Single-Cell Multi-Omics Map of Cell Type-Specific Mechanistic Drivers of Multiple Sclerosis Lesions.

BACKGROUND AND OBJECTIVES: In progressive multiple sclerosis (MS), compartmentalized inflammation plays a pivotal role in the complex pathology of tissue damage. The interplay between epigenetic regulation, transcriptional modifications, and location-specific alterations within white matter (WM) lesions at the single-cell level remains underexplored. METHODS: We examined intracellular and intercellular pathways in the MS brain WM using a novel dataset obtained by integrated single-cell multi-omics techniques from 3 active lesions, 3 chronic active lesions, 3 remyelinating lesions, and 3 control WM of 6 patients with progressive MS and 3 non-neurologic controls. Single-nucleus RNA-seq and ATAC-seq were combined and additionally enriched with newly conducted spatial transcriptomics from 1 chronic active lesion. Functional gene modules were then validated in our previously published bulk tissue transcriptome data obtained from 73 WM lesions of patients with progressive MS and 25 WM of non-neurologic disease controls. RESULTS: Our analysis uncovered an MS-specific oligodendrocyte genetic signature influenced by the KLF/SP gene family. This modulation has potential associations with the autocrine iron uptake signaling observed in transcripts of transferrin and its receptor LRP2. In addition, an inflammatory profile emerged within these oligodendrocytes. We observed unique cellular endophenotypes both at the periphery and within the chronic active lesion. These include a distinct metabolic astrocyte phenotype, the importance of FGF signaling among astrocytes and neurons, and a notable enrichment of mitochondrial genes at the lesion edge populated predominantly by astrocytes. Our study also identified B-cell coexpression networks indicating different functional B-cell subsets with differential location and specific tendencies toward certain lesion types. DISCUSSION: The use of single-cell multi-omics has offered a detailed perspective into the cellular dynamics and interactions in MS. These nuanced findings might pave the way for deeper insights into lesion pathogenesis in progressive MS.

The prevalence and topography of spinal cord demyelination in multiple sclerosis: a retrospective study.

Spinal cord pathology is a major determinant of irreversible disability in progressive multiple sclerosis. The demyelinated lesion is a cardinal feature. The well-characterised anatomy of the spinal cord and new analytic approaches allows the systematic study of lesion topography and its extent of inflammatory activity unveiling new insights into disease pathogenesis. We studied cervical, thoracic, and lumbar spinal cord tissue from 119 pathologically confirmed multiple sclerosis cases. Immunohistochemistry was used to detect demyelination (PLP) and classify lesional inflammatory activity (CD68). Prevalence and distribution of demyelination, staged by lesion activity, was determined and topographical maps were created to identify patterns of lesion prevalence and distribution using mixed models and permutation-based voxelwise analysis. 460 lesions were observed throughout the spinal cord with 76.5% of cases demonstrating at least 1 lesion. The cervical level was preferentially affected by lesions. 58.3% of lesions were inflammatory with 87.9% of cases harbouring at least 1 inflammatory lesion. Topographically, lesions consistently affected the dorsal and lateral columns with relative sparing of subpial areas in a distribution mirroring the vascular network. The presence of spinal cord lesions and the proportion of active lesions related strongly with clinical disease milestones, including time from onset to wheelchair and onset to death. We demonstrate that spinal cord demyelination is common, highly inflammatory, has a predilection for the cervical level, and relates to clinical disability. The topography of lesions in the dorsal and lateral columns and relative sparing of subpial areas points to a role of the vasculature in lesion pathogenesis, suggesting short-range cell infiltration from the blood and signaling molecules circulating in the perivascular space incite lesion development. These findings challenge the notion that end-stage progressive multiple sclerosis is 'burnt out' and an outside-in lesional gradient predominates in the spinal cord. Taken together, this study provides support for long-term targeting of inflammatory demyelination in the spinal cord and nominates vascular dysfunction as a potential target for new therapeutic approaches to limit irreversible disability.

The Impact of Sample Storage on Blood Methylation: Towards Assessing Myelin Gene Methylation as a Biomarker for Progressive Multiple Sclerosis.

One of the major challenges in multiple sclerosis (MS) is to accurately monitor and quantify disability over time. Thus, there is a pressing need to identify new biomarkers for disease progression. Peripheral blood DNA methylation has been demonstrated to be an easily accessible and quantifiable marker in many neurodegenerative diseases. In this study, we aimed to investigate whether methylation patterns that were previously determined in chronic inactive white matter lesions of patients with progressive MS are also reflected in the blood, and whether the latter can serve as a biomarker for disease progression in MS. While our initial analysis revealed differences in the blood methylation state of important myelin-related genes between patients with progressive MS and controls, these findings could not be validated in other independent patient cohorts. Subsequent investigation suggests that sample storage can selectively influence DNA methylation patterns, potentially hindering accurate epigenetic analysis. Therefore, sample storage time should be taken into consideration during the initial sample selection stage in biomarker studies.

What contributes to disability in progressive MS? A brain and cervical cord-matched quantitative MRI study.

BACKGROUND: We assessed the ability of a brain-and-cord-matched quantitative magnetic resonance imaging (qMRI) protocol to differentiate patients with progressive multiple sclerosis (PMS) from controls, in terms of normal-appearing (NA) tissue abnormalities, and explain disability. METHODS: A total of 27 patients and 16 controls were assessed on the Expanded Disability Status Scale (EDSS), 25-foot timed walk (TWT), 9-hole peg (9HPT) and symbol digit modalities (SDMT) tests. All underwent 3T brain and (C2-C3) cord structural imaging and qMRI (relaxometry, quantitative magnetisation transfer, multi-shell diffusion-weighted imaging), using a fast brain-and-cord-matched protocol with brain-and-cord-unified imaging readouts. Lesion and NA-tissue volumes and qMRI metrics reflecting demyelination and axonal loss were obtained. Random forest analyses identified the most relevant volumetric/qMRI measures to clinical outcomes. Confounder-adjusted linear regression estimated the actual MRI-clinical associations. RESULTS: Several qMRI/volumetric differences between patients and controls were observed (p < 0.01). Higher NA-deep grey matter quantitative-T1 (EDSS: beta = 7.96, p = 0.006; 9HPT: beta = -0.09, p = 0.004), higher NA-white matter orientation dispersion index (TWT: beta = -3.21, p = 0.005; SDMT: beta = -847.10, p < 0.001), lower whole-cord bound pool fraction (9HPT: beta = 0.79, p = 0.001) and higher NA-cortical grey matter quantitative-T1 (SDMT = -94.31, p < 0.001) emerged as particularly relevant predictors of greater disability. CONCLUSION: Fast brain-and-cord-matched qMRI protocols are feasible and identify demyelination - combined with other mechanisms - as key for disability accumulation in PMS.

Time is myelin: early cortical myelin repair prevents atrophy and clinical progression in multiple sclerosis.

Cortical myelin loss and repair in multiple sclerosis (MS) have been explored in neuropathological studies, but the impact of these processes on neurodegeneration and the irreversible clinical progression of the disease remains unknown. Here, we evaluated in vivo cortical demyelination and remyelination in a large cohort of people with all clinical phenotypes of MS followed up for 5 years using magnetization transfer imaging (MTI), a technique that has been shown to be sensitive to myelin content changes in the cortex. We investigated 140 people with MS (37 clinically isolated syndrome, 71 relapsing-MS, 32 progressive-MS), who were clinically assessed at baseline and after 5 years and, along with 84 healthy controls, underwent a 3 T-MRI protocol including MTI at baseline and after 1 year. Changes in cortical volume over the radiological follow-up were computed with a Jacobian integration method. Magnetization transfer ratio was employed to calculate for each patient an index of cortical demyelination at baseline and of dynamic cortical demyelination and remyelination over the follow-up period. The three indices of cortical myelin content change were heterogeneous across patients but did not significantly differ across clinical phenotypes or treatment groups. Cortical remyelination, which tended to fail in the regions closer to CSF (-11%, P < 0.001), was extensive in half of the cohort and occurred independently of age, disease duration and clinical phenotype. Higher indices of cortical dynamic demyelination (ß = 0.23, P = 0.024) and lower indices of cortical remyelination (ß = -0.18, P = 0.03) were significantly associated with greater cortical atrophy after 1 year, independently of age and MS phenotype. While the extent of cortical demyelination predicted a higher probability of clinical progression after 5 years in the entire cohort [odds ratio (OR) = 1.2; P = 0.043], the impact of cortical remyelination in reducing the risk of accumulating clinical disability after 5 years was significant only in the subgroup of patients with shorter disease duration and limited extent of demyelination in cortical regions (OR = 0.86, P = 0.015, area under the curve = 0.93). In this subgroup, a 30% increase in cortical remyelination nearly halved the risk of clinical progression at 5 years, independently of clinical relapses. Overall, our results highlight the critical role of cortical myelin dynamics in the cascade of events leading to neurodegeneration and to the subsequent accumulation of irreversible disability in MS. Our findings suggest that early-stage myelin repair compensating for cortical myelin loss has the potential to prevent neuro-axonal loss and its long-term irreversible clinical consequences in people with MS.

The glymphatic system as a potential biomarker and therapeutic target in secondary progressive multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a refractory immune-mediated inflammatory disease of the central nervous system, and some cases of the major subtype, relapsing-remitting (RR), transition to secondary progressive (SP). However, the detailed pathogenesis, biomarkers, and effective treatment strategies for secondary progressive multiple sclerosis have not been established. The glymphatic system, which is responsible for waste clearance in the brain, is an intriguing avenue for investigation and is primarily studied through diffusion tensor image analysis along the perivascular space (DTI-ALPS). This study aimed to compare DTI-ALPS indices between patients with RRMS and SPMS to uncover potential differences in their pathologies and evaluate the utility of the glymphatic system as a possible biomarker. METHODS: A cohort of 26 patients with MS (13 RRMS and 13 SPMS) who met specific criteria were enrolled in this prospective study. Magnetic resonance imaging (MRI), including diffusion MRI, 3D T1-weighted imaging, and relaxation time quantification, was conducted. The ALPS index, a measure of glymphatic function, was calculated using diffusion-weighted imaging data. Demographic variables, MRI metrics, and ALPS indices were compared between patients with RRMS and those with SPMS. RESULTS: The ALPS index was significantly lower in the SPMS group. Patients with SPMS exhibited longer disease duration and higher Expanded Disability Status Scale (EDSS) scores than those with RRMS. Despite these differences, the correlations between the EDSS score, disease duration, and ALPS index were minimal, suggesting that the impact of these clinical variables on ALPS index variations was negligible. DISCUSSION: Our study revealed the potential microstructural and functional differences between RRMS and SPMS related to glymphatic system impairment. Although disease severity and duration vary among subtypes, their influence on ALPS index differences appears to be limited. This highlights the stronger association between SP conversion and changes in the ALPS index. These findings align with those of previous research, indicating the involvement of the glymphatic system in the progression of MS. CONCLUSION: Although the causality remains uncertain, our study suggests that a reduced ALPS index, reflecting glymphatic system dysfunction, may contribute to MS progression, particularly in SPMS. This suggests the potential of the ALPS index as a diagnostic biomarker for SPMS and underscores the potential of the glymphatic system as a therapeutic target to mitigate MS progression. Future studies with larger cohorts and pathological validation are necessary to confirm these findings. This study provides new insights into the pathogenesis of SPMS and the potential for innovative therapeutic strategies.

Serum macrophage migration inhibitory factor levels predict brain atrophy in people with primary progressive multiple sclerosis.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a cytokine linked to multiple sclerosis (MS) progression that is thought to be inhibited by ibudilast. SPRINT-MS was a phase 2 placebo-controlled trial of ibudilast in progressive multiple sclerosis (PMS). OBJECTIVE: To determine whether baseline MIF levels predict imaging outcomes and assess the effects of ibudilast on serum and cerebrospinal fluid (CSF) MIF levels in people with PMS treated with ibudilast. METHODS: Participants in the SPRINT-MS trial were treated with either ibudilast or placebo and underwent brain magnetic resonance imaging (MRI) every 24 weeks over a duration of 96 weeks. MIF was measured in serum and CSF. RESULTS: MIF levels were compared with imaging outcomes in 223 participants from the SPRINT-MS study. In the primary progressive multiple sclerosis (PPMS) cohort, males had higher serum (p < 0.001) and CSF (p = 0.01) MIF levels, as compared with females. Higher baseline serum MIF levels in PPMS were associated with faster brain atrophy (beta = -0.113%, 95% confidence interval (CI): -0.204% to -0.021%; p = 0.016). These findings were not observed in secondary progressive multiple sclerosis (SPMS). Ibudilast did not affect either serum or CSF MIF levels. CONCLUSIONS: Serum MIF levels were associated with male sex and predicted brain atrophy in PPMS, but not SPMS. Ibudilast did not demonstrate an effect on MIF levels, as compared with placebo, although we cannot exclude a functional effect.

High Levels of Perivascular Inflammation and Active Demyelinating Lesions at Time of Death Associated with Rapidly Progressive Multiple Sclerosis Disease Course: A Retrospective Postmortem Cohort Study.

OBJECTIVE: Analysis of postmortem multiple sclerosis (MS) tissues combined with in vivo disease milestones suggests that whereas perivascular white matter infiltrates are associated with demyelinating activity in the initial stages, leptomeningeal immune cell infiltration, enriched in B cells, and associated cortical lesions contribute to disease progression. We systematically examine the association of inflammatory features and white matter demyelination at postmortem with clinical milestones. METHODS: In 269 MS brains, 20 sites were examined using immunohistochemistry for active lesions (ALs) and perivenular inflammation (PVI). In a subset of 22, a detailed count of CD20+ B cells and CD3+ T cells in PVIs was performed. RESULTS: ALs were detected in 22%, whereas high levels of PVI were detected in 52% of cases. ALs were present in 35% of cases with high levels of PVI. Shorter time from onset of progression to death was associated with increased prevalence and higher levels of PVI (both p < 0.0001). Shorter time from onset of progression to wheelchair use was associated with higher prevalence of ALs (odds ratio [OR] = 0.921, 95% confidence interval [CI] = 0.858-0.989, p = 0.0230) and higher level of PVI (OR = 0.932, 95% CI = 0.886-0.981, p = 0.0071). High levels of PVI were associated with meningeal inflammation and increased cortical demyelination and significantly higher levels of B lymphocytes within the PVI. INTERPRETATION: ALs, a feature of early disease stage, persist up to death in a subgroup with high levels of PVI. These features link to a rapid progressive phase and higher levels of meningeal inflammation and B-cell infiltrates, supporting the hypothesis that chronic inflammation drives progression in MS. ANN NEUROL 2024;95:706-719.

Toward identifying key mechanisms of progression in multiple sclerosis.

A major therapeutic goal in the treatment of multiple sclerosis (MS) is to prevent the accumulation of disability over an often decades-long disease course. Disability progression can result from acute relapses as well as from CNS intrinsic parenchymal disintegration without de novo CNS lesion formation. Research focus has shifted to progression not associated with acute inflammation, as it is not sufficiently controlled by currently available treatments. This review outlines how recent advances in the understanding of the pathogenesis of progressive MS have been facilitated by the development of more precise, less static pathogenetic concepts of progressive MS, as well as by new techniques for the analysis of region-specific proteomic and transcriptomic signatures in the human CNS. We highlight key drivers of MS disease progression and potential targets in its treatment.

Gasdermin D activation in oligodendrocytes and microglia drives inflammatory demyelination in progressive multiple sclerosis.

Neuroinflammation coupled with demyelination and neuro-axonal damage in the central nervous system (CNS) contribute to disease advancement in progressive multiple sclerosis (P-MS). Inflammasome activation accompanied by proteolytic cleavage of gasdermin D (GSDMD) results in cellular hyperactivation and lytic death. Using multiple experimental platforms, we investigated the actions of GSDMD within the CNS and its contributions to P-MS. Brain tissues from persons with P-MS showed significantly increased expression of GSDMD, NINJ1, IL-1ß, and -18 within chronic active demyelinating lesions compared to MS normal appearing white matter and nonMS (control) white matter. Conditioned media (CM) from stimulated GSDMD+/+ human macrophages caused significantly greater cytotoxicity of oligodendroglial and neuronal cells, compared to CM from GSDMD-/- macrophages. Oligodendrocytes and CNS macrophages displayed increased Gsdmd immunoreactivity in the central corpus callosum (CCC) of cuprizone (CPZ)-exposed Gsdmd+/+ mice, associated with greater demyelination and reduced oligodendrocyte precursor cell proliferation, compared to CPZ-exposed Gsdmd-/- animals. CPZ-exposed Gsdmd+/+ mice exhibited significantly increased G-ratios and reduced axonal densities in the CCC compared to CPZ-exposed Gsdmd-/- mice. Proteomic analyses revealed increased brain complement C1q proteins and hexokinases in CPZ-exposed Gsdmd-/- animals. [18F]FDG PET imaging showed increased glucose metabolism in the hippocampus and whole brain with intact neurobehavioral performance in Gsdmd-/- animals after CPZ exposure. GSDMD activation in CNS macrophages and oligodendrocytes contributes to inflammatory demyelination and neuroaxonal injury, offering mechanistic and potential therapeutic insights into P-MS pathogenesis.

Association of MRI leptomeningeal enhancement with disability worsening in progressive multiple sclerosis: A clinical and post-mortem study.

BACKGROUND: Leptomeningeal enhancement (LME) has been described as a biomarker of meningeal inflammation in multiple sclerosis (MS). OBJECTIVE: The aim of this study was to (1) assess if LME is predictive of disability worsening in progressive MS (pMS) patients and (2) investigate the pathological substrates of LME in an independent post-mortem MS series. METHODS: In total, 115 pMS patients were imaged yearly with 1.5T MRI, using post-contrast CUBE 3D FLAIR for LME detection. Endpoint: to identify the baseline variables predictive of confirmed disability worsening (CDW) at 24 months follow-up. Post-mortem, inflammation, and structural changes of the leptomeninges were assessed in 12 MS/8 control brains. RESULTS: LME (27% of patients at baseline) was associated with higher EDSS and lower brain volume (nBV). LME was unchanged in most patients over follow-up. LME at baseline MRI was independently associated with higher risk of 24 months CDW (HR 3.05, 95% CI 1.36-6.84, p = 0.007) in a Cox regression, including age, nBV, T2 lesion volume, high-efficacy treatments, and MRI disease activity. Post-mortem, focal structural changes (fibrosis) of the leptomeninges were observed in MS, usually associated with inflammation (Kendall's Tau 0.315, p < 0.0001). CONCLUSIONS: LME is frequently detected in pMS patients using 1.5T MRI and is independently predictive of disability progression. LME could result from both focal leptomeningeal post-inflammatory fibrosis and inflammation.

Real-world challenges in the diagnosis of primary progressive multiple sclerosis.

BACKGROUND AND PURPOSE: Despite the 2017 revisions to the McDonald criteria, diagnosing primary progressive multiple sclerosis (PPMS) remains challenging. To improve clinical practice, the aim was to identify frequent diagnostic challenges in a real-world setting and associate these with the performance of the 2010 and 2017 PPMS diagnostic McDonald criteria. METHODS: Clinical, radiological and laboratory characteristics at the time of diagnosis were retrospectively recorded from designated PPMS patient files. Possible complicating factors were recorded such as confounding comorbidity, signs indicative of alternative diagnoses, possible earlier relapses and/or incomplete diagnostic work-up (no cerebrospinal fluid examination and/or magnetic resonance imaging brain and spinal cord). The percentages of patients fulfilling the 2010 and 2017 McDonald criteria were calculated after censoring patients with these complicating factors. RESULTS: A total of 322 designated PPMS patients were included. Of all participants, it was found that n = 28/322 had confounding comorbidity and/or signs indicative of alternative diagnoses, n = 103/294 had possible initial relapsing and/or uncertainly progressive phenotypes and n = 73/191 received an incomplete diagnostic work-up. When applying the 2010 and 2017 diagnostic PPMS McDonald criteria on n = 118 cases with a full diagnostic work-up and a primary progressive disease course without a better alternative explanation, these were met by 104/118 (88.1%) and 98/118 remaining patients (83.1%), respectively (p = 0.15). CONCLUSION: Accurate interpretation of the initial clinical course, consideration of alternative diagnoses and a full diagnostic work-up are the cornerstones of a PPMS diagnosis. When these conditions are met, the 2010 and 2017 McDonald criteria for PPMS perform similarly, emphasizing the importance of their appropriate application in clinical practice.

Effect of ibudilast on thalamic magnetization transfer ratio and volume in progressive multiple sclerosis.

BACKGROUND: Thalamic volume (TV) is a sensitive biomarker of disease burden of injury in multiple sclerosis (MS) and appears to reflect overall lesion loads. Ibudilast showed significant treatment effect on brain atrophy and magnetization transfer ratio (MTR) of normal-appearing brain tissue but not in new/enlarging T2 lesion in the SPRINT-MS randomized clinical trial. OBJECTIVE: To evaluate the effect of ibudilast on thalamic tissue integrity and volume in the SPRINT-MS. METHODS: A total of 255 participants with progressive MS were randomized to oral ibudilast or placebo, and thalamic MTR and normalized TV over 96 weeks were quantified. Mixed-effect modeling assessed treatment effects on the thalamic MTR and TV, separately. Similarly, the measures were compared between the participants with confirmed disability progression (CDP). RESULTS: Ibudilast's treatment effect was observed compared to placebo for thalamic MTR (p = 0.03) but not for TV (p = 0.68) while TV correlated with T2 lesion volume (p < 0.001). CDP associated with thalamic MTR (p = 0.04) but not with TV (p = 0.7). CONCLUSION: Ibudilast showed an effect on thalamic MTR, which was associated with CDP, suggesting a clinically relevant effect on thalamic tissue integrity. However, the treatment effect was not observed in TV, suggesting that thalamic atrophy is more closely associated with global inflammatory activity than local tissue integrity. CLINICALTRIALS.GOV: NCT01982942.

The Heterogeneous Multiple Sclerosis Lesion: How Can We Assess and Modify a Degenerating Lesion?

Multiple sclerosis (MS) is a heterogeneous disease of the central nervous system that is governed by neural tissue loss and dystrophy during its progressive phase, with complex reactive pathological cellular changes. The immune-mediated mechanisms that promulgate the demyelinating lesions during relapses of acute episodes are not characteristic of chronic lesions during progressive MS. This has limited our capacity to target the disease effectively as it evolves within the central nervous system white and gray matter, thereby leaving neurologists without effective options to manage individuals as they transition to a secondary progressive phase. The current review highlights the molecular and cellular sequelae that have been identified as cooperating with and/or contributing to neurodegeneration that characterizes individuals with progressive forms of MS. We emphasize the need for appropriate monitoring via known and novel molecular and imaging biomarkers that can accurately detect and predict progression for the purposes of newly designed clinical trials that can demonstrate the efficacy of neuroprotection and potentially neurorepair. To achieve neurorepair, we focus on the modifications required in the reactive cellular and extracellular milieu in order to enable endogenous cell growth as well as transplanted cells that can integrate and/or renew the degenerative MS plaque.

Effects of Ibudilast on Retinal Atrophy in Progressive Multiple Sclerosis Subtypes: Post Hoc Analyses of the SPRINT-MS Trial.

BACKGROUND AND OBJECTIVES: Ganglion cell + inner plexiform layer (GCIPL) thinning, measured by optical coherence tomography (OCT), reflects global neurodegeneration in multiple sclerosis (MS). Atrophy of the inner (INL) and outer nuclear layer (ONL) may also be prominent in progressive MS (PMS). The phase 2, SPRINT-MS trial found reduced brain atrophy with ibudilast therapy in PMS. In this post hoc analysis of the SPRINT-MS trial, we investigate (1) retinal atrophy (2) differences in response by subtype and (3) associations between OCT and MRI measures of neurodegeneration. METHODS: In the multicenter, double-blind SPRINT-MS trial, participants with secondary progressive MS (SPMS) or primary progressive MS (PPMS) were randomized to ibudilast or placebo. OCT and MRI data were collected every 24 weeks for 96 weeks. Extensive OCT quality control and algorithmic segmentation produced consistent results across Cirrus HD-OCT and Spectralis devices. Primary endpoints were GCIPL, INL, and ONL atrophy, assessed by linear mixed-effects regression. Secondary endpoints were associations of OCT measures, brain parenchymal fraction, and cortical thickness, assessed by partial Pearson correlations. RESULTS: One hundred thirty-four PPMS and 121 SPMS participants were included. GCIPL atrophy was 79% slower in the ibudilast (-0.07 ± 0.23 µm/y) vs placebo group (-0.32 ± 0.20 µm/y, p = 0.003). This effect predominated in the PPMS cohort (ibudilast: -0.08 ± 0.29 µm/y vs placebo: -0.60 ± 0.29 µm/y, a decrease of 87%, p < 0.001) and was not detected in the SPMS cohort (ibudilast: -0.21 ± 0.28 µm/y vs placebo: -0.14 ± 0.27 µm/y, p = 0.55). GCIPL, INL, and ONL atrophy rates correlated with whole brain atrophy rates across the cohort (r = 0.27, r = 0.26, and r = 0.20, respectively; p < 0.001). Power calculations from these data show future trials of similar size and design have ≥80% power to detect GCIPL atrophy effect sizes of approximately 40%. DISCUSSION: Ibudilast treatment decreased GCIPL atrophy in PMS, driven by the PPMS cohort, with no effect seen in SPMS. Modulated atrophy of retinal layers may be detectable in sample sizes smaller than the SPRINT-MS trial and correlate with whole brain atrophy in PMS, further highlighting their utility as outcomes in PMS. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that ibudilast reduces composite ganglion cell + inner plexiform layer atrophy, without reduction of inner or outer nuclear layer atrophy, in patients with primary progressive MS but not those with secondary progressive MS.

Mechanisms of metabolic stress induced cell death of human oligodendrocytes: relevance for progressive multiple sclerosis.

Oligodendrocyte (OL) injury and loss are central features of evolving lesions in multiple sclerosis. Potential causative mechanisms of OL loss include metabolic stress within the lesion microenvironment. Here we use the injury response of primary human OLs (hOLs) to metabolic stress (reduced glucose/nutrients) in vitro to help define the basis for the in situ features of OLs in cases of MS. Under metabolic stress in vitro, we detected reduction in ATP levels per cell that precede changes in survival. Autophagy was initially activated, although ATP levels were not altered by inhibitors (chloroquine) or activators (Torin-1). Prolonged stress resulted in autophagy failure, documented by non-fusion of autophagosomes and lysosomes. Consistent with our in vitro results, we detected higher expression of LC3, a marker of autophagosomes in OLs, in MS lesions compared to controls. Both in vitro and in situ, we observe a reduction in nuclear size of remaining OLs. Prolonged stress resulted in increased ROS and cleavage of spectrin, a target of Ca2+-dependent proteases. Cell death was however not prevented by inhibitors of ferroptosis or MPT-driven necrosis, the regulated cell death (RCD) pathways most likely to be activated by metabolic stress. hOLs have decreased expression of VDAC1, VDAC2, and of genes regulating iron accumulation and cyclophilin. RNA sequencing analyses did not identify activation of these RCD pathways in vitro or in MS cases. We conclude that this distinct response of hOLs, including resistance to RCD, reflects the combined impact of autophagy failure, increased ROS, and calcium influx, resulting in metabolic collapse and degeneration of cellular structural integrity. Defining the basis of OL injury and death provides guidance for development of neuro-protective strategies.

NfL and GFAP in serum are associated with microstructural brain damage in progressive multiple sclerosis.

BACKGROUND: The potential of neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) as biomarkers of disease activity and severity in progressive forms of multiple sclerosis (MS) is unclear. OBJECTIVE: To investigate the relationship between serum concentrations of NfL, GFAP, and magnetic resonance imaging (MRI) in progressive MS. METHODS: Serum concentrations of NfL and GFAP were measured in 32 healthy controls and 32 patients with progressive MS from whom clinical and MRI data including diffusion tensor imaging (DTI) were obtained during three years of follow-up. RESULTS: Serum concentrations of NfL and GFAP at follow-up were higher in progressive MS patients than in healthy controls and serum NfL correlated with the EDSS score. Decreasing fractional anisotropy (FA) in normal-appearing white matter (NAWM) correlated with worsening EDSS scores and higher serum NfL. Higher serum NfL and increasing T2 lesion volume correlated with worsening paced autitory serial addition test scores. In multivariable regression analyses with serum GFAP and NfL as independent factors and DTI measures of NAWM as dependent factors, we showed that high serum NfL at follow-up was independently associated with decreasing FA and increasing MD in NAWM. Moreover, we found that high serum GFAP was independently associated with decreasing MD in NAWM and with decreasing MD and increasing FA in cortical gray matter. CONCLUSION: Serum concentrations of NfL and GFAP are increased in progressive MS and are associated with distinct microstructural changes in NAWM and CGM.

Thalamic asymmetry in Multiple Sclerosis.

BACKGROUND: Multiple Sclerosis (MS) is a chronic neuroinflammatory disease that affects the central nervous system. Asymmetry is one of the finding in brain MRI of these patients, which is related to the debilitating symptoms of the disease. This study aimed to investigate and compare the thalamic asymmetry in MS patients and its relationship with other MRI and clinical findings of these patients. METHODS: This cross-sectional study conducted on 83 patients with relapse-remitting MS (RRMS), 43 patients with secondary progressive MS (SPMS), and 89 healthy controls. The volumes of total intracranial, total gray matter, total white matter, lesions, thalamus, and also the thalamic asymmetry indices were calculated. The 9-hole peg test (9-HPT) and Expanded Disability Status Scale (EDSS) were assessed as clinical findings. RESULTS: We showed that the normalized whole thalamic volume in healthy subjects was higher than MS patients (both RRMS and SPMS). Thalamic asymmetry index (TAI) was significantly different between RRMS patients and SPMS patients (p = 0.011). The absolute value of TAI was significantly lower in healthy subjects than in RRMS (p < 0.001) and SPMS patients (p < 0.001), and SPMS patients had a higher absolute TAI compared to RRMS patients (p = 0.037). CONCLUSIONS: In this cross-sectional study we showed a relationship between normalized whole thalamic volume and MS subtype. Also, we showed that the asymmetric indices of the thalamus can be related to the progression of the disease. Eventually, we showed that thalamic asymmetry can be related to the disease progression and subtype changes in MS.

Cardiovascular risk factors in secondary progressive multiple sclerosis: A cross-sectional analysis from the MS-STAT2 randomized controlled trial.

BACKGROUND AND PURPOSE: There is increasing evidence that cardiovascular risk (CVR) contributes to disability progression in multiple sclerosis (MS). CVR is particularly prevalent in secondary progressive MS (SPMS) and can be quantified through validated composite CVR scores. The aim was to examine the cross-sectional relationships between excess modifiable CVR, whole and regional brain atrophy on magnetic resonance imaging, and disability in patients with SPMS. METHODS: Participants had SPMS, and data were collected at enrolment into the MS-STAT2 trial. Composite CVR scores were calculated using the QRISK3 software. Prematurely achieved CVR due to modifiable risk factors was expressed as QRISK3 premature CVR, derived through reference to the normative QRISK3 dataset and expressed in years. Associations were determined with multiple linear regressions. RESULTS: For the 218 participants, mean age was 54 years and median Expanded Disability Status Scale was 6.0. Each additional year of prematurely achieved CVR was associated with a 2.7 mL (beta coefficient; 95% confidence interval 0.8-4.7; p = 0.006) smaller normalized whole brain volume. The strongest relationship was seen for the cortical grey matter (beta coefficient 1.6 mL per year; 95% confidence interval 0.5-2.7; p = 0.003), and associations were also found with poorer verbal working memory performance. Body mass index demonstrated the strongest relationships with normalized brain volumes, whilst serum lipid ratios demonstrated strong relationships with verbal and visuospatial working memory performance. CONCLUSIONS: Prematurely achieved CVR is associated with lower normalized brain volumes in SPMS. Future longitudinal analyses of this clinical trial dataset will be important to determine whether CVR predicts future disease worsening.