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Background: Despite highly effective treatment strategies for patients with relapsing-remitting multiple sclerosis (RRMS), long-term neurodegeneration and disease progression are often considerable. Accurate blood-based biomarkers that predict long-term neurodegeneration are lacking. Objective: To assess the predictive value of serum neurofilament-light (sNfL) and serum contactin-1 (sCNTN1) for long-term magnetic resonance imaging (MRI)–derived neurodegeneration in natalizumab-treated patients with RRMS. Methods: sNfL and sCNTN1 were measured in an observational cohort of natalizumab-treated patients with RRMS at baseline (first dose) and at 3 months, Year 1, Year 2, and last follow-up (median = 5.2 years) of treatment. Disability progression was quantified using “EDSS-plus” criteria. Neurodegeneration was measured by calculating annualized percentage brain, ventricular, and thalamic volume change (PBVC, VVC, and TVC, respectively). Linear regression analysis was performed to identify longitudinal predictors of neurodegeneration. Results: In total, 88 patients (age = 37 ± 9 years, 75% female) were included, of whom 48% progressed. Year 1 sNfL level (not baseline or 3 months) was associated with PBVC (standardized (std.) β = −0.26, p = 0.013), VVC (standardized β = 0.36, p Conclusion: Year 1 (but not baseline) sNfL level was predictive for long-term brain atrophy in patients treated with natalizumab. sCNTN1 level did not show a clear predictive value.

Background: Cortical multiple sclerosis lesions are clinically relevant but inconspicuous at conventional clinical MRI. Double inversion recovery (DIR) and phase-sensitive inversion recovery (PSIR) are more sensitive but often unavailable. In the past 2 years, artificial intelligence (AI) was used to generate DIR and PSIR from standard clinical sequences (eg, T1-weighted, T2-weighted, and fluid-attenuated inversion-recovery sequences), but multicenter validation is crucial for further implementation. Purpose: To evaluate cortical and juxtacortical multiple sclerosis lesion detection for diagnostic and disease monitoring purposes on AI-generated DIR and PSIR images compared with MRI-acquired DIR and PSIR images in a multicenter setting. Materials and Methods: Generative adversarial networks were used to generate AI-based DIR (n = 50) and PSIR (n = 43) images. The number of detected lesions between AI-generated images and MRI-acquired (reference) images was compared by randomized blinded scoring by seven readers (all with >10 years of experience in lesion assessment). Reliability was expressed as the intraclass correlation coefficient (ICC). Differences in lesion subtype were determined using Wilcoxon signed-rank tests. Results: MRI scans of 202 patients with multiple sclerosis (mean age, 46 years ± 11 [SD]; 127 women) were retrospectively collected from seven centers (February 2020 to January 2021). In total, 1154 lesions were detected on AI-generated DIR images versus 855 on MRI-acquired DIR images (mean difference per reader, 35.0% ± 22.8; P < .001). On AI-generated PSIR images, 803 lesions were detected versus 814 on MRI-acquired PSIR images (98.9% ± 19.4; P = .87). Reliability was good for both DIR (ICC, 0.81) and PSIR (ICC, 0.75) across centers. Regionally, more juxtacortical lesions were detected on AI-generated DIR images than on MRI-acquired DIR images (495 [42.9%] vs 338 [39.5%]; P < .001). On AI-generated PSIR images, fewer juxtacortical lesions were detected than on MRI-acquired PSIR images (232 [28.9%] vs 282 [34.6%]; P = .02). Conclusion: Artificial intelligence–generated double inversion-recovery and phase-sensitive inversion-recovery images performed well compared with their MRI-acquired counterparts and can be considered reliable in a multicenter setting, with good between-reader and between-center interpretative agreement.

Background: Cognitive training elicits mild-to-moderate improvements in cognitive functioning in people with multiple sclerosis (PwMS), although response heterogeneity limits overall effectiveness. Objective: To identify patient characteristics associated with response and non-response to cognitive training. Methods: Eighty-two PwMS were randomized into a 7-week attention training ( n = 58, age = 48.4 ± 10.2 years) or a waiting-list control group ( n = 24, age = 48.5 ± 9.4 years). Structural and functional magnetic resonance imaging (MRI) was obtained at baseline and post-intervention. Twenty-one healthy controls (HCs, age = 50.27 ± 10.15 years) were included at baseline. Responders were defined with a reliable change index of 1.64 on at least 2/6 cognitive domains. General linear models and logistic regression were applied. Results: Responders ( n = 36) and non-responders ( n = 22) did not differ on demographics, clinical variables and baseline cognition and structural MRI. However, non-responders exhibited a higher baseline functional connectivity (FC) between the default-mode network (DMN) and the ventral attention network (VAN), compared with responders ( p = 0.018) and HCs ( p = 0.001). Conversely, responders exhibited no significant baseline differences in FC compared with HCs. Response to cognitive training was predicted by lower DMN-VAN FC ( p = 0.004) and DMN-frontoparietal FC ( p = 0.029) (Nagelkerke R2 = 0.25). Conclusion: An intact pre-intervention FC is associated with cognitive training responsivity in pwMS, suggesting a window of opportunity for successful cognitive interventions.

Background: Neuroaxonal degeneration is one of the hallmarks of clinical deterioration in progressive multiple sclerosis (PMS). Objective: To elucidate the association between neuroaxonal degeneration and both local cortical and connected white matter (WM) tract pathology in PMS. Methods: Post-mortem in situ 3T magnetic resonance imaging (MRI) and cortical tissue blocks were collected from 16 PMS donors and 10 controls. Cortical neuroaxonal, myelin, and microglia densities were quantified histopathologically. From diffusion tensor MRI, fractional anisotropy, axial diffusivity (AD), radial diffusivity (RD), and mean diffusivity (MD) were quantified in normal-appearing white matter (NAWM) and white matter lesions (WML) of WM tracts connected to dissected cortical regions. Between-group differences and within-group associations were investigated through linear mixed models. Results: The PMS donors displayed significant axonal loss in both demyelinated and normal-appearing (NA) cortices ( p Conclusion: Reduced integrity of connected WM tracts and cortical demyelination both contribute to cortical axonal loss in PMS.

Background Increasing numbers of adolescents seek help for gender-identity questions. Consequently, requests for medical treatments, such as puberty suppression, are growing. However, studies investigating the neurobiological substrate of gender incongruence (when birth-assigned sex and gender identity do not align) are scarce, and knowledge about the effects of puberty suppression on the developing brain of transgender youth is limited. Methods Here we cross-sectionally investigated sex and gender differences in regional fractional anisotropy (FA) as measured by diffusion MR imaging, and the impact of puberty on alterations in the white-matter organization of 35 treatment-naive prepubertal children and 41 adolescents with gender incongruence, receiving puberty suppression. The transgender groups were compared with 79 age-matched, treatment-naive cisgender (when sex and gender align) peers. Results We found that transgender adolescents had lower FA in the bilateral inferior fronto-occipital fasciculus (IFOF), forceps major and corpus callosum than cisgender peers. In addition, average FA values of the right IFOF correlated negatively with adolescents' cumulative dosage of puberty suppressants received. Of note, prepubertal children also showed significant FA group differences in, again, the right IFOF and left cortico-spinal tract, but with the reverse pattern (transgender > cisgender) than was seen in adolescents. Conclusions Importantly, our results of lower FA (indexing less longitudinal organization, fiber coherence, and myelination) in the IFOF of gender-incongruent adolescents replicate prior findings in transgender adults, suggesting a salient neural correlate of gender incongruence. Findings highlight the complexity with which (pubertal) sex hormones impact white-matter development and add important insight into the neurobiological substrate associated with gender incongruence.

The severity and effects of damage to short- and long-range structural connections in multiple sclerosis are unknown. Meijer et al. report that long-range connections are more severely damaged than short-range connections. Damage to long-range connections is more strongly associated with reduced structural network efficiency, abnormal functional connectivity, and impaired cognition., An efficient network such as the human brain features a combination of global integration of information, driven by long-range connections, and local processing involving short-range connections. Whether these connections are equally damaged in multiple sclerosis is unknown, as is their relevance for cognitive impairment and brain function. Therefore, we cross-sectionally investigated the association between damage to short- and long-range connections with structural network efficiency, the functional connectome and cognition. From the Amsterdam multiple sclerosis cohort, 133 patients (age = 54.2 ± 9.6) with long-standing multiple sclerosis and 48 healthy controls (age = 50.8 ± 7.0) with neuropsychological testing and MRI were included. Structural connectivity was estimated from diffusion tensor images using probabilistic tractography (MRtrix 3.0) between pairs of brain regions. Structural connections were divided into short- (length < quartile 1) and long-range (length > quartile 3) connections, based on the mean distribution of tract lengths in healthy controls. To determine the severity of damage within these connections, (i) fractional anisotropy as a measure for integrity; (ii) total number of fibres; and (iii) percentage of tract affected by lesions were computed for each connecting tract and averaged for short- and long-range connections separately. To investigate the impact of damage in these connections for structural network efficiency, global efficiency was computed. Additionally, resting-state functional connectivity was computed between each pair of brain regions, after artefact removal with FMRIB’s ICA-based X-noiseifier. The functional connectivity similarity index was computed by correlating individual functional connectivity matrices with an average healthy control connectivity matrix. Our results showed that the structural network had a reduced efficiency and integrity in multiple sclerosis relative to healthy controls (both P < 0.05). The long-range connections showed the largest reduction in fractional anisotropy (z = −1.03, P < 0.001) and total number of fibres (z = −0.44, P < 0.01), whereas in the short-range connections only fractional anisotropy was affected (z = −0.34, P = 0.03). Long-range connections also demonstrated a higher percentage of tract affected by lesions than short-range connections, independent of tract length (P < 0.001). Damage to long-range connections was more strongly related to structural network efficiency and cognition (fractional anisotropy: r = 0.329 and r = 0.447. number of fibres r = 0.321 and r = 0.278. and percentage of lesions: r = −0.219; r = −0.426, respectively) than damage to short-range connections. Only damage to long-distance connections correlated with a more abnormal functional network (fractional anisotropy: r = 0.226). Our findings indicate that long-range connections are more severely affected by multiple sclerosis-specific damage than short-range connections. Moreover compared to short-range connections, damage to long-range connections better explains network efficiency and cognition.

Background: Coordinated patterns of gray matter morphology can be represented as networks, and network disruptions may explain cognitive dysfunction related to multiple sclerosis (MS). Objective: To investigate whether single-subject gray matter network properties are related to impaired cognition in MS. Methods: We studied 148 MS patients (99 female) and 33 healthy controls (HC, 21 female). Seven network parameters were computed and compared within MS between cognitively normal and impaired subjects, and associated with performance on neuropsychological tests in six cognitive domains with regression models. Analyses were controlled for age, gender, whole-brain gray matter volumes, and education level. Results: Compared to MS subjects with normal cognition, MS subjects with cognitive impairment showed a more random network organization as indicated by lower lambda values (all p Conclusion: Our findings indicate that MS subjects with a more randomly organized gray matter network show worse cognitive functioning, suggesting that single-subject gray matter graphs may capture neurological dysfunction due to MS.

Background: Cortical lesions are highly inconspicuous on magnetic resonance imaging (MRI). Double inversion recovery (DIR) has a higher sensitivity than conventional clinical sequences (i.e. T1, T2, FLAIR) but is difficult to acquire, leading to overseen cortical lesions in clinical care and clinical trials. Objective: To evaluate the usability of artificially generated DIR (aDIR) images for cortical lesion detection compared to conventionally acquired DIR (cDIR). Methods: The dataset consisted of 3D-T1 and 2D-proton density (PD) T2 images of 73 patients (49RR, 20SP, 4PP) at 1.5 T. Using a 4:1 train:test-ratio, a fully convolutional neural network was trained to predict 3D-aDIR from 3D-T1 and 2D-PD/T2 images. Randomized blind scoring of the test set was used to determine detection reliability, precision and recall. Results: A total of 626 vs 696 cortical lesions were detected on 15 aDIR vs cDIR images (intraclass correlation coefficient (ICC) = 0.92). Compared to cDIR, precision and recall were 0.84 ± 0.06 and 0.76 ± 0.09, respectively. The frontal and temporal lobes showed the largest differences in discernibility. Conclusion: Cortical lesions can be detected with good reliability on artificial DIR. The technique has potential to broaden the availability of DIR in clinical care and provides the opportunity of ex post facto implementation of cortical lesions imaging in existing clinical trial data.

Cortical lesions are important in multiple sclerosis, but notoriously hard to detect with MRI. Bouman et al. compare all hitherto suggested sequences to histopathology, and report that double inversion recovery and phase-sensitive inversion recovery outperform conventional clinical sequences, but complement each other., Cortical demyelinating lesions are clinically important in multiple sclerosis, but notoriously difficult to visualize with MRI. At clinical field strengths, double inversion recovery MRI is most sensitive, but still only detects 18% of all histopathologically validated cortical lesions. More recently, phase-sensitive inversion recovery was suggested to have a higher sensitivity than double inversion recovery, although this claim was not histopathologically validated. Therefore, this retrospective study aimed to provide clarity on this matter by identifying which MRI sequence best detects histopathologically-validated cortical lesions at clinical field strength, by comparing sensitivity and specificity of the thus far most commonly used MRI sequences, which are T2, fluid-attenuated inversion recovery (FLAIR), double inversion recovery and phase-sensitive inversion recovery. Post-mortem MRI was performed on non-fixed coronal hemispheric brain slices of 23 patients with progressive multiple sclerosis directly after autopsy, at 3 T, using T1 and proton-density/T2-weighted, as well as FLAIR, double inversion recovery and phase-sensitive inversion recovery sequences. A total of 93 cortical tissue blocks were sampled from these slices. Blinded to histopathology, all MRI sequences were consensus scored for cortical lesions. Subsequently, tissue samples were stained for proteolipid protein (myelin) and scored for cortical lesion types I–IV (mixed grey matter/white matter, intracortical, subpial and cortex-spanning lesions, respectively). MRI scores were compared to histopathological scores to calculate sensitivity and specificity per sequence. Next, a retrospective (unblinded) scoring was performed to explore maximum scoring potential per sequence. Histopathologically, 224 cortical lesions were detected, of which the majority were subpial. In a mixed model, sensitivity of T1, proton-density/T2, FLAIR, double inversion recovery and phase-sensitive inversion recovery was 8.9%, 5.4%, 5.4%, 22.8% and 23.7%, respectively (20, 12, 12, 51 and 53 cortical lesions). Specificity of the prospective scoring was 80.0%, 75.0%, 80.0%, 91.1% and 88.3%. Sensitivity and specificity did not significantly differ between double inversion recovery and phase-sensitive inversion recovery, while phase-sensitive inversion recovery identified more lesions than double inversion recovery upon retrospective analysis (126 versus 95; P

The ratio between T1-weighted (T1w) and T2-weighted (T2w) sequences (T1w/T2w-ratio) has been proposed to enhance myelin sensitivity and specificity.1 However, studies assessing its histopathological substrates in multiple sclerosis (MS) found contradictory results. One study reported lower T1w/T2w-ratio in demyelinated versus normal-appearing cortex, but neurite density was not investigated.2 In another study, T1w/T2w-ratio correlated with dendrite but not myelin density; however, only normal-appearing cortex was evaluated.3 Both myelin and neurite density could influence T1w/T2w-ratio.4 Since T1w/T2w-ratio is derived from commonly acquired sequences and seems sensitive to cortical damage, its pathological validation is strongly needed before its wider application. In this post-mortem MRI/histopathology study, we evaluated normal-appearing and demyelinated cortices from non-neurological controls (nNC) and patients with MS (PwMS) to define whether myelin and/or neurite density were associated with T1w/T2w-ratio. ### Donors PwMS were collected by the Netherlands Brain Bank (http://www.brainbank.nl/), while nNC by the Normal Aging Brain Collection Amsterdam (http://nabca.eu/). ### MRI The following sequences were acquired during a 3T whole-brain in situ MRI scan: sagittal three-dimensional FLAIR; sagittal three-dimensional T1w fast spoiled gradient echo; axial T2w spin echo. See online supplemental methods for scan geometry. ### Supplementary data [jnnp-2020-324391supp001.pdf] White matter (WM) lesion volume (LV) was automatically quantified on FLAIR.5 T1w/T2w-ratio was obtained with an in-house pipeline adapted from Ganzetti et al 1 (online supplemental figure). Native three-dimensional T1w and T2w sequences underwent bias field correction (SPM12). Unbiased images were calibrated adjusting the intensity histograms using the lowest and highest intensity peaks derived from eye and temporal muscle masks on T1w and T2w images. T2w sequences were coregistered on T1w images and the T1w/T2w-ratio was calculated. From three-dimensional T1w images, grey matter (GM) and WM maps were created (SPM12). GM maps were thresholded at 0.5 to limit partial voluming and masked to include the cortex and remove cerebellum and basal ganglia. ### Histopathology After …

We aimed to study if patients with dementia with Lewy bodies (DLB) who have concomitant Alzheimer's disease (AD) pathology (detected antemortem by cerebrospinal fluid [CSF] biomarkers) have additional loss of gray matter volume. Ninety-eight DLB patients were divided into a “pure DLB” (DLB/AD−, n = 62) and a “mixed DLB” group (DLB/AD+, n = 36) and matched for age and symptom duration to 84 AD patients and 75 controls. We compared visual atrophy ratings, and in a subset, we analyzed cortical thickness and subcortical gray matter volumes. DLB/AD+ patients had more pronounced medial temporal lobe atrophy (MTA) compared to DLB/AD− (mean [total] MTA score 2.5 vs. 1.3, p = 0.02). Global and parietal atrophy scores were comparable between the 3 dementia groups and differed from controls. MTA score was associated with CSF Aβ-42, while posterior cortical and global atrophy scores were associated with CSF tau. Cortical thinning was found in DLB/AD−, DLB/AD+, and AD compared to controls. Concomitant AD pathology seems to cause additional (hippocampal) atrophy in DLB, and this may contribute to a more devastating disease course in DLB/AD+ patients.

To understand the heterogeneity of functional connectivity results reported in the literature, we analyzed the separate effects of grey and white matter damage on functional connectivity and networks in multiple sclerosis. For this, we employed a biophysical thalamo‐cortical model consisting of interconnected cortical and thalamic neuronal populations, informed and amended by empirical diffusion MRI tractography data, to simulate functional data that mimic neurophysiological signals. Grey matter degeneration was simulated by decreasing within population connections and white matter degeneration by lowering between population connections, based on lesion predilection sites in multiple sclerosis. For all simulations, functional connectivity and functional network organization are quantified by phase synchronization and network integration, respectively. Modeling results showed that both cortical and thalamic grey matter damage induced a global increase in functional connectivity, whereas white matter damage induced an initially increased connectivity followed by a global decrease. Both white and especially grey matter damage, however, induced a decrease in network integration. These empirically informed simulations show that specific topology and timing of structural damage are nontrivial aspects in explaining functional abnormalities in MS. Insufficient attention to these aspects likely explains contradictory findings in multiple sclerosis functional imaging studies so far.

Purpose The purpose of this study was to present an effective algorithm for computing the discrete polar Stockwell transform (PST), investigate its unique multiscale and multi-orientation features, and explore potentially new applications including denoising and tissue segmentation. Theory and methods We investigated PST responses using both synthetic and MR images. Moreover, we compared the features of PST with both Gabor and Morlet wavelet transforms, and compared the PST with two wavelet approaches for denoising using MRI. Using a synthetic image, we also tested the edge effect of PST through signal-padding. Then, we constructed a partially supervised classifier using radial, marginal PST spectra of T2-weighted MRI, acquired from postmortem brains with multiple sclerosis. The classification involved three histology-verified tissue types: normal appearing white matter (NAWM), lesion, or other, along with 5-fold cross-validation. Results The PST generated a series of images with varying orientations or rotation-invariant scales. Radial frequencies highlighted image structures of different size, and angular frequencies enhanced structures by orientation. Signal-padding helped suppress boundary artifacts but required attention to incidental artifacts. In comparison, the Gabor transform produced more redundant images and the wavelet spectra appeared less spatially smooth than the PST. In addition, the PST demonstrated lower root-mean-square errors than other transforms in denoising and achieved a 93% accuracy for NAWM pixels (296/317), and 88% accuracy for lesion pixels (165/188) in MRI segmentation. Conclusions The PST is a unique local spectral density-assessing tool which is sensitive to both structure orientations and scales. This may facilitate multiple new applications including advanced characterization of tissue structure in standard MRI.

[Background] Mitochondrial dysfunction is increasingly recognized as an important feature of multiple sclerosis (MS) pathology and may be relevant for clinical disease progression. However, it is unknown whether mitochondrial DNA (mtDNA) levels in the cerebrospinal fluid (CSF) associate with disease progression and therapeutic response. [Objectives] To evaluate whether CSF concentrations of mtDNA in MS patients can serve as a marker of ongoing neuropathology and may be helpful to differentiate between MS disease subtypes. To explore the effect of disease-modifying therapies on mtDNA levels in the CSF. [Methods] CSF mtDNA was measured using a digital polymerase chain reaction (PCR) CSF mtDNA in two independent MS cohorts. The cohorts included 92 relapsing-remitting multiple sclerosis (RRMS) patients, 40 progressive multiple sclerosis (PMS) patients (27 secondary progressive and 13 primary progressive), 50 various neurologic disease controls, and 5 healthy controls. [Results] Patients with PMS showed a significant increase in CSF mtDNA compared to non-inflammatory neurologic disease controls. Patients with higher T2 lesion volumes and lower normalized brain volumes showed increased concentration of mtDNA. Patients treated with fingolimod had significantly lower mtDNA copy levels at follow-up compared to baseline. [Conclusion] Our results showed a non-specific elevation of concentration of mtDNA in PMS patients. mtDNA concentrations respond to fingolimod and may be used to monitor biological effect of this treatment, This work has been funded, in part, by the Ministerio de Economia y Competitividad of Spain (SAF2014-56644) and by CIBERNED grants to R.T. and P.P.

Objective To explore the added value of dynamic functional connectivity (dFC) of the default mode network (DMN) during resting-state (RS), during an information processing speed (IPS) task, and the within-subject difference between these conditions, on top of conventional brain measures in explaining IPS in people with multiple sclerosis (pwMS). Methods In 29 pwMS and 18 healthy controls, IPS was assessed with the Letter Digit Substitution Test and Stroop Card I and combined into an IPS-composite score. White matter (WM), grey matter (GM) and lesion volume were measured using 3 T MRI. WM integrity was assessed with diffusion tensor imaging. During RS and task-state fMRI (i.e. symbol digit modalities task, IPS), stationary functional connectivity (sFC; average connectivity over the entire time series) and dFC (variation in connectivity using a sliding window approach) of the DMN was calculated, as well as the difference between both conditions (i.e. task-state minus RS; ΔsFC-DMN and ΔdFC-DMN). Regression analysis was performed to determine the most important predictors for IPS. Results Compared to controls, pwMS performed worse on IPS-composite (p = 0.022), had lower GM volume (p, Highlights • Problems with information processing speed occur often in multiple sclerosis (MS) • Dynamics in brain communication can reflect information transfer within the brain • With fMRI, dynamic communication can be measured, which increases upon task demands • This increase in dynamics explains information processing speed in MS

Contains fulltext : 190740.pdf (Publisher’s version ) (Open Access) BACKGROUND AND PURPOSE: Precise in vivo measurement of deep GM volume change is a highly demanded prerequisite for an adequate evaluation of disease progression and new treatments. However, quantitative data on the reproducibility of deep GM structure volumetry are not yet available. In this paper we aim to investigate this reproducibility using a large multicenter dataset. MATERIALS AND METHODS: We have assessed the reproducibility of 2 automated segmentation software packages (FreeSurfer and the FMRIB Integrated Registration and Segmentation Tool) by quantifying the volume changes of deep GM structures by using back-to-back MR imaging scans from the Alzheimer Disease Neuroimaging Initiative's multicenter dataset. Five hundred sixty-two subjects with scans at baseline and 1 year were included. Reproducibility was investigated in the bilateral caudate nucleus, putamen, amygdala, globus pallidus, and thalamus by carrying out descriptives as well as multilevel and variance component analysis. RESULTS: Median absolute back-to-back differences varied between GM structures, ranging from 59.6-156.4 muL for volume change, and 1.26%-8.63% for percentage volume change. FreeSurfer had a better performance for the outcome of longitudinal volume change for the bilateral amygdala, putamen, left caudate nucleus (P < .005), and right thalamus (P < .001). For longitudinal percentage volume change, Freesurfer performed better for the left amygdala, bilateral caudate nucleus, and left putamen (P < .001). Smaller limits of agreement were found for FreeSurfer for both outcomes for all GM structures except the globus pallidus. Our results showed that back-to-back differences in 1-year percentage volume change were approximately 1.5-3.5 times larger than the mean measured 1-year volume change of those structures. CONCLUSIONS: Longitudinal deep GM atrophy measures should be interpreted with caution. Furthermore, deep GM atrophy measurement techniques require substantially improved reproducibility, specifically when aiming for personalized medicine.

Background: We conducted a multi-modal optical coherence tomography (OCT) and magnetoencephalography (MEG) study to test whether there is a relationship between retinal layer integrity and electrophysiological activity and connectivity (FC) in the visual network influenced by optic neuritis (ON) in patients with multiple sclerosis (MS). Methods: One hundred and two MS patients were included in this MEG/OCT study. Retinal OCT data were collected from the optic discs, macular region, and segmented. Neuronal activity and FC in the visual cortex was estimated from source-reconstructed resting-state MEG data by computing relative power and the phase lag index (PLI). Generalized estimating equations (GEE) were used to account for intereye within-patient dependencies. Results: There was a significant relationship for both relative power and FC in the visual cortex with retinal layer thicknesses. The findings were influenced by the presence of MSON, particularly for connectivity in the alpha bands and the outer macular layers. In the absence of MSON, this relationship was dominated by the lower frequency bands (theta, delta) and inner and outer retinal layers. Conclusion: These results suggest that visual cortex FC more than activity alters in the presence of MSON, which may guide the understanding of FC plasticity effects following MSON.

ObjectiveTo determine which pathologic process could be responsible for the acceleration of cognitive decline during the course of multiple sclerosis (MS), using longitudinal structural MRI, which was related to cognitive decline in relapsing-remitting MS (RRMS) and progressive MS (PMS).MethodsA prospective cohort of 230 patients with MS (179 RRMS and 51 PMS) and 59 healthy controls was evaluated twice with 5-year (mean 4.9, SD 0.94) interval during which 22 patients with RRMS converted to PMS. Annual rates of cortical and deep gray matter atrophy as well as lesion volume increase were computed on longitudinal (3T) MRI data and correlated to the annual rate of cognitive decline as measured using an extensive cognitive evaluation at both time points.ResultsThe deep gray matter atrophy rate did not differ between PMS and RRMS (−0.82%/year vs −0.71%/year, p = 0.11), while faster cortical atrophy was observed in PMS (−0.87%/year vs −0.48%/year, p < 0.01). Similarly, faster cognitive decline was observed in PMS compared to RRMS (p < 0.01). Annual cognitive decline was related to the rate of annual lesion volume increase in stable RRMS (r = −0.17, p = 0.03) to the rate of annual deep gray matter atrophy in converting RRMS (r = 0.50, p = 0.02) and annual cortical atrophy in PMS (r = 0.35, p = 0.01).ConclusionsThese results indicate that cortical atrophy and cognitive decline accelerate together during the course of MS. Substrates of cognitive decline shifted from worsening lesional pathology in stable RRMS to deep gray matter atrophy in converting RRMS and to accelerated cortical atrophy in PMS only.

Post-mortem in situ MRI has been used as an intermediate between brain histo(patho)logy and in vivo imaging. However, it is not known how comparable post-mortem in situ is to ante-mortem imaging. We report the unique situation of a patient with familial early-onset Alzheimer’s disease due to a PSEN1 mutation, who underwent ante-mortem brain MRI and post-mortem in situ imaging only 4 days apart. T1-weighted and diffusion MRI was performed at 3-Tesla at both time points. Visual atrophy rating scales, brain volume, cortical thickness and diffusion measures were derived from both scans and compared. Post-mortem visual atrophy scores decreased 0.5–1 point compared with ante-mortem, indicating an increase in brain volume. This was confirmed by quantitative analysis; showing a 27% decrease of ventricular and 7% increase of whole-brain volume. This increase was more pronounced in the cerebellum and supratentorial white matter than in grey matter. Furthermore, axial and radial diffusivity decreased up to 60% post-mortem whereas average fractional anisotropy of white matter increased approximately 10%. This unique case study shows that the process of dying affects several imaging markers. These changes need to be taken into account when interpreting post-mortem MRI to make inferences on the in vivo situation., Post-mortem brain imaging is used in many neurodegenerative disorders to validate imaging markers with immunohistopathology. This unique case study evaluates whether post-mortem brain imaging is a valid proxy for linking histopathology to the in vivo situation and demonstrates that MRI measurements significantly alter after death., Graphical Abstract Graphical Abstract

Cortical microstructural abnormalities are associated with clinical and cognitive deterioration in multiple sclerosis. Using diffusion tensor MRI, a higher fractional anisotropy has been found in cortical lesions versus normal-appearing cortex in multiple sclerosis. The pathological substrates of this finding have yet to be definitively elucidated. By performing a combined post-mortem diffusion tensor MRI and histopathology study, we aimed to define the histopathological substrates of diffusivity abnormalities in multiple sclerosis cortex. Sixteen subjects with multiple sclerosis and 10 age- and sex-matched non-neurological control donors underwent post-mortem in situ at 3 T MRI, followed by brain dissection. One hundred and ten paraffin-embedded tissue blocks (54 from multiple sclerosis patients, 56 from non-neurological controls) were matched to the diffusion tensor sequence to obtain regional diffusivity measures. Using immunohistochemistry and silver staining, cortical density of myelin, microglia, astrocytes and axons, and density and volume of neurons and glial cells were evaluated. Correlates of diffusivity abnormalities with histological markers were assessed through linear mixed-effects models. Cortical lesions (77% subpial) were found in 27/54 (50%) multiple sclerosis cortical regions. Multiple sclerosis normal-appearing cortex had a significantly lower fractional anisotropy compared to cortex from non-neurological controls (P = 0.047), whereas fractional anisotropy in demyelinated cortex was significantly higher than in multiple sclerosis normal-appearing cortex (P = 0.012) but not different from non-neurological control cortex (P = 0.420). Compared to non-neurological control cortex, both multiple sclerosis normal-appearing and demyelinated cortices showed a lower density of axons perpendicular to the cortical surface (P = 0.012 for both) and of total axons (parallel and perpendicular to cortical surface) (P = 0.028 and 0.012). In multiple sclerosis, demyelinated cortex had a lower density of myelin (P = 0.004), parallel (P = 0.018) and total axons (P = 0.029) versus normal-appearing cortex. Regarding the pathological substrate, in non-neurological controls, cortical fractional anisotropy was positively associated with density of perpendicular, parallel, and total axons (P = 0.031 for all). In multiple sclerosis, normal-appearing cortex fractional anisotropy was positively associated with perpendicular and total axon density (P = 0.031 for both), while associations with myelin, glial and total cells and parallel axons did not survive multiple comparison correction. Demyelinated cortex fractional anisotropy was positively associated with density of neurons, and total cells and negatively with microglia density, without surviving multiple comparison correction. Our results suggest that a reduction of perpendicular axons in normal-appearing cortex and of both perpendicular and parallel axons in demyelinated cortex may underlie the substrate influencing cortical microstructural coherence and being responsible for the different patterns of fractional anisotropy changes occurring in multiple sclerosis cortex.

Introduction Atrophy of the spinal cord is known to occur in multiple sclerosis (MS). The mean upper cervical cord area (MUCCA) can be used to measure this atrophy. Currently, several (semi-)automated methods for MUCCA measurement exist, but validation in clinical magnetic resonance (MR) images is lacking. Methods Five methods to measure MUCCA (SCT-PropSeg, SCT-DeepSeg, NeuroQLab, Xinapse JIM and ITK-SNAP) were investigated in a predefined upper cervical cord region. First, within-scanner reproducibility and between-scanner robustness were assessed using intra-class correlation coefficient (ICC) and Dice's similarity index (SI) in scan-rescan 3DT1-weighted images (brain, including cervical spine using a head coil) performed on three 3 T MR machines (GE MR750, Philips Ingenuity, Toshiba Vantage Titan) in 21 subjects with MS and 6 healthy controls (dataset A). Second, sensitivity of MUCCA measurement to lesions in the upper cervical cord was assessed with cervical 3D T1-weighted images (3 T GE HDxT using a head-neck-spine coil) in 7 subjects with MS without and 14 subjects with MS with cervical lesions (dataset B), using ICC and SI with manual reference segmentations. Results In dataset A, MUCCA differed between MR machines (p 0.176). However, there was an effect of method for both volumetric and voxel wise agreement of the segmentations (both p, Highlights • Mean upper cervical cord area (MUCCA) was obtained with five different methods. • MUCCA was determined in a unique scan-rescan multi-vendor MR study. • Reproducibility: MUCCA did not differ between scan-rescan images for any method. • Robustness: MUCCA differed between methods and between scanners. • Performance of MUCCA methods was not affected by the presence of lesions.

ObjectiveThe clinical course of multiple sclerosis (MS) is variable and largely unpredictable pointing to an urgent need for markers to monitor disease activity and progression. Recent evidence revealed that tissue transglutaminase (TG2) is altered in patient-derived monocytes. We hypothesize that blood cell–derived TG2 messenger RNA (mRNA) can potentially be used as biomarker in patients with MS.MethodsIn peripheral blood mononuclear cells (PBMCs) from 151 healthy controls and 161 patients with MS, TG2 mRNA was measured and correlated with clinical and MRI parameters of disease activity (annualized relapse rate, gadolinium-enhanced lesions, and T2 lesion volume) and disease progression (Expanded Disability Status Scale [EDSS], normalized brain volume, and hypointense T1 lesion volume).ResultsPBMC-derived TG2 mRNA levels were significantly associated with disease progression, i.e., worsening of the EDSS over 2 years of follow-up, normalized brain volume, and normalized gray and white matter volume in the total MS patient group at baseline. Of these, in patients with relapsing-remitting MS, TG2 expression was significantly associated with worsening of the EDSS scores over 2 years of follow-up. In the patients with primary progressive (PP) MS, TG2 mRNA levels were significantly associated with EDSS, normalized brain volume, and normalized gray and white matter volume at baseline. In addition, TG2 mRNA associated with T1 hypointense lesion volume in the patients with PP MS at baseline.ConclusionPBMC-derived TG2 mRNA levels hold promise as biomarker for disease progression in patients with MS.Classification of EvidenceThis study provides Class II evidence that in patients with MS, PBMC-derived TG2 mRNA levels are associated with disease progression.

Background: Body fluid and structural imaging biomarkers give information on neurodegeneration. The relationship over time is not known in multiple sclerosis. Objective: To investigate the temporal relationship of elevated cerebrospinal fluid (CSF) neurofilament (Nf) protein levels, a biomarker for axonal loss, with magnetic resonance imaging (MRI) atrophy measures. Methods: In patients with multiple sclerosis, CSF Nf heavy chain (NfH) phosphoform levels were quantified at baseline and dichotomised into ‘normal’ and ‘high’. Atrophy was assessed by MRI at baseline and 15-year follow-up using SIENAX and FreeSurfer software. Results: High baseline CSF NfH SMI35 levels predicted pronounced atrophy at 15-year follow-up (odds ratio (OR): 36, p 80% is reached with 14–50 patients. Conclusion: These data suggest that high CSF NfH levels are an early predictor of later brain and spinal cord atrophy using structural imaging biomarkers and can be investigated in reasonably sized patient cohorts.

Background: Cortical gray matter (GM) demyelination is frequent and clinically relevant in multiple sclerosis (MS). Quantitative magnetic resonance imaging (qMRI) sequences such as magnetization transfer ratio (MTR) and quantitative R2* (qR2*) can capture pathological subtleties missed by conventional magnetic resonance imaging (MRI) sequences. Although differences in MTR and qR2* have been reported between lesional and non-lesional tissue, differences between lesion types or lesion types and myelin density matched normal-appearing gray matter (NAGM) have not been found or investigated. Objective: Identify quantitative differences in histopathologically verified GM lesion types and matched NAGM at ultra-high field strength. Methods: Using 7T post-mortem MRI, MRI lesions were marked on T2 images and co-registered to the calculated MTR and qR2* maps for further evaluation. In all, 15 brain slices were collected, containing a total of 74 cortical GM lesions and 45 areas of NAGM. Results: Intracortical lesions had lower MTR and qR2* values compared to NAGM. Type I lesions showed lower MTR than type III lesions. Type III lesions showed lower MTR than matched NAGM, and type I and IV lesions showed lower qR2* than matched NAGM. Conclusion: qMRI at 7T can provide additional information on extent of cortical pathology, especially concerning subpial lesions. This may be relevant for monitoring disease progression and potential treatment effects.

BACKGROUND AND PURPOSE: White matter hyperintensities of presumed vascular origin in elderly patients with hypertension may be part of a general cerebral perfusion deficit, involving not only the white matter hyperintensities but also the surrounding normal-appearing white matter and gray matter. We aimed to study the relation between white matter hyperintensity volume and CBF and assess whether white matter hyperintensities are related to a general perfusion deficit. MATERIALS AND METHODS: In 185 participants of the Prevention of Dementia by Intensive Vascular Care trial between 72 and 80 years of age with systolic hypertension, white matter hyperintensity volume and CBF were derived from 3D FLAIR and arterial spin-labeling MR imaging, respectively. We compared white matter hyperintensity CBF, normal-appearing white matter CBF, and GM CBF across quartiles of white matter hyperintensity volume and assessed the continuous relation between these CBF estimates and white matter hyperintensity volume by using linear regression. RESULTS: Mean white matter hyperintensity CBF was markedly lower in higher quartiles of white matter hyperintensity volume, and white matter hyperintensity volume and white matter hyperintensity CBF were negatively related (standardized β = −0.248, P = .001) in linear regression. We found no difference in normal-appearing white matter or GM CBF across quartiles of white matter hyperintensity volume or any relation between white matter hyperintensity volume and normal-appearing white matter CBF (standardized β = −0.065, P = .643) or GM CBF (standardized β = −0.035, P = .382) in linear regression. CONCLUSIONS: Higher white matter hyperintensity volume in elderly individuals with hypertension was associated with lower perfusion within white matter hyperintensities, but not with lower perfusion in the surrounding normal-appearing white matter or GM. These findings suggest that white matter hyperintensities in elderly individuals with hypertension relate to local microvascular alterations rather than a general cerebral perfusion deficit.

BACKGROUND AND PURPOSE: Natalizumab treatment strongly affects relapsing-remitting multiple sclerosis, possibly by restraining white matter damage. This study investigated changes in white matter diffusivity in patients with relapsing-remitting multiple sclerosis during their first year of natalizumab treatment by using diffusion tensor imaging. MATERIALS AND METHODS: The study included patients with relapsing-remitting multiple sclerosis initiating natalizumab at baseline ( n = 22), patients with relapsing-remitting multiple sclerosis continuing interferon-β or glatiramer acetate ( n = 17), and healthy controls ( n = 12). Diffusion tensor imaging parameters were analyzed at baseline and month 12. We measured the extent and severity of white matter damage with diffusion tensor imaging parameters such as fractional anisotropy, comparing the patient groups with healthy controls at both time points. RESULTS: The extent and severity of white matter damage were reduced significantly in the natalizumab group with time (fractional anisotropy–based extent, 56.8% to 47.2%; severity, z = −0.67 to −0.59; P = .02); this reduction was not observed in the interferon-β/glatiramer acetate group (extent, 41.4% to 39.1%, and severity, z = −0.64 to −0.67; P = .94). Cognitive performance did not change with time in the patient groups but did correlate with the severity of damage ( r = 0.53, P = CONCLUSIONS: In patients with relapsing-remitting multiple sclerosis starting natalizumab treatment, the extent and severity of white matter damage were reduced significantly in the first year of treatment. These findings may aid in explaining the large observed clinical effect of natalizumab in relapsing-remitting multiple sclerosis.

Objective Abnormalities in segregative and integrative properties of brain networks have been observed in multiple sclerosis (MS) and are related to clinical functioning. This study aims to investigate the micro-scale correlates of macro-scale network measures of segregation and integration in MS. Methods Eight MS patients underwent post-mortem in situ whole-brain diffusion tensor (DT) imaging and subsequent brain dissection. Macro-scale structural network topology was derived from DT data using graph theory. Clustering coefficient and mean white matter (WM) fiber length were measures of nodal segregation and integration. Thirty-three tissue blocks were collected from five cortical brain regions. Using immunohistochemistry micro-scale tissue properties were evaluated, including, neuronal size, neuronal density, axonal density and total cell density. Nodal network properties and tissue properties were correlated. Results A negative correlation between clustering coefficient and WM fiber length was found. Higher clustering coefficient was associated with smaller neuronal size and lower axonal density, and vice versa for fiber length. Higher whole-brain WM lesion load was associated with higher whole-brain clustering, shorter whole-brain fiber length, lower neuronal size and axonal density. Conclusion Structural network properties on MRI associate with neuronal size and axonal density, suggesting that macro-scale network measures may grasp cortical neuroaxonal degeneration in MS. Electronic supplementary material The online version of this article (10.1007/s00415-018-9130-2) contains supplementary material, which is available to authorized users.

Background/objective: Endurance exercise can improve memory function in persons with multiple sclerosis (pwMS), but the effects on hippocampal functioning are currently unknown. We investigated the effects of a running intervention on memory and hippocampal functional connectivity in pwMS. Methods/results: Memory and resting-state functional magnetic resonance imaging (fMRI) data were collected in a running intervention (n = 15) and waitlist group (n = 14). Visuospatial memory improvement was correlated to increased connectivity between the hippocampus and the default-mode network (DMN) in the intervention group only. Conclusion: As a result of endurance exercise, improvements in visuospatial memory may be mediated by a stronger functional embedding of the hippocampus in the DMN. The non-for-profit organization Move to Sport (www.movetosport.be) initiated the study. The authors acknowledge Prof. Dr P. Parizel (UZA Antwerp) for facilitation of neuroimaging at UZA Wilrijk and Novartis and the MS Network Limburg for funding related operational costs.

Post-mortem in situ MRI has been used as an intermediate between brain histo(patho)logy and in vivo imaging. However, it is not known how comparable post-mortem in situ is to ante-mortem imaging. We report the unique situation of a patient with familial early-onset Alzheimer’s disease due to a PSEN1 mutation, who underwent ante-mortem brain MRI and post-mortem in situ imaging only 4 days apart. T1-weighted and diffusion MRI was performed at 3-Tesla at both time points. Visual atrophy rating scales, brain volume, cortical thickness and diffusion measures were derived from both scans and compared. Post-mortem visual atrophy scores decreased 0.5–1 point compared with ante-mortem, indicating an increase in brain volume. This was confirmed by quantitative analysis; showing a 27% decrease of ventricular and 7% increase of whole-brain volume. This increase was more pronounced in the cerebellum and supratentorial white matter than in grey matter. Furthermore, axial and radial diffusivity decreased up to 60% post-mortem whereas average fractional anisotropy of white matter increased approximately 10%. This unique case study shows that the process of dying affects several imaging markers. These changes need to be taken into account when interpreting post-mortem MRI to make inferences on the in vivo situation.

Background The aim of the present study was to determine trajectories of cognitive and cortical changes over time in middle‐aged patients with type 1 diabetes mellitus (T1DM) and proliferative retinopathy. Methods Twenty‐five patients and 25 controls underwent neuropsychological assessment and neuroimaging twice in a mean (±SD) of 3.56 ± 0.65 and 3.94 ± 0.91 years, respectively (P = 0.098). Cognitive assessment included the domains of general cognitive ability, memory, information processing speed, executive functions, attention, and motor and psychomotor speed. Symmetrized percentage change in local cortical thickness, surface area, and volume was determined using the FreeSurfer 6 vertex‐wise general linear model method. Analyses were performed uncorrected and corrected for baseline systolic blood pressure and depressive symptoms. Results In patients versus controls, accelerated executive function decline was accompanied by, but not related to, lower left frontal and temporal surface area, left parietal and right frontal thickness, and bilateral frontal and right posterior cingulate volume (family‐wise error [FWE]‐corrected P

Background: The biological mechanism of depression in multiple sclerosis (MS) is not well understood. Based on work in major depressive disorder, fronto-limbic disconnection might be important. Objective: To investigate structural and functional fronto-limbic changes in depressed MS (DMS) and non-depressed MS (nDMS) patients. Methods: In this retrospective study, 22 moderate-to-severe DMS patients (disease duration 8.2 ± 7.7 years), 21 nDMS patients (disease duration 15.3 ± 8.3 years), and 12 healthy controls underwent neuropsychological testing and magnetic resonance imaging (MRI; 1.5 T). Brain volumes (white matter (WM), gray matter, amygdala, hippocampus, thalamus), lesion load, fractional anisotropy (FA) of fronto-limbic tracts, and resting-state functional connectivity (FC) between limbic and frontal areas were measured and compared between groups. Regression analysis was performed to relate MRI measures to the severity of depression. Results: Compared to nDMS patients, DMS patients (shorter disease duration) had lower WM volume ( p Conclusion: DMS patients showed more pronounced (MS) damage, that is, structural and functional changes in temporo-frontal regions, compared to nDMS patients, suggestive of fronto-limbic disconnection.

Grey matter atrophy is common in multiple sclerosis. However, in contrast with other neurodegenerative diseases, it is unclear whether grey matter atrophy in multiple sclerosis is a diffuse ‘global’ process or develops, instead, according to distinct anatomical patterns. Using source-based morphometry we searched for anatomical patterns of co-varying cortical thickness and assessed their relationships with white matter pathology, physical disability and cognitive functioning. Magnetic resonance imaging was performed at 3 T in 208 patients with long-standing multiple sclerosis (141 females; age = 53.7 ± 9.6 years; disease duration = 20.2 ± 7.1 years) and 60 age- and sex-matched healthy controls. Spatial independent component analysis was performed on cortical thickness maps derived from 3D T 1 -weighted images across all subjects to identify co-varying patterns. The loadings, which reflect the presence of each cortical thickness pattern in a subject, were compared between patients with multiple sclerosis and healthy controls with generalized linear models. Stepwise linear regression analyses were used to assess whether white matter pathology was associated with these loadings and to identify the cortical thickness patterns that predict measures of physical and cognitive dysfunction. Ten cortical thickness patterns were identified, of which six had significantly lower loadings in patients with multiple sclerosis than in controls: the largest loading differences corresponded to the pattern predominantly involving the bilateral temporal pole and entorhinal cortex, and the pattern involving the bilateral posterior cingulate cortex. In patients with multiple sclerosis, overall white matter lesion load was negatively associated with the loadings of these two patterns. The final model for physical dysfunction as measured with Expanded Disability Status Scale score (adjusted R 2 = 0.297; P < 0.001) included the predictors age, overall white matter lesion load, the loadings of two cortical thickness patterns (bilateral sensorimotor cortex and bilateral insula), and global cortical thickness. The final model predicting average cognition (adjusted R 2 = 0.469; P < 0.001) consisted of age, the loadings of two cortical thickness patterns (bilateral posterior cingulate cortex and bilateral temporal pole), overall white matter lesion load and normal-appearing white matter integrity. Although white matter pathology measures were part of the final clinical regression models, they explained limited incremental variance (to a maximum of 4%). Several cortical atrophy patterns relevant for multiple sclerosis were found. This suggests that cortical atrophy in multiple sclerosis occurs largely in a non-random manner and develops (at least partly) according to distinct anatomical patterns. In addition, these cortical atrophy patterns showed stronger associations with clinical (especially cognitive) dysfunction than global cortical atrophy.

BACKGROUND AND PURPOSE: The considerable clinical effect of natalizumab in patients with relapsing-remitting multiple sclerosis might be explained by its possible beneficial effect on axonal functioning. In this longitudinal study, the effect of natalizumab on absolute concentrations of total N-acetylaspartate, a marker for neuronal integrity, and other brain metabolites is investigated in patients with relapsing-remitting multiple sclerosis by using MR spectroscopic imaging. MATERIALS AND METHODS: In this explorative observational study, 25 patients with relapsing-remitting multiple sclerosis initiating natalizumab treatment were included and scanned every 6 months for 18 months. Additionally 18 matched patients with relapsing-remitting multiple sclerosis continuing treatment with interferon-β or glatiramer acetate were included along with 12 healthy controls. Imaging included short TE 2D-MR spectroscopic imaging with absolute metabolite quantification of total N-acetylaspartate, creatine and phosphocreatine, choline-containing compounds, myo-inositol, and glutamate. Concentrations were determined for lesional white matter, normal-appearing white matter, and gray matter. RESULTS: At baseline in both patient groups, lower concentrations of total N-acetylaspartate and creatine and phosphocreatine were found in lesional white matter compared with normal-appearing white matter and additionally lower glutamate in lesional white matter of patients receiving natalizumab. In those patients, a significant yearly metabolite increase was found for lesional white matter total N-acetylaspartate (7%, P < .001), creatine and phosphocreatine (6%, P = .042), and glutamate (10%, P = .028), while lesion volumes did not change. In patients receiving interferon-β/glatiramer acetate, no significant change was measured in lesional white matter for any metabolite, while whole-brain normalized lesion volumes increased. CONCLUSIONS: Patients treated with natalizumab showed an increase in total N-acetylaspartate, creatine and phosphocreatine, and glutamate in lesional white matter. These increasing metabolite concentrations might be a sign of enhanced axonal metabolism.