The comparative effects of bone marrow mesenchymal stem cells and supernatant transplantation on demyelination and inflammation in cuprizone model.

BACKGROUND: Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS) with inflammation and immune dysfunction. OBJECTIVES: We compared the remyelination and immunomodulation properties of mesenchymal stem cells (MSCs) with their conditioned medium (CM) in the cuprizone model. METHODS: Twenty-four C57BL/ 6 mice were divided into four groups. After cuprizone demyelination, MSCs and their CM were injected into the right lateral ventricle of mice. The expression level of IL-1ß, TNF-α, and BDNF genes was evaluated using the qRT-PCR. APC antibody was used to assess the oligodendrocyte population using the immunofluorescent method. The remyelination and axonal repair were studied by specific staining of the LFB and electron microscopy techniques. RESULTS: Transplantation of MSCs and CM increased the expression of the BDNF gene and decreased the expression of IL-1ß and TNF-α genes compared to the cuprizone group, and these effects in the cell group were more than CM. Furthermore, cell transplantation resulted in a significant improvement in myelination and axonal repair, which was measured by luxol fast blue and transmission electron microscope images. The cell group had a higher number of oligodendrocytes than other groups. CONCLUSIONS: According to the findings, injecting MSCs intraventricularly versus cell-conditioned medium can be a more effective approach to improving chronic demyelination in degenerative diseases like MS.

Targeting of KOR by famotidine promotes OPC maturation differentiation and CNS remyelination via STAT3 signaling pathway.

This study aims to identify FDA-approved drugs that can target the kappa-opioid receptor (KOR) for the treatment of demyelinating diseases. Demyelinating diseases are characterized by myelin sheath destruction or formation that results in severe neurological dysfunction. Remission of this disease is largely dependent on the differentiation of oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes (OLGs) in demyelinating lesions. KOR is an important regulatory protein and drug target for the treatment of demyelinating diseases. However, no drug targeting KOR has been developed due to the long clinical trials for drug discovery. Here, a structure-based virtual screening was applied to identify drugs targeting KOR among 1843 drugs of FDA-approved drug libraries, and famotidine was screen out by its high affinity cooperation with KOR as well as the clinical safety. We discovered that famotidine directly promoted OPC maturation and remyelination using the complementary in vitro and in vivo models. Administration of famotidine was not only effectively enhanced CNS myelinogenesis, but also promoted remyelination. Mechanically speaking, famotidine promoted myelinogenesis or remyelination through KOR/STAT3 signaling pathway. In general, our study provided evidence of new clinical applicability of famotidine for the treatment of demyelinating diseases for which there is currently no effective therapy.

Improving explanation of motor disability with diffusion-based graph metrics at onset of the first demyelinating event.

BACKGROUND: Conventional magnetic resonance imaging (MRI) does not account for all disability in multiple sclerosis. OBJECTIVE: The objective was to assess the ability of graph metrics from diffusion-based structural connectomes to explain motor function beyond conventional MRI in early demyelinating clinically isolated syndrome (CIS). METHODS: A total of 73 people with CIS underwent conventional MRI, diffusion-weighted imaging and clinical assessment within 3 months from onset. A total of 28 healthy controls underwent MRI. Structural connectomes were produced. Differences between patients and controls were explored; clinical associations were assessed in patients. Linear regression models were compared to establish relevance of graph metrics over conventional MRI. RESULTS: Local efficiency (p = 0.045), clustering (p = 0.034) and transitivity (p = 0.036) were reduced in patients. Higher assortativity was associated with higher Expanded Disability Status Scale (EDSS) (ß = 74.9, p = 0.026) scores. Faster timed 25-foot walk (T25FW) was associated with higher assortativity (ß = 5.39, p = 0.026), local efficiency (ß = 27.1, p = 0.041) and clustering (ß = 36.1, p = 0.032) and lower small-worldness (ß = -3.27, p = 0.015). Adding graph metrics to conventional MRI improved EDSS (p = 0.045, ΔR2 = 4) and T25FW (p < 0.001, ΔR2 = 13.6) prediction. CONCLUSION: Graph metrics are relevant early in demyelination. They show differences between patients and controls and have relationships with clinical outcomes. Segregation (local efficiency, clustering, transitivity) was particularly relevant. Combining graph metrics with conventional MRI better explained disability.

Small-molecule-induced epigenetic rejuvenation promotes SREBP condensation and overcomes barriers to CNS myelin regeneration.

Remyelination failure in diseases like multiple sclerosis (MS) was thought to involve suppressed maturation of oligodendrocyte precursors; however, oligodendrocytes are present in MS lesions yet lack myelin production. We found that oligodendrocytes in the lesions are epigenetically silenced. Developing a transgenic reporter labeling differentiated oligodendrocytes for phenotypic screening, we identified a small-molecule epigenetic-silencing-inhibitor (ESI1) that enhances myelin production and ensheathment. ESI1 promotes remyelination in animal models of demyelination and enables de novo myelinogenesis on regenerated CNS axons. ESI1 treatment lengthened myelin sheaths in human iPSC-derived organoids and augmented (re)myelination in aged mice while reversing age-related cognitive decline. Multi-omics revealed that ESI1 induces an active chromatin landscape that activates myelinogenic pathways and reprograms metabolism. Notably, ESI1 triggered nuclear condensate formation of master lipid-metabolic regulators SREBP1/2, concentrating transcriptional co-activators to drive lipid/cholesterol biosynthesis. Our study highlights the potential of targeting epigenetic silencing to enable CNS myelin regeneration in demyelinating diseases and aging.

The level of cytokines in tears as a novel indicator of demyelinating diseases.

INTRODUCTION: A novel research objective is to identify new molecules in more readily accessible biological fluids that could be used in the diagnosis of multiple sclerosis (MS) and other demyelinating disorders. AIM: To compare the level of selected cytokines in tears between patients with MS or other demyelinating disorder and healthy controls. MATERIAL AND METHODS: 84 patients with diagnosed MS during remission or with other demyelinating disease of the CNS and 70 healthy controls were enrolled in the study. Tears were collected without any stimulation and stored till the day of assessment. The concentration of selected cytokines was measured by the Bio-Plex Pro Human cytokine screening panel 27 cytokines assay according to the manufacturer's instructions. Statistical analysis was performed with Statistica 13. RESULTS: IL-1b level was significantly lower in the study group compared to the control group [3,6 vs 8.71, p < 0.001]. The same pattern was observed for IL-6 [3,1 vs 5.26, p = 0.027] and IL-10 [1,7 vs 10.92, p < 0.001] (Table 1). In the study group, IL-1RA (p = 0.015), IL-5 (p = 0.04), IL-9 (p = 0.014), and IL-15 (p = 0.037) showed significant correlations with age. In the total sample, IL-1Ra (p = 0.016) and IFN-g (p = 0.041) were significantly correlated with age, while in the control group, IL-8 (p = 0.09), MIP-1a (p = 0.009), and RANTES (p = 0.031) showed significant correlations. CONCLUSIONS: Our results show that MS and other demyelination diseases lead to decrease in the overall level of cytokines in tears. Further research is needed to determine the role of tear fluid in the assessment of demyelinating disorders like MS.

The Smoothened agonist SAG Modulates the Male and Female Peripheral Immune Systems Differently in an Immune Model of Central Nervous System Demyelination.

Both Hedgehog and androgen signaling pathways are known to promote myelin regeneration in the central nervous system. Remarkably, the combined administration of agonists of each pathway revealed their functional cooperation towards higher regeneration in demyelination models in males. Since multiple sclerosis, the most common demyelinating disease, predominates in women, and androgen effects were reported to diverge according to sex, it seemed essential to assess the existence of such cooperation in females. Here, we developed an intranasal formulation containing the Hedgehog signaling agonist SAG, either alone or in combination with testosterone. We show that SAG promotes myelin regeneration and presumably a pro-regenerative phenotype of microglia, thus mimicking the effects previously observed in males. However, unlike in males, the combined molecules failed to cooperate in the demyelinated females, as shown by the level of functional improvement observed. Consistent with this observation, SAG administered in the absence of testosterone amplified peripheral inflammation by presumably activating NK cells and thus counteracting a testosterone-induced reduction in Th17 cells when the molecules were combined. Altogether, the data uncover a sex-dependent effect of the Hedgehog signaling agonist SAG on the peripheral innate immune system that conditions its ability to cooperate or not with androgens in the context of demyelination.

5Z-7-Oxozaenol attenuates cuprizone-induced demyelination in mice through microglia polarization regulation.

INTRODUCTION: Demyelination is a key factor in axonal degeneration and neural loss, leading to disability in multiple sclerosis (MS) patients. Transforming growth factor beta activated kinase 1 (TAK1) is a critical molecule involved in immune and inflammatory signaling pathways. Knockout of microglia TAK1 can inhibit autoimmune inflammation of the brain and spinal cord and improve the outcome of MS. However, it is unclear whether inhibiting TAK1 can alleviate demyelination. METHODS: Eight-week-old male c57bl/6j mice were randomly divided into five groups: (a) the control group, (b) the group treated with cuprizone (CPZ) only, (c) the group treated with 5Z-7-Oxozaenol (OZ) only, and (d) the group treated with both cuprizone and 15 µg/30 µg OZ. Demyelination in the mice of this study was induced by administration of CPZ (ig) at a daily dose of 400 mg/kg for consecutive 5 weeks. OZ was intraperitoneally administered at mentioned doses twice a week, starting from week 3 after beginning cuprizone treatment. Histology, rotarod test, grasping test, pole test, Western blot, RT-PCR, and ELISA were used to evaluate corpus callosum demyelination, behavioral impairment, oligodendrocyte differentiation, TAK1 signaling pathway expression, microglia, and related cytokines. RESULTS: Our results demonstrated that OZ protected against myelin loss and behavior impairment caused by CPZ. Additionally, OZ rescued the loss of oligodendrocytes in CPZ-induced mice. OZ inhibited the activation of JNK, p65, and p38 pathways, transformed M1 polarized microglia into M2 phenotype, and increased brain-derived neurotrophic factor (BDNF) expression to attenuate demyelination in CPZ-treated mice. Furthermore, OZ reduced the expression of proinflammatory cytokines and increases anti-inflammatory cytokines in CPZ-treated mice. CONCLUSION: These findings suggest that inhibiting TAK1 may be an effective approach for treating demyelinating diseases.

Assessment of dietary intake and its inflammatory potential in persons with pediatric-onset multiple sclerosis.

OBJECTIVE: To compare diet and the modified dietary inflammatory index (mDII) between individuals with pediatric-onset multiple sclerosis (PoMS), monophasic acquired demyelinating syndromes (monoADS), and controls. METHODS: The association between diet, mDII, and disease status was examined in 131 individuals with PoMS/monoADS/controls (38/45/48) using logistic regression. RESULTS: The associations between diet and PoMS were modest, reaching significance for whole grain intake (adjusted odds ratio, aOR=0.964, 95 % confidence intervals, CI:0.934-0.995) but not mDII (aOR=1.20, 95 %CI:0.995-1.46) versus controls. No findings for monoADS reached significance versus controls. CONCLUSIONS: Individuals with PoMS, but not monoADS, had lower dietary whole grain intake than controls.

Predicting the conversion from clinically isolated syndrome to multiple sclerosis: An explainable machine learning approach.

INTRODUCTION: Predicting the conversion of clinically isolated syndrome (CIS) to clinically definite multiple sclerosis (CDMS) is critical to personalizing treatment planning and benefits for patients. The aim of this study is to develop an explainable machine learning (ML) model for predicting this conversion based on demographic, clinical, and imaging data. METHOD: The ML model, Extreme Gradient Boosting (XGBoost), was employed on the public dataset of 273 Mexican mestizo CIS patients with 10-year follow-up. The data was divided into a training set for cross-validation and feature selection, and a holdout test set for final testing. Feature importance was determined using the SHapley Additive Explanations library (SHAP). Then, two experiments were conducted to optimize the model's performance by selectively adding variables and selecting the most contributive variables for the final model. RESULTS: Nine variables including age, gender, schooling, motor symptoms, infratentorial and periventricular lesion at imaging, oligoclonal band in cerebrospinal fluid, lesion and symptoms types were significant. The model achieved an accuracy of 83.6 %, AUC of 91.8 %, sensitivity of 83.9 %, and specificity of 83.4 % in cross-validation. In the final testing, the model achieved an accuracy of 78.3 %, AUC of 85.8 %, sensitivity of 75 %, and specificity of 81.1 %. Finally, a web-based demo of the model was created for testing purposes. CONCLUSION: The model, focusing on feature selection and interpretability, effectively stratifies risk for treatment decisions and disability prevention in MS patients. It provides a numerical risk estimate for CDMS conversion, enhancing transparency in clinical decision-making and aiding in patient care.

Automated whole slide morphometry of sural nerve biopsy using machine learning.

AIM: The morphometry of sural nerve biopsies, such as fibre diameter and myelin thickness, helps us understand the underlying mechanism of peripheral neuropathies. However, in current clinical practice, only a portion of the specimen is measured manually because of its labour-intensive nature. In this study, we aimed to develop a machine learning-based application that inputs a whole slide image (WSI) of the biopsied sural nerve and automatically performs morphometric analyses. METHODS: Our application consists of three supervised learning models: (1) nerve fascicle instance segmentation, (2) myelinated fibre detection and (3) myelin sheath segmentation. We fine-tuned these models using 86 toluidine blue-stained slides from various neuropathies and developed an open-source Python library. RESULTS: Performance evaluation showed (1) a mask average precision (AP) of 0.861 for fascicle segmentation, (2) box AP of 0.711 for fibre detection and (3) a mean intersection over union (mIoU) of 0.817 for myelin segmentation. Our software identified 323,298 nerve fibres and 782 fascicles in 70 WSIs. Small and large fibre populations were objectively determined based on clustering analysis. The demyelination group had large fibres with thinner myelin sheaths and higher g-ratios than the vasculitis group. The slope of the regression line from the scatter plots of the diameters and g-ratios was higher in the demyelination group than in the vasculitis group. CONCLUSION: We developed an application that performs whole slide morphometry of human biopsy samples. Our open-source software can be used by clinicians and pathologists without specific machine learning skills, which we expect will facilitate data-driven analysis of sural nerve biopsies for a more detailed understanding of these diseases.

Clinical and paraclinical characteristics of optic neuritis in the context of the McDonald criteria 2017.

Optic neuritis is often an initial symptom in multiple sclerosis (MS) or clinically isolated syndrome (CIS), yet comprehensive studies using the 2017 McDonald criteria for MS are scarce. Patient records from our academic centre (2010-2018) were reviewed. Using the 2017 McDonald criteria, three groups were formed: MS optic neuritis (optic neuritis with confirmed MS), CIS optic neuritis (optic neuritis without confirmed MS) and suspected optic neuritis (sON). We compared clinical and paraclinical findings among the groups to identify predictors for CIS- or MS-optic neuritis. The study included 129 MS, 108 CIS, and 44 sON cases. The combination of visual impairment, dyschromatopsia, and retrobulbar pain was observed in 47% of MS patients, 42% of CIS patients, and 30% of sON patients. Dyschromatopsia was the strongest indicator of MS or CIS diagnosis in the backward regression model. 56% of MS patients had relative afferent pupillary defect, 61% optic nerve anomalies within magnetic resonance imaging, and 81% abnormal visual evoked potentials. Our results emphasize the challenges in diagnosing optic neuritis, as not all patients with objectively diagnosed MS exhibit the triad of typical symptoms. To address potentially missing clinical features, incorporating additional paraclinical findings is proposed.

Leukodystrophy with Macrocephaly, Refractory Epilepsy, and Severe Hyponatremia-The Neonatal Type of Alexander Disease.

INTRODUCTION: Alexander disease (AxD) is a rare neurodegenerative condition that represents the group of leukodystrophies. The disease is caused by GFAP mutation. Symptoms usually occur in the infantile age with macrocephaly, developmental deterioration, progressive quadriparesis, and seizures as the most characteristic features. In this case report, we provide a detailed clinical description of the neonatal type of AxD. METHOD: Next-Generation Sequencing (NGS), including a panel of 49 genes related to Early Infantile Epileptic Encephalopathy (EIEE), was carried out, and then Whole Exome Sequencing (WES) was performed on the proband's DNA extracted from blood. CASE DESCRIPTION: In the first weeks of life, the child presented with signs of increased intracranial pressure, which led to ventriculoperitoneal shunt implementation. Recurrent focal-onset motor seizures with secondary generalization occurred despite phenobarbital treatment. Therapy was modified with multiple anti-seizure medications. In MRI contrast-enhanced lesions in basal ganglia, midbrain and cortico-spinal tracts were observed. During the diagnostic process, GLUT-1 deficiency, lysosomal storage disorders, organic acidurias, and fatty acid oxidation defects were excluded. The NGS panel of EIEE revealed no abnormalities. In WES analysis, GFAP missense heterozygous variant NM_002055.5: c.1187C>T, p.(Thr396Ile) was detected, confirming the diagnosis of AxD. CONCLUSION: AxD should be considered in the differential diagnosis in all neonates with progressive, intractable seizures accompanied by macrocephaly.

Radiologically isolated syndromes: to treat or not to treat?

The widespread use of magnetic resonance imaging (MRI) has led to increased detection of individuals exhibiting asymptomatic brain and spinal cord lesions suggestive of multiple sclerosis (MS), defined as "radiologically isolated syndrome" (RIS). Specific criteria have been proposed and updated over time to identify individuals with RIS. Moreover, a younger age, the presence of infratentorial, spinal cord or gadolinium-enhancing lesions, as well as of cerebrospinal fluid-specific oligoclonal bands have been recognized as relevant risk factors for the occurrence of a first clinical event. Recent randomized controlled trials conducted in individuals with RIS have shown that dimethyl fumarate and teriflunomide significantly reduce the occurrence of clinical events in this population. These findings support the notion that early treatment initiation may positively influence the prognosis of these patients. However, several aspects should be taken into account before treating individuals with RIS in the real-world clinical setting, including an accurate identification of individuals with RIS to avoid misdiagnosis, a precise stratification of their risk of experiencing a first clinical event and further data supporting favorable balance between benefits and risks, even in the long term. This commentary provides an overview of the latest updates in RIS diagnosis, prognosis, and emerging treatment evidence.

Serum neurofilament light for detecting disease activity in individual patients in multiple sclerosis: A 48-week prospective single-center study.

BACKGROUND: Serum neurofilament light (sNfL) reflects neuroaxonal damage and is now used as an outcome in treatment trials of relapsing-remitting multiple sclerosis (RRMS). However, the diagnostic properties of sNfL for monitoring disease activity in individual patients warrant further investigations. METHOD: Patients with suspected relapse and/or contrast-enhancing lesions (CELs) were consecutively included and performed magnetic resonance imaging (MRI) of the brain at baseline and weeks 28 and 48. Serum was obtained at baseline and 2, 4, 8, 16, 24, and 48 weeks. Neurofilament light concentration was measured using Single molecule array technology. RESULTS: We included 44 patients, 40 with RRMS and 4 with clinically isolated syndrome. The median sNfL level peaked at 2 weeks post-baseline (14.6 ng/L, interquartile range (IQR); 9.3-31.6) and reached nadir at 48 weeks (9.1 ng/L, IQR; 5.5-15.0), equivalent to the median sNfL of controls (9.1 ng/L, IQR; 7.4-12). A baseline Z-score of more than 1.1 (area under the curve; 0.78, p < 0.0001) had a sensitivity of 81% and specificity of 70% to detect disease activity. CONCLUSION: One out of five patients with relapse and/or CELs did not change significantly in post-baseline sNfL levels. The utility of repeated sNfL measurements to monitor disease activity is complementary rather than a substitute for clinical and MRI measures.

Matching proposed clinical and MRI criteria of aggressive multiple sclerosis to serum and cerebrospinal fluid markers of neuroaxonal and glial injury.

BACKGROUND: Definitions of aggressive MS employ clinical and MR imaging criteria to identify highly active, rapidly progressing disease courses. However, the degree of overlap between clinical and radiological parameters and biochemical markers of CNS injury is not fully understood. Aim of this cross-sectional study was to match clinical and MR imaging hallmarks of aggressive MS to serum/CSF markers of neuroaxonal and astroglial injury (neurofilament light chain (sNfL, cNfL), and glial fibrillary acidic protein (sGFAP, cGFAP)). METHODS: We recruited 77 patients with relapsing-remitting MS (RRMS) and 22 patients with clinically isolated syndrome. NfL and GFAP levels in serum and CSF were assessed using a single-molecule-array HD-1-analyzer. A general linear model with each biomarker as a dependent variable was computed. Clinical and imaging criteria of aggressive MS, as recently proposed by the ECTRIMS Consensus Group, were modeled as independent variables. Other demographic, clinical or laboratory parameters, were modeled as covariates. Analyses were repeated in a homogenous subgroup, consisting only of newly diagnosed, treatment-naïve RRMS patients presenting with an acute relapse. RESULTS: After adjusting for covariates and multiplicity of testing, sNfL and cNfL concentrations were strongly associated with the presence of ≥2 gadolinium-enhancing lesions (psNfL = 0.00008; pcNfL = 0.004) as well as the presence of infratentorial lesions on MRI (psNfL = 0.0003; pcNfL < 0.004). No other clinical and imaging criteria of aggressive MS correlated significantly with NfL or GFAP in serum and CSF. In the more homogeneous subgroup, sNfL still was associated with the presence of ≥2 gadolinium-enhancing lesions (psNfL = 0.001), presence of more than 20 T2-lesions (psNfL = 0.049) as well as the presence of infratentorial lesions on MRI (psNfL = 0.034), while cNfL was associated with the presence of ≥2 gadolinium-enhancing lesions (psNfL = 0.011) and presence of more than 20 T2-lesions (psNfL = 0.029). CONCLUSIONS: Among proposed risk factors for an aggressive disease course, MRI findings but not clinical characteristics correlated with sNfL and cNfL as a marker of neuroaxonal injury and should be given appropriate weight considering MS prognosis and therapy. No significant correlation was detected for GFAP alone.

Long-term in vivo three-photon imaging reveals region-specific differences in healthy and regenerative oligodendrogenesis.

The generation of new myelin-forming oligodendrocytes in the adult central nervous system is critical for cognitive function and regeneration following injury. Oligodendrogenesis varies between gray and white matter regions, suggesting that local cues drive regional differences in myelination and the capacity for regeneration. However, the layer- and region-specific regulation of oligodendrocyte populations is unclear due to the inability to monitor deep brain structures in vivo. Here we harnessed the superior imaging depth of three-photon microscopy to permit long-term, longitudinal in vivo three-photon imaging of the entire cortical column and subcortical white matter in adult mice. We find that cortical oligodendrocyte populations expand at a higher rate in the adult brain than those of the white matter. Following demyelination, oligodendrocyte replacement is enhanced in the white matter, while the deep cortical layers show deficits in regenerative oligodendrogenesis and the restoration of transcriptional heterogeneity. Together, our findings demonstrate that regional microenvironments regulate oligodendrocyte population dynamics and heterogeneity in the healthy and diseased brain.

CD4+ T cell mitochondrial genotype in Multiple Sclerosis: a cross-sectional and longitudinal analysis.

Multiple Sclerosis (MS) is a chronic autoimmune demyelinating disease of the central nervous system (CNS), with a largely unknown etiology, where mitochondrial dysfunction likely contributes to neuroaxonal loss and brain atrophy. Mirroring the CNS, peripheral immune cells from patients with MS, particularly CD4+ T cells, show inappropriate mitochondrial phenotypes and/or oxidative phosphorylation (OxPhos) insufficiency, with a still unknown contribution of mitochondrial DNA (mtDNA). We hypothesized that mitochondrial genotype in CD4+ T cells might influence MS disease activity and progression. Thus, we performed a retrospective cross-sectional and longitudinal study on patients with a recent diagnosis of either Clinically Isolated Syndrome (CIS) or Relapsing-Remitting MS (RRMS) at two timepoints: 6 months (VIS1) and 36 months (VIS2) after disease onset. Our primary outcomes were the differences in mtDNA extracted from CD4+ T cells between: (I) patients with CIS/RRMS (PwMS) at VIS1 and age- and sex-matched healthy controls (HC), in the cross-sectional analysis, and (II) different diagnostic evolutions in PwMS from VIS1 to VIS2, in the longitudinal analysis. We successfully performed mtDNA whole genome sequencing (mean coverage: 2055.77 reads/base pair) in 183 samples (61 triplets). Nonetheless, mitochondrial genotype was not associated with a diagnosis of CIS/RRMS, nor with longitudinal diagnostic evolution.