinically and with MRI (planned at 3 months, 1,3 and 5 years). Current MRI criteria are highly specific for the development of clinically definite multiple sclerosis (CDMS) but have limited sensitivity and are complex. Presented is the evaluation of simplified MRI criteria in my London CIS cohort and in a multicentre CIS cohort. Results from the presented studies show that the MRI criteria can be simplified (dissemination in space: 2 or more lesions in separate but characteristic locations, dissemination in time: an early new T2 lesion) and still maintain high specificity, with improved sensitivity and accuracy. The prognostic role of early MRI was investigated in the optic neuritis (ON) subgroup, as 80% of my cohort presented with ON and some studies have suggested that such a presentation is associated with more benign disease. Whereas baseline lesion number significantly predicted conversion to CDMS and increased disability at 5 years, other MRI parameters, namely baseline lesion location (periventricular lesions increasing the hazard of CDMS and spinal cord and infratentorial lesions increasing the odds of greater disability at 5 years) and lesion activity (new T2 lesion at 3 month follow-up), were stronger predictors. No non-conventional MRI parameters (spectroscopy, magnetisation transfer ratio or atrophy measures) had a significant prognostic role. Overall early MRI findings can aid diagnosis and help identify the CIS patients at greatest risk of conversion to CDMS and subsequent disability, which in turn can help direct treatment and clinical follow-up in specialist MS clinics

MRI quantification of multiple sclerosis pathology

Background: Multiple sclerosis (MS) is a chronic neuroinflammatory and neurodegenerative disease and a common cause of neurologic disability. MS pathology is characterized by demyelination, neuroaxonal loss and atrophy. Magnetic Resonance Imaging (MRI) is an essential tool in diagnosing and monitoring MS, but its clinical value could be even further expanded by more advanced and quantitative MRI methods, which may also provide additional pathophysiological insights. Purpose: The overall aim of this thesis was to quantify MS pathology using volumetric brain MRI, ultra-high field brain and cervical spinal cord MRI as well as a newly developed rapid myelin imaging technique in relation to cognitive and physical MS disability. Study I, a prospective 17-year longitudinal study of 37 MS participants with 23 age/sex- matched healthy controls for comparison at the last follow-up. Longitudinal volumetric brain 1.5 Tesla MRI during the second half of the study showed that lesion accumulation and corpus callosum atrophy were the most strongly associated neuroanatomical correlates of cognitive disability, with the lesion fraction being an independent predictor of cognitive performance 8.5 years later. Study II, a prospective cross-sectional study of 35 MS participants and 11 age-matched healthy controls using 3 and 7 Tesla MRI. The study demonstrated involvement of both grey and white matter in MS, not only the brain but also the cervical spinal cord, associated with MS disability. Lesions appeared in proximity to the cerebrospinal fluid (CSF), with special predilection to the periventricular and grey matter surrounding the central canal in secondary progressive MS. Study III, a prospective in vivo (71 MS participants and 21 age/sex-matched healthy controls) and ex vivo (brain tissue from 3 MS donors) study at 3 Tesla, showed that a new clinically approved and feasible rapid myelin imaging technique correlates well with myelin stainings and produces robust in vivo myelin quantification that is related to both current and future cognitive and physical disability in MS. Study IV, an in-depth topographical analysis based on Study III, mapped the distribution of demyelination, both in vivo and ex vivo, in the periventricular and perilesional regions of the brain. A gradient of demyelination with predominance near the CSF spaces was seen. Measures of clinical disability were consistently and more strongly associated with the myelin content in normal-appearing tissue compared to the intralesional myelin content. Conclusions: Lesions and atrophy contribute to cognitive and physical disability in MS but to a varying degree, likely dependent on the relative involvement of white vs. grey matter. Both focal lesions/demyelination as well as diffuse demyelination in normal-appearing white matter shows an apparent gradient from the CSF, which differ between relapsing-remitting and progressive MS subtypes/phases. The growing utility and clinical availability of advanced and quantitative MRI techniques holds promise for improved monitoring of MS pathology and likely represents a vital tool for assessing the efficacy of potential remyelinating/reparative therapies in MS