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Highlights • Leptomeningeal enhancement (LME) is a nonspecific imaging feature. • LME can be present in neoplastic, infectious, and primary neuroinflammatory diseases. • The presence of LME is associated with worse outcome measures in multiple sclerosis. • We recommend the ascertainment of LME in clinical practice. • Neuroinflammatory animal models can be used to investigate the pathophysiology of LME., Background The lack of systematic evidence on leptomeningeal enhancement (LME) on MRI in neurological diseases, including multiple sclerosis (MS), hampers its interpretation in clinical routine and research settings. Purpose To perform a systematic review and meta-analysis of MRI LME in MS and other neurological diseases. Materials and Methods In a comprehensive literature search in Medline, Scopus, and Embase, out of 2292 publications, 459 records assessing LME in neurological diseases were eligible for qualitative synthesis. Of these, 135 were included in a random-effects model meta-analysis with subgroup analyses for MS. Results Of eligible publications, 161 investigated LME in neoplastic neurological (n = 2392), 91 in neuroinfectious (n = 1890), and 75 in primary neuroinflammatory diseases (n = 4038). The LME-proportions for these disease classes were 0.47 [95%-CI: 0.37–0.57], 0.59 [95%-CI: 0.47–0.69], and 0.26 [95%-CI: 0.20–0.35], respectively. In a subgroup analysis comprising 1605 MS cases, LME proportion was 0.30 [95%-CI 0.21–0.42] with lower proportions in relapsing-remitting (0.19 [95%-CI 0.13–0.27]) compared to progressive MS (0.39 [95%-CI 0.30–0.49], p = 0.002) and higher proportions in studies imaging at 7 T (0.79 [95%-CI 0.64–0.89]) compared to lower field strengths (0.21 [95%-CI 0.15–0.29], p

As the news of approval of COVID-19 vaccination emerge, neurologists across the globe ponder upon whether to use immunotherapies in patients with Multiple Sclerosis (MS). This paper highlights the mechanism of various disease modifying therapies (DMTs) as well as the recently approved Pfizer and Moderna vaccines for COVID-19 as well as guidelines as introduced by National Multiple Sclerosis Society. As their mechanisms counteract each other at the molecular level, we believe further evidence and data might lay the foundation to formulate much needed recommendations for the usage of these medications while vaccinating MS patients on DMTs.

Background Several concerns exist on the immunogenicity of SARS-CoV-2 vaccines in multiple sclerosis (MS) subjects due to their immunomodulating disease modifying therapies (DMTs). Here we report a comparison of the humoral response to BNT162b2-mRNA coronavirus (COVID)-19 vaccine and the immunological phenotype in a cohort of 125 MS subjects undergoing different DMTs, with no history of SARS-CoV-2 infection. Methods We collected serum and blood samples at the first day of vaccine (T0) and 21 days after the second vaccine dose (T1) from 125 MS subjects, undergoing eight different DMTs. Sera were tested using the Elecsys anti-SARS-CoV-2-IgG assay for the detection of IgG antibodies to SARS-CoV-2 spike protein. The anti-spike IgG titres from MS subjects were compared with 24 age- and sex-matched healthy controls (HC). Percentage and absolute number of B and T lymphocytes were evaluated by cytofluorimetric analysis in the same study cohort. Results When compared with SARS-CoV-2 IgG levels in HC (n = 24, median 1089 (IQR 652.5–1625) U/mL), we observed an increased secretion of SARS-CoV-2 IgG in interferon-beta 1a (IFN)-treated MS subjects (n = 22, median 1916 (IQR 1024–2879) U/mL) and an impaired humoral response in MS subjects undergoing cladribine (CLAD) (n = 10, median 396.9 (IQR 37.52–790.9) U/mL), fingolimod (FTY) (n = 19, median 7.9 (IQR 4.8–147.6) U/mL) and ocrelizumab (OCRE) (n = 15, median 0.67 (IQR 0.4–5.9) U/mL) treatment. Moreover, analysis of geometric mean titre ratio (GMTR) between different DMT's groups of MS subjects revealed that, when compared with IFN-treated MS subjects, intrinsic antibody production was impaired in teriflunomide (TERI)-, natalizumab (NAT)-, CLAD-, FTY- and OCRE-, while preserved in DMF- and GA-treated MS subjects. Conclusion Humoral response to BNT162b2-mRNA-vaccine was increased in IFN-treated MS subjects while clearly blunted in those under CLAD, FTY and OCRE treatment. This suggests that the DMTs could have a key role in the protection from SARS-CoV-2 related disease and complication in MS subjects, underlying a novel aspect that should be considered in the selection of the most appropriate therapy under COVID-19 pandemic.

Highlights • 7 T MRI improves cortical lesion detection compared to 3 T MRI. • Type II-IV intracortical lesion detection benefit most from 7 T MRI. • Progressive multiple sclerosis patients have four times more cortical lesions. • 7 T MRI has modest sensitivity towards cortical lesions compared with histopathology. • Guidelines for image acquisition and cortical lesion segmentation at 7 T are missing., Background Cortical lesions are abundant in multiple sclerosis (MS), yet difficult to visualize in vivo. Ultra-high field (UHF) MRI at 7 T and above provides technological advances suited to optimize the detection of cortical lesions in MS. Purpose To provide a narrative and quantitative systematic review of the literature on UHF MRI of cortical lesions in MS. Methods A systematic search of all literature on UHF MRI of cortical lesions in MS published before September 2020. Quantitative outcome measures included cortical lesion numbers reported using 3 T and 7 T MRI and between 7 T MRI sequences, along with sensitivity of UHF MRI towards cortical lesions verified by histopathology. Results 7 T MRI detected on average 52 ± 26% (mean ± 95% confidence interval) more cortical lesions than the best performing image contrast at 3 T, with the largest increase in type II-IV intracortical lesion detection. Across all studies, the mean cortical lesion number was 17 ± 6 per patient. In progressive MS cohorts, approximately four times more cortical lesions were reported than in CIS/early RRMS, and RRMS. Yet, there was no difference in lesion type ratio between these MS subtypes. Furthermore, superiority of one MRI sequence over another could not be established from available data. Post-mortem lesion detection with UHF MRI agreed only modestly with pathological examinations. Mean pro- and retrospective sensitivity was 33 ± 6% and 71 ± 10%, respectively, with the highest sensitivity towards type I and type IV lesions. Conclusion UHF MRI improves cortical lesion detection in MS considerably compared to 3 T MRI, particularly for type II-IV lesions. Despite modest sensitivity, 7 T MRI is still capable of visualizing all aspects of cortical lesion pathology and could potentially aid clinicians in diagnosing and monitoring MS, and progressive MS in particular. However, standardization of acquisition and segmentation protocols is needed.

Highlights • Impaired eye movements in multiple sclerosis (MS) and functional connectivity (FC) • Eye movements related to altered FC of the oculomotor brain network. • Lower (beta band) and higher (theta/delta band) FC related to abnormal eye movements. • Regional changes were more informative than whole-network measures. • Eye movement parameters also related to disability and cognitive dysfunction., Background Impaired eye movements in multiple sclerosis (MS) are common and could represent a non-invasive and accurate measure of (dys)functioning of interconnected areas within the complex brain network. The aim of this study was to test whether altered saccadic eye movements are related to changes in functional connectivity (FC) in patients with MS. Methods Cross-sectional eye movement (pro-saccades and anti-saccades) and magnetoencephalography (MEG) data from the Amsterdam MS cohort were included from 176 MS patients and 33 healthy controls. FC was calculated between all regions of the Brainnetome atlas in six conventional frequency bands. Cognitive function and disability were evaluated by previously validated measures. The relationships between saccadic parameters and both FC and clinical scores in MS patients were analysed using multivariate linear regression models. Results In MS pro- and anti-saccades were abnormal compared to healthy controls A relationship of saccadic eye movements was found with FC of the oculomotor network, which was stronger for regional than global FC. In general, abnormal eye movements were related to higher delta and theta FC but lower beta FC. Strongest associations were found for pro-saccadic latency and FC of the precuneus (beta band β = -0.23, p = .006), peak velocity and FC of the parietal eye field (theta band β = -0.25, p = .005) and gain and FC of the inferior frontal eye field (theta band β = -0.25, p = .003). Pro-saccadic latency was also strongly associated with disability scores and cognitive dysfunction. Conclusions Impaired saccadic eye movements were related to functional connectivity of the oculomotor network and clinical performance in MS. This study also showed that, in addition to global network connectivity, studying regional changes in MEG studies could yield stronger correlations.

Highlights • Thalamus atrophy is associated with cognitive impairment in multiple sclerosis. • This was confirmed by automated and manual segmentations, but effect sizes varied. • The algorithms work in a multi-center setting. • Automated techniques exhibit proportional bias with respect to thalamus size. • Differences between vendors can affect the robustness of these associations., Background and rationale Thalamus atrophy has been linked to cognitive decline in multiple sclerosis (MS) using various segmentation methods. We investigated the consistency of the association between thalamus volume and cognition in MS for two common automated segmentation approaches, as well as fully manual outlining. Methods Standardized neuropsychological assessment and 3-Tesla 3D-T1-weighted brain MRI were collected (multi-center) from 57 MS patients and 17 healthy controls. Thalamus segmentations were generated manually and using five automated methods. Agreement between the algorithms and manual outlines was assessed with Bland-Altman plots; linear regression assessed the presence of proportional bias. The effect of segmentation method on the separation of cognitively impaired (CI) and preserved (CP) patients was investigated through Generalized Estimating Equations; associations with cognitive measures were investigated using linear mixed models, for each method and vendor. Results In smaller thalami, automated methods systematically overestimated volumes compared to manual segmentations [ρ=(-0.42)-(-0.76); p-values

Highlights • LGN correlates with structural markers of the anterior and posterior visual pathway. • LGN volume may reduce after an episode of ON, shown in four patients. • LGN volume does not change over time in the absence of ON episodes. • Our findings argue against occult neurodegeneration in the visual pathway in NMO., Objectives Lateral geniculate nucleus (LGN) volume is reduced after optic neuritis (ON) in neuromyelitis optica spectrum disorders (NMOSD). We aimed at a longitudinal assessment of LGN volume in NMOSD. Methods Twenty-nine patients with aquaporin 4-IgG seropositive NMOSD (age: 47.8 ± 14.6 years (y), female: n = 27, history of ON (NMO-ON): n = 17, median time since ON: 3[1.2–12.1]y) and 18 healthy controls (HC; age: 39.3 ± 15.8y; female: n = 13) were included. Median follow-up was 4.1[1.1–4.7]y for patients and 1.7[0.9–3.2]y for HC. LGN volume was measured using a multi-atlas-based approach of automated segmentation on 3 Tesla magnetic resonance images. Retinal optical coherence tomography and probabilistic tractography of the optic radiations (OR) were also performed. Results At baseline, NMO-ON patients had lower LGN volumes (395.4 ± 48.9 mm3) than patients without ON (NMO-NON: 450.7 ± 55.6 mm3; p = 0.049) and HC (444.5 ± 61.5 mm3, p = 0.025). LGN volume was associated with retinal neuroaxonal loss and microstructural OR damage. Longitudinally, there was no change in LGN volumes in the absence of ON, neither in all patients (B = −0.6, SE = 1.4, p = 0.670), nor in NMO-ON (B = −0.8, SE = 1.6, p = 0.617) and NMO-NON (B = 1.7, SE = 3.5, p = 0.650). However, in four patients with new ON during follow-up, LGN volume was reduced at last visit (median time since ON: 2.6 [1.8–3.9] y) compared to the measurement before ON (352 ± 52.7 vs. 371.1 ± 55.9 mm3; t = −3.6, p = 0.036). Conclusion Although LGN volume is reduced after ON in NMOSD, this volume loss is not progressive over longer follow-up or independent of ON. Thus, our findings -at least in this relatively small cohort- do not support occult neurodegeneration of the afferent visual pathway in NMOSD.

Objectives This study reports on the feasibility of the SitLess with MS trial, an intervention targeting sedentary behavior in individuals with multiple sclerosis (MS). Design Single group, pre-post intervention design. Setting Community. Participants Participants (N=41) with mild to moderate disability from MS. Intervention The intervention was 15 weeks, with a 7-week follow-up, and included 2 stages: SitLess and MoveMore. During the SitLess stage, participants were encouraged to break up prolonged sitting bouts over a 7-week period, whereas the MoveMore stage promoted increased steps per day and interrupting sitting over a 7-week period. The intervention was delivered through weekly one-on-one coaching sessions via telerehabilitation and an accompanying newsletter based on social-cognitive theory. Activity was monitored throughout the program using a Fitbit. Main Outcome Measures Process (eg, recruitment) and resource and management (eg, personnel requirements) metrics were assessed, along with efficacy outcomes (eg, effect). Progression criteria were set a priori and were related to safety, fatigue, satisfaction, and attrition. Sedentary behavior, measured using the ActivPal, was reported pre- and postintervention, as well as 7 weeks postintervention. Effect sizes (pre to post, pre to 7 weeks post) were calculated for the sedentary behavior outcomes (eg, time sitting, transitions from sitting to standing, number of long sitting bouts). Experiences with the intervention were explored through an online survey. Results Forty-one participants enrolled, 39 of whom completed the intervention. All participants but 1 were satisfied with the experience. Pre-post intervention effect sizes for change in total sedentary time, number of transitions from sit to stand, and number of long (>30 min) sedentary bouts were 0.34, 0.02, and 0.39 respectively. All a priori progression criteria were met. Conclusions The SitLess with MS program, a novel intervention that emphasized and facilitated sitting less and moving more, was feasible and resulted in small changes in sedentary behavior in individuals with MS.

Objective To examine the safety, feasibility, and response to functional electrical stimulation (FES) cycling protocols requiring differing levels of effort in people with multiple sclerosis (MS) who are nonambulatory. Design Pilot study with pre-post intervention testing. Setting Outpatient clinic setting of a long-term acute care hospital. Participants Individuals (N=10) with MS (6 men; mean age 58.6±9.86y) who use a wheelchair for community mobility. Participants’ Expanded Disability Status Scale score ranged from 6.5 to 8.5 (median 7.5). Intervention Participants performed 3 or 4 FES cycling protocols requiring different levels of volitional effort during 6-8 testing sessions. Main Outcome Measures The primary outcome was safety, measured by adverse events and increase in MS symptoms, all assessed throughout, immediately post- and 1 day postsession. FES cycling performance for each protocol was also recorded. Exploratory outcome measures collected before and after all testing sessions included functional assessment of MS, MS Impact Scale, Exercise Self Efficacy Scale, Patient Health Questionnaire-9 item, and the Zarit Caregiver Burden Scale. Results All participants (4 women, 6 men) completed all testing sessions. There were no serious adverse events or differences in vitals or symptoms between protocols. Two participants had an isolated episode of mild hypotension. Changes in pain, spasticity, and fatigue were minimal. Five participants were able to cycle for 30 minutes and completed interval training protocols requiring increasing difficulty. The remainder cycled for, Highlights • People with multiple sclerosis who are nonambulatory can safely perform functional electrical stimulation (FES) cycling using parameters requiring more effort. • FES cycling may offer an opportunity for increased exercise stimulus or neuromuscular training if individuals are adequately challenged using an appropriate cycling protocol.

Highlights • T1ρ and RAFF4 - MRI protocols sensitive to slow motional regimes – are able to detect changes in T2w-defined normally appearing white matter of patients with multiple sclerosis. • RAFF4, T1ρ and T2ρ showed differences in hippocampus in patients with multiple sclerosis despite the absence of alterations in resting-state functional MRI metrics. • Hence, RAFF4, T1ρ and T2ρ hold promise as potential non-invasive tools for monitoring MS activity and eventually for the evaluation of therapeutic effects., Even though MRI visualization of white matter lesions is pivotal for the diagnosis and management of multiple sclerosis (MS), the issue of detecting diffuse brain tissue damage beyond the apparent T2-hyperintense lesions continues to spark considerable interest. Motivated by the notion that rotating frame MRI methods are sensitive to slow motional regimes critical for tissue characterization, here we utilized novel imaging protocols of rotating frame MRI on a clinical 3 Tesla platform, including adiabatic longitudinal, T1ρ, and transverse, T2ρ, relaxation methods, and Relaxation Along a Fictitious Field (RAFF) in the rotating frame of rank 4 (RAFF4), in 10 relapsing-remitting multiple sclerosis patients and 10 sex- and age-matched healthy controls. T1ρ, T2ρ and RAFF4 relaxograms extracted from the whole white matter exhibited a significant shift towards longer relaxation time constants in MS patients as compared to controls. T1ρ and RAFF4 detected alterations even when considering only regions of normally appearing white matter (NAWM), while other MRI metrics such as T1w/T2w ratio and diffusion tensor imaging measures failed to find group differences. In addition, RAFF4, T2ρ and, to a lesser extent, T1ρ showed differences in subcortical grey matter structures, mainly hippocampus, whereas no functional changes in this region were detected in resting-state functional MRI metrics. We conclude that rotating frame MRI techniques are exceptionally sensitive methods for the detection of subtle abnormalities not only in NAWM, but also in deep grey matter in MS, where they surpass even highly sensitive measures of functional changes, which are often suggested to precede detectable structural alterations. Such abnormalities are consistent with a wide spectrum of different, but interconnected pathological features of MS, including the loss of neuronal cells and their axons, decreased levels of myelin even in NAWM, and altered iron content.

Background multiple sclerosis (MS) patients are treated with immunomodulatory treatments that can influence their ability to develop a protective antibody response to the SARS-CoV-2 vaccine. Vaccine efficacy is important for treatment decision and for patients’ reassurance. The main objective is to assess antibody response to SARS-CoV-2 vaccine in MS patients treated with cladribine. Methods Serology response was tested in 97 participants, 67 MS patients and 30 healthy controls (HCs), using two independent methods, 2–3 weeks following the second dose of the BNT162b2 vaccine. Results HCs (n = 30) and MS patients treated with cladribine (n = 32) had 100% positive serology response against the SARS-CoV-2 spike protein following the second vaccine dose (mean S1/S2-IgG and RBD-IgG:284.5 ± 104.9, 13,041±9411 AU/mL and 226.3 ± 121.4, 10,554±11,405 AU/mL respectively). Comparable findings were observed for untreated MS patients, and interferon beta-1a-treated MS patients (mean S1/S2-IgG: 282.1 ± 100.1, 276.9 ± 94.31 AU/mL respectively). No correlation was found between lymphocyte counts, treatment duration, or time between cladribine dose and vaccination, and serology response or antibody titers. Conclusion and relevance Cladribine treated MS patients are able to produce antibodies to the SARS-CoV-2 mRNA vaccine. In the era of the COVID-19 pandemic, it is reassuring and important for both patients and physicians and will allow to develop consensus guidelines.

Objective To investigate the rich-club organization in clinically isolated syndrome (CIS) and multiple sclerosis (MS), and to characterize its relationships with physical disabilities and cognitive impairments. Methods We constructed high-resolution white matter (WM) structural networks in 41 CIS, 32 MS and 35 healthy controls (HCs) using diffusion MRI and deterministic tractography. Group differences in rich-club organization, global and local network metrics were investigated. The relationship between the altered network metrics, brain lesions and clinical variables including EDSS, MMSE, PASAT, disease duration were calculated. Additionally, reproducibility analysis was performed using different parcellation schemes. Results Compared with HCs, MS patients exhibited a decreased strength in all types of connections (rich-club: p, Highlights • The rich-club organization was disrupted in MS patients and preserved in CIS. • The disrupted rich-club connectivity correlated with cognitive impairment in MS. • The rich-club results are reproducible across data analysis methods.

Background Only recently has the first disease-modifying therapy been approved for children with multiple sclerosis (MS) and practice patterns including substantial off-label use have evolved. Understanding attitudes towards treatment of paediatric MS and whether this has changed due to the ongoing COVID-19 pandemic is vital to guide future therapeutic trials and for developing guidelines that reflect practice. Methods We performed an online survey within the International Paediatric Multiple Sclerosis Study Group between July and September 2020. The survey was sent to 130 members from 25 countries and consisted of five sections: demographic data, treatment, disease modifying therapies and COVID-19, outcome and three patient cases. Results The survey was completed by 66 members (51%), both paediatric neurologists and adult neurologists. Fingolimod and β-interferons were the most frequently used disease-modifying therapies, especially among paediatric neurologists. Almost a third (31%) of respondents had altered their prescribing practice due to COVID-19, in particular at the beginning of the pandemic. Conclusions The survey results indicate a tendency of moving from the traditional escalation therapy starting with injectables towards an early start with newer, highly effective disease modifying therapies. The COVID-19 pandemic only slightly affected prescribing patterns and treatment choices in paediatric MS.

Highlights • COVID-19 severity was in line with that of the general population and MS datasets • Most patients did not require hospitalization • There was no association between duration of exposure to ocrelizumab and COVID-19 • Patients with known COVID-19 risk factors had more severe COVID-19 • Case fatality rates were comparable with other MS cohorts, Background : There are limited data on the impact of coronavirus disease (COVID-19) caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) on people with multiple sclerosis (MS). Objective : To better understand SARS-CoV-2 infection in ocrelizumab-treated people with MS. Methods :Internal Roche/Genentech data sources: Cases of COVID-19 from ongoing Roche/Genentech clinical trials and from post-marketing use of ocrelizumab until July 31, 2020 were identified and assessed using descriptive statistics. External real-world data (RWD) source: An MS COVID-19 cohort and an ocrelizumab-treated MS COVID-19 cohort were identified and assessed from the OPTUM® de-identified COVID-19 Electronic Health Record (EHR) database. Results :Roche/Genentech clinical trial data: There were 51 (1.3%) suspected or confirmed cases of COVID-19 identified from 4,000 patients ongoing in nine Roche/Genentech clinical trials. Of these, 26 (51%) were confirmed COVID-19 and 25 (49%) were suspected COVID-19. Sixteen (31.4%) patients were hospitalized. COVID-19 severity was mild-to-moderate in most patients (35, 68.6%). Ten (19.6%) patients had severe disease and there were three (5.9%) fatal cases. Most patients (43, 84.3%) recovered or were recovering. There was no association apparent between duration of exposure to ocrelizumab and COVID-19. Among COVID-19 patients with previous serum immunoglobulin status (27/51, 52.9%), all (27/27, 100%) had IgG levels within the normal range. Roche/Genentech post-marketing safety database data: There were 307 post-marketing cases of COVID-19 in the Roche/Genentech global safety database. Of these, 263 (85.7%) were confirmed and 44 (14.3%) were suspected COVID-19. 100 (32.6%) patients were hospitalized. COVID-19 was asymptomatic, mild or moderate in 143 (46.6%) patients, severe in 52 (16.9%) patients, and critical in 15 (4.9%) patients. There were 17 (5.5%) fatal cases. Information on severity was not reported in 80 (26.1%) cases. Most patients (211, 68.7%) recovered or were recovering at the time of the report. External RWD data source: As of July 13, 2020, the OPTUM® database included EHRs for almost 1.2 million patients with suspected COVID-19, 130,500 of whom met the criteria for confirmed/clinically diagnosed COVID-19. A total of 357 patients with MS with confirmed COVID-19 were identified. Forty-eight (13.4%) were treated with ocrelizumab, of whom 12 (25.0%) were hospitalized and one died (2.1%). Similar rates of hospitalization, invasive ventilation, and death were observed in the ocrelizumab-treated and non-ocrelizumab-treated MS cohorts. Across the Roche/Genentech and RWD sources assessed, age, male sex, and the presence of comorbidities such as hypertension were associated with a more severe disease course of COVID-19. There was a higher number of comorbidities present in hospitalized versus non-hospitalized patients. Conclusions : This assessment provides evidence that COVID-19 in ocrelizumab-treated people with MS is predominantly mild to moderate in severity with most patients not requiring hospitalization; in line with data reported from the general population and MS datasets. Risk factors known to be associated with severe COVID-19 outcomes in the general population also appear to influence COVID-19 severity in ocrelizumab-treated people with MS. Case fatality rates for ocrelizumab-treated people with MS were within published ranges for the general population and other MS cohorts.

Highlights • Central and whole brain atrophy are faster in MS patients with disability progression. • These measures can be reliably assessed on clinically-available FLAIR images. • They are meaningful even with longitudinal scanner and field strength changes., Background Greater brain atrophy is associated with disability progression (DP) in patients with multiple sclerosis (PwMS). However, methodological challenges limit its routine clinical use. Objective To determine the feasibility of atrophy measures as markers of DP in PwMS scanned across different MRI field strengths. Methods A total of 980 PwMS were scanned on either 1.5 T or 3.0 T MRI scanners. Demographic and clinical data were retrospectively collected, and the presence of DP was determined according to standard clinical trial criteria. Lateral ventricular volume (LVV) change was measured with the NeuroSTREAM technique on clinical routine T2-FLAIR images. Percent brain volume change (PBVC) was measured using SIENA and ventricular cerebrospinal fluid (vCSF) % change was measured using VIENA and SIENAX algorithms on 3D T1-weighted images (WI). Stable vs. DP PwMS were compared using analysis of covariance (ANCOVA). Mixed modeling determined the effect of MRI scanner change on MRI-derived atrophy measures. Results Longitudinal LVV analysis was successful in all PwMS. SIENA-based PBVC and VIENA-based changes failed in 37.6% of cases, while SIENAX-based vCSF failed in 12.9% of cases. PwMS with DP (n = 241) had significantly greater absolute (20.9% vs. 8.7%, d = 0.66, p

Highlights • Macroscopic and microscopic diffusion properties discriminate between MS lesion types. • The number and volume of lesions with larger diffusion changes are associated with worse clinical outcomes. • Diffusion MRI provides useful information of the pathological heterogeneity in plaques., Diffusion magnetic resonance imaging can reveal quantitative information about the tissue changes in multiple sclerosis. The recently developed multi-compartment spherical mean technique can map different microscopic properties based only on local diffusion signals, and it may provide specific information on the underlying microstructural modifications that arise in multiple sclerosis. Given that the lesions in multiple sclerosis may reflect different degrees of damage, we hypothesized that quantitative diffusion maps may help characterize the severity of lesions “in vivo” and correlate these to an individual’s clinical profile. We evaluated this in a cohort of 59 multiple sclerosis patients (62% female, mean age 44.7 years), for whom demographic and disease information was obtained, and who underwent a comprehensive physical and cognitive evaluation. The magnetic resonance imaging protocol included conventional sequences to define focal lesions, and multi-shell diffusion imaging was used with b-values of 1000, 2000 and 3000 s/mm2 in 180 encoding directions. Quantitative diffusion properties on a macro- and micro-scale were used to discriminate distinct types of lesions through a k-means clustering algorithm, and the number and volume of those lesion types were correlated with parameters of the disease. The combination of diffusion tensor imaging metrics (fractional anisotropy and radial diffusivity) and multi-compartment spherical mean technique values (microscopic fractional anisotropy and intra-neurite volume fraction) differentiated two type of lesions, with a prediction strength of 0.931. The B-type lesions had larger diffusion changes compared to the A-type lesions, irrespective of their location (P

Cortical gray matter (GM) damage is now widely recognized in multiple sclerosis (MS). The standard MRI does not reliably detect cortical GM lesions, although cortical volume loss can be measured. In this study, we demonstrate that the gradient echo MRI can reliably and quantitatively assess cortical GM damage in MS patients using standard clinical scanners. High resolution multi-gradient echo MRI was used for regional mapping of tissue-specific MRI signal transverse relaxation rate values (R2*) in 10 each relapsing–remitting, primary-progressive and secondary-progressive MS subjects. A voxel spread function method was used to correct artifacts induced by background field gradients. R2* values from healthy controls (HCs) of varying ages were obtained to establish baseline data and calculate ΔR2* values – age-adjusted differences between MS patients and HC. Thickness of cortical regions was also measured in all subjects. In cortical regions, ΔR2* values of MS patients were also adjusted for changes in cortical thickness. Symbol digit modalities (SDMT) and paced auditory serial addition (PASAT) neurocognitive tests, as well as Expanded Disability Status Score, 25-foot timed walk and nine-hole peg test results were also obtained on all MS subjects. We found that ΔR2* values were lower in multiple cortical GM and normal appearing white matter (NAWM) regions in MS compared with HC. ΔR2* values of global cortical GM and several specific cortical regions showed significant (p, Highlights • We report a novel method to detect abnormalities in cortical gray matter in MS based on measuring R2*. • Results correlated well with cognitive test results in MS patients. • This represents a potentially quantitative biomarker of cortical disease in MS.

Background Cortical, thalamic and hippocampal gray matter atrophy in relapsing–remitting MS (RRMS) is associated cognitive deficits. However, the role of interconnecting white matter pathways including the fornix, cingulum, and uncinate fasciculus (UF) is less well studied. Objective To assess MS damage to a hippocampal–thalamic–prefrontal network and the relative contributions of its components to specific cognitive domains. Methods We calculated diffusion tensor fractional anisotropy (FA) in the fornix, cingulum and UF as well as thalamic and hippocampal volumes in 27 RRMS patients and 20 healthy controls. A neuropsychological battery was administered and 4 core tests known to be sensitive to MS changes were used to assess cognitive impairment. To determine the relationships between structure and cognition, all tests were grouped into 4 domains: attention/executive function, processing speed, verbal memory, and spatial memory. Univariate correlations with structural measures and depressive symptoms identified potential contributors to cognitive performance and subsequent linear regression determined their relative effects on performance in each domain. For significant predictors, we also explored the effects of laterality and axial versus radial diffusivity. Results RRMS patients had worse performance on the Symbol Digit Modalities Test, but no significant impairment in the 4 cognitive domains. RRMS had reduced mean FA of all 3 pathways and reduced thalamic and hippocampal volumes compared to controls. In RRMS we found that thalamic volume and BDI predicted attention/executive function, UF FA predicted processing speed, thalamic volume predicted verbal memory, and UF FA and BDI predicted spatial memory. Conclusions Hippocampal–thalamic–prefrontal disruption affects cognitive performance in early RRMS with mild to minimal cognitive impairment, confirming both white and gray matter involvement in MS and demonstrating utility in assessing functional networks to monitor cognition., Highlights • We detect limbic white matter and gray matter insult in early RRMS patients with mild to minimal cognitive impairment. • Thalamic atrophy and uncinate fasciculus microstructural changes are associated with cognitive performance in early RRMS. • Depressive symptomatology also independently predicts cognitive performance

Objectives Conventional magnetic resonance imaging (MRI) is used to diagnose and monitor inflammatory disease in relapsing remitting (RR) multiple sclerosis (MS). In the less common primary progressive (PP) form of MS, in which focal inflammation is less evident, biomarkers are still needed to enable evaluation of novel therapies in clinical trials. Our objective was to characterize the association — across the brain and cervical spinal cord — between clinical disability measures in PPMS and two potential biomarkers (one for myelin, and one for atrophy, both resulting from the same imaging technique). Methods Multi-component driven equilibrium single pulse observation of T1 and T2 (mcDESPOT) MRI of the brain and cervical spinal cord were obtained for 15 PPMS patients and 11 matched controls. Data were analysed to estimate the signal related to myelin water (VFM), as well as volume measurements. MS disability was assessed using the Multiple Sclerosis Functional Composite score, which includes measures of cognitive processing (Paced Auditory Serial Addition Test), manual dexterity (9-Hole Peg Test) and ambulatory function (Timed 25-Foot Walk); and the Expanded Disability Status Scale. Results Brain and spinal cord volumes were different in PPMS compared to controls, particularly ventricular (+ 46%, p = 0.0006) and cervical spinal cord volume (− 16%, p = 0.0001). Brain and spinal cord myelin (VFM) were also reduced in PPMS (brain: − 11%, p = 0.01; spine: − 19%, p = 0.000004). Cognitive processing correlated with brain ventricular volume (p = 0.009). Manual dexterity correlated with brain ventricular volume (p = 0.007), and both brain and spinal cord VFM (p = 0.01 and 0.06, respectively). Ambulation correlated with spinal cord volume (p = 0.04) and spinal cord VFM (p = 0.04). Interpretation In this study we demonstrated that mcDESPOT can be used to measure myelin and atrophy in the brain and spinal cord. Results correlate well with clinical disability scores in PPMS representing cognitive, fine motor and ambulatory disability., Highlights • A biomarker is needed to evaluate therapies for primary progressive MS (PPMS). • We used novel MRI providing both general atrophy and specific myelin measures. • We studied brain and cervical spinal cord in PPMS and healthy controls. • Brain and spinal cord volume and myelin measures were reduced in PPMS vs controls. • MRI measures described cognitive, fine motor and ambulatory disability scores.

Brain iron homeostasis is known to be disturbed in multiple sclerosis (MS), yet little is known about the association of common gene variants linked to iron regulation and pathological tissue changes in the brain. In this study, we investigated the association of genetic determinants linked to iron regulation with deep gray matter (GM) magnetic susceptibility in both healthy controls (HC) and MS patients. Four hundred (400) patients with MS and 150 age- and sex-matched HCs were enrolled and obtained 3 T MRI examination. Three (3) single nucleotide polymorphisms (SNPs) associated with iron regulation were genotyped: two SNPs in the human hereditary hemochromatosis protein gene HFE: rs1800562 (C282Y mutation) and rs1799945 (H63D mutation), as well as the rs1049296 SNP in the transferrin gene (C2 mutation). The effects of disease and genetic status were studied using quantitative susceptibility mapping (QSM) voxel-based analysis (VBA) and region-of-interest (ROI) analysis of the deep GM. The general linear model framework was used to compare groups. Analyses were corrected for age and sex, and adjusted for false discovery rate. We found moderate increases in susceptibility in the right putamen of participants with the C282Y (+ 6.1 ppb) and H63D (+ 6.9 ppb) gene variants vs. non-carriers, as well as a decrease in thalamic susceptibility of progressive MS patients with the C282Y mutation (left: − 5.3 ppb, right: − 6.7 ppb, p, Highlights • Magnetic susceptibility and common gene variants linked to iron were investigated. • The C282Y and H63D alleles were associated with putamen and thalamus susceptibility changes. • Dependent on allele status, men and women differed in deep GM susceptibility in MS.

Objective White matter atrophy occurs independently of lesions in multiple sclerosis. In contrast to lesion detection, the quantitative assessment of white matter atrophy in individual patients has been regarded as a major challenge. We therefore tested the hypothesis that white matter atrophy (WMA) is present at the very beginning of multiple sclerosis (MS) and in virtually each individual patient. To find a new sensitive and robust marker for WMA we investigated the relationship between cortical surface area, white matter volume (WMV), and whole-brain-surface-averaged rectified cortical extrinsic curvature. Based on geometrical considerations we hypothesized that cortical curvature increases if WMV decreases and the cortical surface area remains constant. Methods In total, 95 participants were enrolled: 30 patients with early and advanced relapsing–remitting MS; 30 age-matched control subjects; 30 patients with Alzheimer's disease (AD) and 5 patients with clinically isolated syndrome (CIS). Results 29/30 MS and 5/5 CIS patients showed lower WMV than expected from their intracranial volume (average reduction 13.0%, P, Highlights • We suggest cortical curvature as marker for selective white matter atrophy (WMA). • This geometric marker is more specific and sensitive than volumetric measures. • It is not confounded by intra-cranial volume, age, and gender. • WMA seems to be a characteristic symptom in early multiple sclerosis. • WMA can be also detected in patients with a clinical isolated symptom.

Background: Multiple sclerosis is a disorder related to demyelination of axons. Iron is an essential cofactor in myelin synthesis. Previously, we described two children (males of mixed ancestry) with relapsing-remitting multiple sclerosis (RRMS) where long-term remission was achieved by regular iron supplementation. A genetic defect in iron metabolism was postulated, suggesting that more advanced genetic studies could shed new light on disease pathophysiology related to iron. Methods: Whole exome sequencing (WES) was performed to identify causal pathways. Blood tests were performed over a 10 year period to monitor the long-term effect of a supplementation regimen. Clinical wellbeing was assessed quarterly by a pediatric neurologist and regular feedback was obtained from the schoolteachers. Results: WES revealed gene variants involved in iron absorption and transport, in the transmembrane protease, serine 6 (TMPRSS6) and transferrin (TF) genes; multiple genetic variants in CUBN, which encodes cubilin (a receptor involved in the absorption of vitamin B12 as well as the reabsorption of transferrin-bound iron and vitamin D in the kidneys); SLC25A37 (involved in iron transport into mitochondria) and CD163 (a scavenger receptor involved in hemorrhage resolution). Variants were also found in COQ3, involved with synthesis of Coenzyme Q10 in mitochondria. Neither of the children had the HLA-DRB1*1501 allele associated with increased genetic risk for MS, suggesting that the genetic contribution of iron-related genetic variants may be instrumental in childhood MS. In both children the RRMS has remained stable without activity over the last 10 years since initiation of nutritional supplementation and maintenance of normal iron levels, confirming the role of iron deficiency in disease pathogenesis in these patients. Conclusion: Our findings highlight the potential value of WES to identify heritable risk factors that could affect the reabsorption of transferrin-bound iron in the kidneys causing sustained iron loss, together with inhibition of vitamin B12 absorption and vitamin D reabsorption (CUBN) and iron transport into mitochondria (SLC25A37) as the sole site of heme synthesis. This supports a model for RRMS in children with an apparent iron-deficient biochemical subtype of MS, with oligodendrocyte cell death and impaired myelination possibly caused by deficits of energy- and antioxidant capacity in mitochondria. Keywords: Pediatric onset multiple sclerosis, Genetic variants, Whole exome sequencing, Iron deficiency, Oxidative stress, Mitochondria

Cognitive impairment is as an important feature of Multiple Sclerosis (MS), and might be even more relevant to patients than mobility restrictions. Compared to the multitude of studies investigating memory deficits or basic cognitive slowing, executive dysfunction is a rarely studied cognitive domain in MS, and its neural correlates remain largely unexplored. Even rarer are topological studies on specific cognitive functions in MS. Here we used several structural MRI parameters – including cortical thinning and T2 lesion load – to investigate neural correlates of executive dysfunction, both on a global and a regional level by means of voxel- and vertex-wise analyses. Forty-eight patients with relapsing-remitting MS and 48 healthy controls participated in the study. Five executive functions were assessed, i.e. verbal and figural fluency, working memory, interference control and set shifting. Patients scored lower than controls in verbal and figural fluency only, and displayed widespread cortical thinning. On a global level, cortical thickness independently predicted verbal fluency performance, when controlling for lesion volume and central brain atrophy estimates. On a regional level, cortical thinning in the anterior cingulate region correlated with deficits in verbal and figural fluency and did so in a lateralised manner: Left-sided thinning was related to reduced verbal – but not figural – fluency, whereas the opposite pattern was observed for right-sided thinning. We conclude that executive dysfunction in MS patients can specifically affect verbal and figural fluency. The observed lateralised clinico-anatomical correlation has previously been described in brain-damaged patients with large focal lesions only, for example after stroke. Based on focal grey matter atrophy, we here show for the first time comparable lateralised findings in a white matter disease with widespread pathology., Highlights • Executive dysfunction in Multiple Sclerosis (MS) can specifically affect fluency. • Fluency deficits in MS correlate with thinning of the anterior cingulate cortex. • This correlation seems lateralised and modality-specific.

Aims Multiple sclerosis (MS) leaves a signature on the phosphorylation and thus proton binding capacity of axonal neurofilament (Nf) proteins. The proton binding capacity in a tissue is the major determinant for exchange between bound and free protons and thus the magnetisation transfer ratio (MTR). This study investigated whether the MTR of non-lesional white matter (NLWM) was related to the brain tissue concentration of neurofilament phosphoforms. Methods Unfixed post-mortem brain slices of 12 MS patients were analysed using MTR, T1 at 1.5 T. Blocks containing NLWM were processed for embedding in paraffin and inspected microscopically. Adjacent tissue was microdissected, homogenised and specific protein levels were quantified by ELISA for the Nf heavy chain (NfH) phosphoforms, glial fibrillary acidic protein (GFAP), S100B and ferritin. Results Averaged hyperphosphorylated NfH (SMI34) but not phosphorylated NfH (SMI35) levels were different between individual patients NLWM. The concentration of hyperphosphorylated NfH-SMI34 correlated with T1 (R = 0.70, p = 0.0114) and — inversely — with MTR (R =−0.73, p = 0.0065). NfH-SMI35 was not correlated to any of the MR indices. Conclusions Post-translational modifications of axonal proteins such as phosphorylation of neurofilaments occur in NLWM and may precede demyelination. The resulting change of proton mobility influences MTR and T1. This permits the in vivo detection of these subtle tissue changes on a proteomic level in patients with MS., Highlights ► Neurofilaments are phosphorylated ► Protein phosphorylation competes with the free proton binding capacity ► Magnetisation transfer depends on magnetisation exchange between macromolecular bound and free protons ► In multiple sclerosis the phosphorylation status of neurofilaments is changed in otherwise normal appearing axons ► In vivo assessment of early axonal pathology is possible using magnetisation transfer

AimsAxonal pathology extends to the axonal cytoarchitecture leaving its signature on axoskeletal proteins. This study investigated whether neurofilament (NfH) phosphorylation would relate to the dynamics of axonal pathology in multiple sclerosis (MS).MethodsNfH phosphoforms (SMI32, SMI34, SMI35) were quantified by ELISA from microdissected samples of control and MS brain and spinal cord. Individual axons were analysed by electron microscopy, densitometrically and morphologically in adjacent tissue sections. Experiments were carried out pre- and post enzymatic dephosphorylation.ResultsIn control tissue a rostro-caudal gradient of NfH indicated an increase in axonal density from the brain gray matter towards the spinal cord. The highest levels of phosphorylated and hyperphosphorylated NfH were found in acute lesions of brain and spinal cord, in contrast to chronic lesions where levels were lower than in white matter, consistent with axonal loss. Dephosphorylated NfH was higher, but less densly packed in MS white matter axons compared to control tissue.ConclusionsThe findings suggest that a less organised/compact axoskeleton or impaired axonal transport may represent an early sign of axonal pathology within the normal appearing white matter in MS. Subsequently a proportional increase of dephosphorylated NfH, aberrant phosphorylation and/or aggregation may occur whilst the protein is transported through the white matter towards the MS plaque, where hyperphosphorylated NfH dominates.

Histochemical and MRI studies have demonstrated that MS (multiple sclerosis) patients have abnormal deposition of iron in both gray and white matter structures. Data is emerging indicating that this iron could partake in pathogenesis by various mechanisms, e.g., promoting the production of reactive oxygen species and enhancing the production of proinflammatory cytokines. Iron chelation therapy could be a viable strategy to block iron-related pathological events or it can confer cellular protection by stabilizing hypoxia inducible factor 1α, a transcription factor that normally responds to hypoxic conditions. Iron chelation has been shown to protect against disease progression and/or limit iron accumulation in some neurological disorders or their experimental models. Data from studies that administered a chelator to animals with experimental autoimmune encephalomyelitis, a model of MS, support the rationale for examining this treatment approach in MS. Preliminary clinical studies have been performed in MS patients using deferoxamine. Although some side effects were observed, the large majority of patients were able to tolerate the arduous administration regimen, i.e., 6–8 h of subcutaneous infusion, and all side effects resolved upon discontinuation of treatment. Importantly, these preliminary studies did not identify a disqualifying event for this experimental approach. More recently developed chelators, deferasirox and deferiprone, are more desirable for possible use in MS given their oral administration, and importantly, deferiprone can cross the blood–brain barrier. However, experiences from other conditions indicate that the potential for adverse events during chelation therapy necessitates close patient monitoring and a carefully considered administration regimen., Abnormal iron deposition begins early in the disease course of multiple sclerosis, and this iron may facilitate pathogenesis. Iron chelation is an intervention strategy that can ameliorate iron-related pathogenic events, but it has associated risks.