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Abstract In chronic stages of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalitis (EAE), connexin (Cx)43 gap junction channel proteins are overexpressed because of astrogliosis. To elucidate the role of increased Cx43, the central nervous system (CNS)-permeable Cx blocker INI-0602 was therapeutically administered. C57BL6 mice with chronic EAE initiated by MOG35-55 received INI-0602 (40 mg/kg) or saline intraperitoneally every other day from days post-immunization (dpi) 17–50. Primary astroglia were employed to observe calcein efflux responses. In INI-0602-treated mice, EAE clinical signs improved significantly in the chronic phase, with reduced demyelination and decreased CD3+ T cells, Iba-1+ and F4/80+ microglia/macrophages, and C3+GFAP+ reactive astroglia infiltration in spinal cord lesions. Flow cytometry analysis of CD4+ T cells from CNS tissues revealed significantly reduced Th17 and Th17/Th1 cells (dpi 24) and Th1 cells (dpi 50). Multiplex array of cerebrospinal fluid showed significantly suppressed IL-6 and significantly increased IL-10 on dpi 24 in INI-0602-treated mice, and significantly suppressed IFN-γ and MCP-1 on dpi 50 in the same group. In vitro INI-0602 treatment inhibited ATP-induced calcium propagations of Cx43+/+ astroglial cells to similar levels of those of Cx43−/− cells. Astroglial Cx43 hemichannels represent a novel therapeutic target for chronic EAE and MS.

Background: Treatment for ocular myasthenia gravis (OMG) has not yet been well established. Few reports have been published on the clinical practice and outcomes of OMG. Objectives: We investigated treatment of OMG and its outcomes in Japan. Design: We performed a retrospective cross-sectional survey of OMG patients from eight hospitals in Japan. Methods: Clinical information, including sex, age at onset, initial symptoms, autoantibodies, clinical course, treatment history, complications, and outcomes, was obtained. In addition, we recorded the total number of patients with MG and OMG separately. Results: In total, 135 patients with OMG (67 men, 68 women) were included. Treatment of OMG was not simple and involved various immunotherapeutic strategies. Eight patients went into remission spontaneously without immunotherapy. A total of 117 patients showed improvements after treatment, whereas 10 patients showed refractory responses to treatment. Overall outcomes were good; however, symptoms persisted in 60.7% of patients even after treatment. Among 90 patients who received immunotherapy, only two showed a refractory response. Meanwhile, for 45 patients who did not receive immunotherapy, 8 were refractory. Thus, the rate of refractory disease in the group with immunotherapy was significantly lower ( p = 0.001, u-test) than in the group without immunotherapy. The proportion of generalized MG patients among all MG cases was low in medical centers where immunotherapy for OMG was frequently performed. Conclusion: Although the overall prognosis for patients with OMG was good, symptoms remained in more than half of the patients. Immunotherapy, including corticosteroids, may be beneficial for patients with OMG. Plain language summary Is immunosuppressive therapy beneficial for myasthenia gravis patients with ocular symptoms only? Patients with ocular myasthenia gravis (OMG) have only eye symptoms for more than 2 years. Whether this condition is an initial stage of the disease before eventually progressing to generalized myasthenia gravis (gMG) is still uncertain. Different from gMG, OMG is not life-threatening. But eye symptoms often cause troublesome problems in life. Doctors have treated OMG patients similarly to patients with gMG. There is no standard clinical practice for OMG. In this study, we examined how patients with OMG were treated at eight different specialist centers in Japan. In 135 patients with OMG, 8 patients became symptom free without treatment, 117 patients showed improvements after treatment, whereas 10 patients did not get well. Overall outcomes were good, but symptoms remained in 60.7% of patients even after treatment. Among 90 patients who received one or more immunotherapies, only 2 did not get well. Meanwhile, for 45 patients who did not receive immunotherapy, 8 remained ill. We found that treatment of OMG was not simple and often needed multiple immunotherapies. Administering immunotherapy, including corticosteroids, may be beneficial for patients with OMG.

Background Reperfusion therapy for acute ischemic stroke efficacy is highly time dependent; therefore, stroke centers are required to further reduce the delays from hospital arrival to treatment efficiently. We developed a visual task management application, Task Calculation Stroke (Task Calc. Stroke: TCS), to facilitate hospital acute ischemic stroke treatment by supporting parallel staff task completion. We evaluated TCS for the reduction of reperfusion therapy delays and improvement of clinical outcomes. Methods In this multicenter cohort study, patients were directly admitted to 4 comprehensive stroke centers in Japan and given intravenous tissue plasminogen activator and/or mechanical thrombectomy from June 2018 to December 2020. The research team visited each facility and instructed the staff on TCS use for acute ischemic stroke (training stage), after which the staff used TCS independently (TCS stage). We then compared door‐to‐needle time for intravenous tissue plasminogen activator, door‐to‐puncture time for mechanical thrombectomy, and clinical outcomes at discharge according to the modified Rankin Scale among patients treated before training (original stage), during the training stage, or the TCS stage. Results During the study period, 316 patients with acute ischemic stroke received reperfusion therapy; of these, 246 received intravenous tissue plasminogen activator and 162 mechanical thrombectomy (including 92 receiving both the treatments). The mean door‐to‐needle time was significantly reduced from 58.0 minutes in the original stage to 54.6 minutes in the training stage (P=0.049) and 47.8 minutes in the TCS stage (P

We previously reported a novel secondary progressive multiple sclerosis (SPMS) model, progressive experimental autoimmune encephalomyelitis (pEAE), in oligodendroglia-specific Cx47-inducible conditional knockout (Cx47 icKO) mice. Based on our prior study showing the efficacy of iguratimod (IGU), an antirheumatic drug, for acute EAE treatment, we aimed to elucidate the effect of IGU on the SPMS animal model. We induced pEAE by immunizing Cx47 icKO mice with myelin oligodendrocyte glycoprotein peptide 35–55. IGU was orally administered from 17 to 50 days post-immunization. We also prepared a primary mixed glial cell culture and measured cytokine levels in the culture supernatant after stimulation with designated cytokines (IL-1α, C1q, TNF-α) and lipopolysaccharide. A migration assay was performed to evaluate the effect of IGU on the migration ability of T cells toward mixed glial cell cultures. IGU treatment ameliorated the clinical signs of pEAE, decreased the demyelinated area, and attenuated glial inflammation on immunohistochemical analysis. Additionally, IGU decreased the intrathecal IL-6 level and infiltrating Th17 cells. The migration assay revealed reduced Th17 cell migration and IL-6 levels in the culture supernatant after IGU treatment. Collectively, IGU successfully mitigated the clinical signs of pEAE by suppressing Th17 migration through inhibition of IL-6 production by proinflammatory-activated glial cells.

Abstract Macrophages expressing C–C chemokine receptor type 2 (CCR2) infiltrate the central and peripheral neural tissues of amyotrophic lateral sclerosis (ALS) patients. To identify the functional role of CCR2+ macrophages in the pathomechanisms of ALS, we used an ALS animal model, mutant Cu/Zn superoxide dismutase 1 G93A (mSOD1)-transgenic (Tg) mice. To clarify the CCR2 function in the model, we generated SOD1 G93A/CCR2 Red fluorescence protein (RFP)/Wild type (WT) /CX3CR1 Green fluorescence protein (GFP)/WT -Tg mice, which heterozygously express CCR2-RFP and CX3CR1-GFP, and SOD1 G93A/CCR2 RFP/RFP-Tg mice, which lack CCR2 protein expression and present with a CCR2-deficient phenotype. In mSOD1-Tg mice, mSOD1 accumulated in the sciatic nerve earlier than in the spinal cord. Furthermore, spinal cords of SOD1 G93A/CCR2 RFP/WT/CX3CR1 GFP/WT mice showed peripheral macrophage infiltration that emerged at the end-stage, whereas in peripheral nerves, macrophage infiltration started from the pre-symptomatic stage. Before disease onset, CCR2+ macrophages harboring mSOD1 infiltrated sciatic nerves earlier than the lumbar cord. CCR2-deficient mSOD1-Tg mice showed an earlier onset and axonal derangement in the sciatic nerve than CCR2-positive mSOD1-Tg mice. CCR2-deficient mSOD1-Tg mice showed an increase in deposited mSOD1 in the sciatic nerve compared with CCR2-positive mice. These findings suggest that CCR2+ and CX3CR1+ macrophages exert neuroprotective functions in mSOD1 ALS via mSOD1 clearance from the peripheral nerves.

Abstract Background Brain astroglia are activated preceding the onset of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We characterized the effects of brain astroglia on spinal cord inflammation, focusing on astroglial connexin (Cx)43, because we recently reported that Cx43 has a critical role in regulating neuroinflammation. Methods Because glutamate aspartate transporter (GLAST)+ astroglia are enriched in the brain gray matter, we generated Cx43 fl/fl ;GLAST-CreER T2/+ mice that were brain gray matter astroglia-specific Cx43 conditional knockouts (Cx43 icKO). EAE was induced by immunization with myelin oligodendroglia glycoprotein (MOG) 35–55 peptide 10 days after tamoxifen injection. Cx43 fl/fl mice were used as controls. Results Acute and chronic EAE signs were significantly milder in Cx43 icKO mice than in controls whereas splenocyte MOG-specific responses were unaltered. Histologically, Cx43 icKO mice showed significantly less demyelination and fewer CD45+ infiltrating immunocytes, including F4/80+ macrophages, and Iba1+ microglia in the spinal cord than controls. Microarray analysis of the whole cerebellum revealed marked upregulation of anti-inflammatory A2-specific astroglia gene sets in the pre-immunized phase and decreased proinflammatory A1-specific and pan-reactive astroglial gene expression in the onset phase in Cx43 icKO mice compared with controls. Astroglia expressing C3, a representative A1 marker, were significantly decreased in the cerebrum, cerebellum, and spinal cord of Cx43 icKO mice compared with controls in the peak phase. Isolated Cx43 icKO spinal microglia showed more anti-inflammatory and less proinflammatory gene expression than control microglia in the pre-immunized phase. In particular, microglial expression of Ccl2, Ccl5, Ccl7, and Ccl8 in the pre-immunized phase and of Cxcl9 at the peak phase was lower in Cx43 icKO than in controls. Spinal microglia circularity was significantly lower in Cx43 icKO than in controls in the peak phase. Significantly lower interleukin (IL)-6, interferon-γ, and IL-10 levels were present in cerebrospinal fluid from Cx43 icKO mice in the onset phase compared with controls. Conclusions The ablation of Cx43 in brain gray matter astroglia attenuates EAE by promoting astroglia toward an anti-inflammatory phenotype and suppressing proinflammatory activation of spinal microglia partly through depressed cerebrospinal fluid proinflammatory cytokine/chemokine levels. Brain astroglial Cx43 might be a novel therapeutic target for MS.

Abstract HLA genotype-clinical phenotype correlations are not established for multiple sclerosis (MS) and neuromyelitis optica spectrum disorders (NMOSD). We studied HLA-DRB1/DPB1 genotype–phenotype correlations in 528 MS and 165 NMOSD cases using Japan MS/NMOSD Biobank materials. HLA-DRB1*04:05, DRB1*15:01 and DPB1*03:01 correlated with MS susceptibility and DRB1*01:01, DRB1*09:01, DRB1*13:02 and DPB1*04:01 were protective against MS. HLA-DRB1*15:01 was associated with increased optic neuritis and cerebellar involvement and worsened visual and pyramidal functional scale (FS) scores, resulting in higher progression index values. HLA-DRB1*04:05 was associated with younger onset age, high visual FS scores, and a high tendency to develop optic neuritis. HLA-DPB1*03:01 increased brainstem and cerebellar FS scores. By contrast, HLA-DRB1*01:01 decreased spinal cord involvement and sensory FS scores, HLA-DRB1*09:01 decreased annualized relapse rate, brainstem involvement and bowel and bladder FS scores, and HLA-DRB1*13:02 decreased spinal cord and brainstem involvement. In NMOSD, HLA-DRB1*08:02 and DPB1*05:01 were associated with susceptibility and DRB1*09:01 was protective. Multivariable analysis revealed old onset age, long disease duration, and many relapses as independent disability risks in both MS and NMOSD, and HLA-DRB1*15:01 as an independent risk only in MS. Therefore, both susceptibility and protective alleles can influence the clinical manifestations in MS, while such genotype–phenotype correlations are unclear in NMOSD.

BackgroundAlzheimer’s disease (AD) is the most common condition of all neurodegenerative diseases and is characterized by various cognitive dysfunctions. Recent resting-state functional magnetic resonance imaging (rs-fMRI) studies have revealed the physiological dynamics of functionally connected brain networks, which are called resting-state networks (RSNs). Associations between impairments of RSNs and various neuropsychiatric diseases, such as AD, have been reported. Acetylcholinesterase inhibitors (AChEIs) have been used as a pharmacological treatment for mild-to-moderate moderate AD, and short-term improvements in cognitive functions and RSNs in restricted areas have been reported.ObjectiveWe aimed to characterize AChEI-related RSN changes by acquiring two sets of rs-fMRI data separated by approximately 3 to 6 months.MethodsSeventeen patients with AD and nine healthy subjects participated in this study. Independent component analysis was performed on the rs-fMRI data of AChEI-responsive and non-responsive AD patients, stratified according to change in Mini-Mental State Examination (MMSE) scores after 3 to 6 months of AChEI therapy. In addition, a region of interest-based analysis of the rs-fMRI data before therapy was performed to explore the functional connectivity (FC) changes associated with AchEI therapy.ResultsResponders showed a significantly greater increase in MMSE scores, especially for orientation for time, than that of non-responders following AChEI therapy. A subtraction map of MMSE score differences (responders minus non-responders) in the independent component analysis revealed higher FC of the dorsal attention network in responders compared with that in non-responders. Moreover, in the region of interest analysis of untreated status data, the dorsal attention network showed significant negative FC with the right planum temporale, which belongs to the ventral attention network, proportional to MMSE score change.ConclusionThe negative correlation of the FC of the dorsal attention network and right planum temporale before AChEI therapy and MMSE score change may be a biomarker of the therapeutic effect of AChEIs for AD.

Abstract Objective To characterize the frequency and patterns of optic, trigeminal, and facial nerve involvement by neuroimaging and electrophysiology in IgG4 anti‐neurofascin 155 antibody‐positive (NF155+) chronic inflammatory demyelinating polyneuropathy (CIDP). Methods Thirteen IgG4 NF155+ CIDP patients with mean onset age of 34 years (11 men) were subjected to neurological examination, blink reflex, and visual‐evoked potential (VEP) testing, and axial and/or coronal T2‐weighted head magnetic resonance imaging (MRI). Results Among 13 patients, facial sensory impairment, facial weakness, and apparent visual impairment were observed in three (23.1%), two (15.4%), and two (15.4%) patients, respectively. All 12 patients tested had blink reflex abnormalities: absent and/or delayed R1 in 11 (91.7%), and absent and/or delayed R2 in 10 (83.3%). R1 latencies had strong positive correlations with serum anti‐NF155 antibody levels (r = 0.9, P ≤ 0.0001 on both sides) and distal and F wave latencies of the median and ulnar nerves. Absent and/or prolonged VEPs were observed in 10/13 (76.9%) patients and 17/26 (65.4%) eyes. On MRI, hypertrophy, and high signal intensity of trigeminal nerves were detected in 9/13 (69.2%) and 10/13 (76.9%) patients, respectively, whereas optic nerves were normal in all patients. The intra‐orbital trigeminal nerve width on coronal sections showed a significant positive correlation with disease duration. Interpretation Subclinical demyelination frequently occurs in the optic, trigeminal, and facial nerves in IgG4 NF155+ CIDP, suggesting that both central and peripheral myelin structures of the cranial nerves are involved in this condition, whereas nerve hypertrophy only develops in myelinated peripheral nerve fibers.

Abstract Objective To identify genetic factors associated with susceptibility to and clinical features of neuromyelitis optica spectrum disorders (NMOSD). Methods Genome‐wide single nucleotide polymorphism (SNP) genotyping was conducted in 211 Japanese patients with NMOSD fulfilling the 2006 criteria with or without anti‐aquaporin‐4 (AQP4) antibody and 1,919 Japanese healthy controls (HCs). HLA‐DRB1 and HLA‐DPB1 alleles were genotyped in 184 NMOSD cases and 317 HCs. Multiple sclerosis (MS) risk alleles outside the major histocompatibility complex (MHC) region were tested in NMOSD and MS genetic burden (MSGB) scores were compared between HCs and NMOSD. Results A SNP (rs1964995) in the MHC region was associated with NMOSD susceptibility (odds ratio (OR) = 2.33, P = 4.07 × 10−11). HLA‐DRB1*08:02 (OR = 2.86, P = 3.03 × 10−4) and HLA‐DRB1*16:02 (OR = 8.39, P = 1.92 × 10−3) were risk alleles for NMOSD susceptibility whereas HLA‐DRB1*09:01 was protective (OR = 0.27, P = 1.06 × 10−5). Three MS risk variants were associated with susceptibility and MSGB scores were significantly higher in NMOSD than in HCs (P = 0.0095). A SNP in the KCNMA1 (potassium calcium‐activated channel subfamily M alpha 1) gene was associated with disability score with genome‐wide significance (rs1516512, P = 2.33 × 10−8) and transverse myelitis (OR = 1.77, P = 0.011). KCNMA1 was immunohistochemically detected in the perivascular endfeet of astrocytes and its immunoreactivity was markedly diminished in active spinal cord lesions in NMOSD. Interpretation Specific HLA‐DRB1 alleles confer NMOSD susceptibility and KCNMA1 is associated with disability and transverse myelitis in NMOSD.

Abstract Background Previous research indicated that nitric oxide synthase (NOS) is the key molecule for S-nitrosylation of ryanodine receptor 1 (RyR1) in DMD model mice (mdx mice) and that both neuronal NOS (nNOS) and inducible NOS (iNOS) might contribute to the reaction because nNOS is mislocalized in the cytoplasm and iNOS expression is higher in mdx mice. We investigated the effect of iNOS on RyR1 S-nitrosylation in mdx mice and whether transgenic expression of truncated dystrophin reduced iNOS expression in mdx mice or not. Methods Three- to 4-month-old C57BL/6 J, mdx, and transgenic mdx mice expressing exon 45–55-deleted human dystrophin (Tg/mdx mice) were used. We also generated two double mutant mice, mdx iNOS KO and Tg/mdx iNOS KO to reveal the iNOS contribution to RyR1 S-nitrosylation. nNOS and iNOS expression levels in skeletal muscle of these mice were assessed by immunohistochemistry (IHC), qRT-PCR, and Western blotting. Total NOS activity was measured by a citrulline assay. A biotin-switch method was used for detection of RyR1 S-nitrosylation. Statistical differences were assessed by one-way ANOVA with Tukey-Kramer post-hoc analysis. Results mdx and mdx iNOS KO mice showed the same level of RyR1 S-nitrosylation. Total NOS activity was not changed in mdx iNOS KO mice compared with mdx mice. iNOS expression was undetectable in Tg/mdx mice expressing exon 45–55-deleted human dystrophin, but the level of RyR1 S-nitrosylation was the same in mdx and Tg/mdx mice. Conclusion Similar levels of RyR1 S-nitrosylation and total NOS activity in mdx and mdx iNOS KO demonstrated that the proportion of iNOS in total NOS activity was low, even in mdx mice. Exon 45–55-deleted dystrophin reduced the expression level of iNOS, but it did not correct the RyR1 S-nitrosylation. These results indicate that iNOS was not involved in RyR1 S-nitrosylation in mdx and Tg/mdx mice muscles.

Abstract Background Progressive multifocal leukoencephalopathy (PML) caused by JC virus (JCV) is a rare but serious complication of some disease-modifying drugs used to treat multiple sclerosis (MS). Japanese MS patients treated with fingolimod were reported to be 10 times more likely to develop PML than equivalent patients in other countries. The strongest susceptibility human leukocyte antigen (HLA) class II alleles for MS are distinct between races (DRB1*15:01 for Caucasians and DRB1*04:05 and DRB1*15:01 for Japanese); therefore, we investigated whether HLA class II alleles modulate anti-JCV antibody serostatus in Japanese MS patients with and without fingolimod. Methods We enrolled 128 Japanese patients with MS, in whom 64 (50%) were under fingolimod treatment at sampling, and examined the relationship between HLA class II alleles and anti-JCV antibody serostatus. Serum anti-JCV antibody positivity and index were measured using a second-generation two-step assay and HLA-DRB1 and -DPB1 alleles were genotyped. Results HLA-DRB1*15 carriers had a lower frequency of anti-JCV antibody positivity (57% vs 78%, p = 0.015), and lower antibody index (median 0.42 vs 1.97, p = 0.037) than non-carriers. Among patients without HLA-DRB1*15, DRB1*04 carriers had a higher seropositivity rate than non-carriers (84% vs 54%, p = 0.030), and DPB1*04:02 carriers had a higher anti-JCV antibody index than non-carriers (3.20 vs 1.34, p = 0.008) although anti-JCV antibody-positivity rates did not differ. Patients treated with fingolimod had a higher antibody index than other patients (1.46 vs 0.64, p = 0.039) and treatment period had a positive correlation with antibody index (p = 0.018). Multivariate logistic regression analysis revealed that age was positively associated, and HLA-DRB1*15 was negatively associated with anti-JCV antibody positivity (odds ratio [OR] = 1.06, p = 0.006, and OR = 0.37, p = 0.028, respectively). Excluding HLA-DRB1*15-carriers, DRB1*04 was an independent risk factor for the presence of anti-JCV antibody (OR = 5.50, p = 0.023). Conclusions HLA-DRB1*15 is associated with low anti-JCV antibody positive rate and low JCV antibody index, and in the absence of DRB1*15, DRB1*04 carriers are associated with a high antibody positive rate in Japanese, suggesting the effects of two susceptible HLA-DRB1 alleles on anti-JCV antibody serostatus differ.

It remains unclear whether epileptogenic networks in focal epilepsy develop on physiological networks. This work aimed to explore the association between the rapid spread of ictal fast activity (IFA), a proposed biomarker for epileptogenic networks, and the functional connectivity or networks of healthy subjects. We reviewed 45 patients with focal epilepsy who underwent electrocorticographic (ECoG) recordings to identify the patients showing the rapid spread of IFA. IFA power was quantified as normalized beta-gamma band power. Using published resting-state functional magnetic resonance imaging databases, we estimated resting-state functional connectivity of healthy subjects (RSFC-HS) and resting-state networks of healthy subjects (RSNs-HS) at the locations corresponding to the patients' electrodes. We predicted the IFA power of each electrode based on RSFC-HS between electrode locations (RSFC-HS-based prediction) using a recently developed method, termed activity flow mapping. RSNs-HS were identified using seed-based and atlas-based methods. We compared IFA power with RSFC-HS-based prediction or RSNs-HS using non-parametric correlation coefficients. RSFC and seed-based RSNs of each patient (RSFC-PT and seed-based RSNs-PT) were also estimated using interictal ECoG data and compared with IFA power in the same way as RSFC-HS and seed-based RSNs-HS. Spatial autocorrelation-preserving randomization tests were performed for significance testing. Nine patients met the inclusion criteria. None of the patients had reflex seizures. Six patients showed pathological evidence of a structural etiology. In total, we analyzed 49 seizures (2–13 seizures per patient). We observed significant correlations between IFA power and RSFC-HS-based prediction, seed-based RSNs-HS, or atlas-based RSNs-HS in 28 (57.1%), 21 (42.9%), and 28 (57.1%) seizures, respectively. Thirty-two (65.3%) seizures showed a significant correlation with either seed-based or atlas-based RSNs-HS, but this ratio varied across patients: 27 (93.1%) of 29 seizures in six patients correlated with either of them. Among atlas-based RSNs-HS, correlated RSNs-HS with IFA power included the default mode, control, dorsal attention, somatomotor, and temporal-parietal networks. We could not obtain RSFC-PT and RSNs-PT in one patient due to frequent interictal epileptiform discharges. In the remaining eight patients, most of the seizures showed significant correlations between IFA power and RSFC-PT-based prediction or seed-based RSNs-PT. Our study provides evidence that the rapid spread of IFA in focal epilepsy can arise from physiological RSNs. This finding suggests an overlap between epileptogenic and functional networks, which may explain why functional networks in patients with focal epilepsy frequently disrupt.

Multiple sclerosis (MS), the most prevalent inflammatory disease of the central nervous system (CNS), is characterized by damaged to myelin sheaths and oligodendrocytes. Because MS patients have variable clinical courses and disease severities, it is important to identify biomarkers that predict disease activity and severity. In this study, we assessed the frequencies of serum autoantibodies against mature oligodendrocytes in MS patients using a tissue-based immunofluorescence assay (IFA) to determine whether anti-oligodendrocyte antibodies are associated with the clinical features of MS patients and whether they might be a biomarker to assess CNS tissue damage in MS patients. We assessed the binding of serum autoantibodies to mouse oligodendrocytes expressing Nogo-A, a reliable mature oligodendrocyte marker, by IFA with mouse brain and sera from 147 MS patients, comprising 103 relapsing–remitting MS (RRMS), 22 secondary progressive MS (SPMS), and 22 primary progressive MS (PPMS) patients, 38 neuromyelitis optica spectrum disorder (NMOSD) patients, 23 other inflammatory neurological disorder (OIND) patients, and 39 healthy controls (HCs). Western blotting (WB) was performed using extracted mouse cerebellum proteins and IgG from anti-oligodendrocyte antibody-positive MS patients. Tissue-based IFA showed that anti-oligodendrocyte antibodies were positive in 3/22 (13.6%) PPMS and 1/22 (4.5%) SPMS patients but not in RRMS, NMOSD, and OIND patients or HCs. WB demonstrated the target CNS proteins recognized by serum anti-oligodendrocyte antibodies were approximately 110 kDa and/or 150 kDa. Compared with anti-oligodendrocyte antibody-negative MS patients, MS patients with anti-oligodendrocyte antibodies were significantly older at the time of serum sampling, scored significantly higher on the Expanded Disability Status Scale and the Multiple Sclerosis Severity Score, and had a higher frequency of mental disturbance. Although the clinical significance of anti-oligodendrocyte antibodies is still unclear because of their low frequency, anti-oligodendrocyte autoantibodies are potential biomarkers for monitoring the disease pathology and progression in MS.

Abstract Objective To characterize the CSF cytokine profile in chronic inflammatory demyelinating polyneuropathy (CIDP) patients with IgG4 anti‐neurofascin 155 (NF155) antibodies (NF155+ CIDP) or those lacking anti‐NF155 antibodies (NF155− CIDP). Methods Twenty‐eight CSF cytokines/chemokines/growth factors were measured by multiplexed fluorescent immunoassay in 35 patients with NF155+ CIDP, 36 with NF155− CIDP, and 28 with non‐inflammatory neurological disease (NIND). Results CSF CXCL8/IL‐8, IL‐13, TNF‐α, CCL11/eotaxin, CCL2/MCP‐1, and IFN‐γ were significantly higher, while IL‐1β, IL‐1ra, and G‐CSF were lower, in NF155+ CIDP than in NIND. Compared with NF155− CIDP, CXCL8/IL‐8 and IL‐13 were significantly higher, and IL‐1β, IL‐1ra, and IL‐6 were lower, in NF155+ CIDP. CXCL8/IL‐8, IL‐13, CCL11/eotaxin, CXCL10/IP‐10, CCL3/MIP‐1α, CCL4/MIP‐1β, and TNF‐α levels were positively correlated with markedly elevated CSF protein, while IL‐13, CCL11/eotaxin, and IL‐17 levels were positively correlated with increased CSF cell counts. IL‐13, CXCL8/IL‐8, CCL4/MIP‐1β, CCL3/MIP‐1α, and CCL5/RANTES were decreased by combined immunotherapies in nine NF155+ CIDP patients examined longitudinally. By contrast, NF155− CIDP had significantly increased IFN‐γ compared with NIND, and exhibited positive correlations of IFN‐γ, CXCL10/IP‐10, and CXCL8/IL‐8 with CSF protein. Canonical discriminant analysis of cytokines/chemokines revealed that NF155+ and NF155− CIDP were separable, and that IL‐4, IL‐10, and IL‐13 were the three most significant discriminators. Interpretation Intrathecal upregulation of type 2 helper T (Th2) cell cytokines is characteristic of IgG4 NF155+ CIDP, while type 1 helper T cell cytokines are increased in CIDP regardless of the presence or absence of anti‐NF155 antibodies, suggesting that overproduction of Th2 cell cytokines is unique to NF155+ CIDP.

Abstract Background We previously reported that Vδ2+Vγ9+ γδ T cells were significantly decreased in multiple sclerosis (MS) patients without disease-modifying therapies (untreated MS) and were negatively correlated with Expanded Disability Status Scale (EDSS) scores, suggesting protective roles of Vδ2+Vγ9+ γδ T cells. Interferon-β (IFN-β) is one of the first-line disease-modifying drugs for MS. However, no previous studies have reported changes in γδ T cell subsets under IFN-β treatment. Therefore, we aimed to clarify the effects of the long-term usage of IFN-β on γδ T cell subsets in MS patients. Methods Comprehensive flow cytometric immunophenotyping was performed in 35 untreated MS and 21 MS patients on IFN-β for more than 2 years (IFN-β-treated MS) including eight super-responders fulfilling no evidence of disease activity criteria, and 44 healthy controls (HCs). Results The percentages of Vδ2+Vγ9+ cells in γδ T cells were significantly lower in untreated and IFN-β-treated MS patients than in HCs. By contrast, the percentages of Vδ1−Vδ2−Vγ9− cells in γδ T cells were markedly higher in IFN-β-treated MS patients than in HCs and untreated MS patients (both p < 0.001). A significant negative correlation between the percentages of Vδ2+Vγ9+ cells in γδ T cells and EDSS scores was confirmed in untreated MS but not evident in IFN-β-treated MS. Moreover, class-switched memory B cells were decreased in IFN-β-treated MS compared with HCs (p < 0.001) and untreated MS patients (p = 0.006). Interestingly, the percentages of Vδ1−Vδ2−Vγ9− cells in γδ T cells were negatively correlated with class-switched memory B cell percentages in all MS patients (r = − 0.369, p = 0.005), and the percentages of Vδ1−Vδ2−Vγ9− cells in Vδ1−Vδ2− γδ T cells were negatively correlated with EDSS scores only in IFN-β super-responders (r = − 0.976, p < 0.001). Conclusions The present study suggests that long-term usage of IFN-β increases Vδ1−Vδ2−Vγ9− γδ T cells, which are associated with a better outcome, especially in IFN-β super-responders. Thus, increased Vδ1−Vδ2−Vγ9− cells together with decreased class-switched memory B cells may contribute to the suppression of disease activity in MS patients under IFN-β treatment.

Chronic inflammatory demyelinating polyneuropathy (CIDP) is an immune-mediated demyelinating disease of the peripheral nervous system (PNS). A small number of CIDP patients harbors autoantibodies against nodal/paranodal proteins, such as neurofascin 155 (NF155), contactin 1, and contactin-associated protein 1. In most cases, the predominant immunoglobulin (IgG) subclass is IgG4. Node/paranode antibody-positive CIDP demonstrates distinct features compared with antibody-negative CIDP, including a poor response to intravenous immunoglobulin. The neuropathology of biopsied sural nerve shows Schwann cell terminal loop detachment from axons without macrophage infiltration or inflammation. This is partly attributable to IgG4, which blocks protein–protein interactions without inducing inflammation. Anti-NF155 antibody-positive (NF155+) CIDP is unique because of the high frequency of subclinical demyelinating lesions in the central nervous system (CNS). This is probably because NF155 coexists in the PNS and CNS. Such cases showing demyelinating lesions in both the CNS and PNS are now termed combined central and peripheral demyelination (CCPD). NF155+ CIDP/CCPD commonly presents hypertrophy of spinal nerve roots and cranial nerves, such as trigeminal and oculomotor nerves, and extremely high levels of cerebrospinal fluid (CSF) protein, which indicates nerve root inflammation. In the CSF, the CXCL8/IL8, IL13, TNFα, CCL11/eotaxin, CCL2/MCP1, and IFNγ levels are significantly higher and the IL1β, IL1ra, and GCSF levels are significantly lower in NF155+ CIDP than in non-inflammatory neurological diseases. Even compared with anti-NF155 antibody-negative (NF155−) CIDP, the CXCL8/IL8 and IL13 levels are significantly higher and the IL1β and IL1ra levels are significantly lower than those in NF155+ CIDP. Canonical discriminant analysis revealed NF155+ and NF155− CIDP to be separable with IL4, IL10, and IL13, the three most significant discriminators, all of which are required for IgG4 class switching. Therefore, upregulation of both Th2 and Th1 cytokines and downregulation of macrophage-related cytokines are characteristic of NF155+ CIDP, which explains spinal root inflammation and the lack of macrophage infiltration in the sural nerves. All Japanese patients with NF155+ CIDP/CCPD have one of two specific human leukocyte antigen (HLA) haplotypes, which results in a significantly higher prevalence of HLA-DRB1*15:01-DQB1*06:02 compared with healthy Japanese controls. This indicates an involvement of specific HLA class II molecules and relevant T cells in addition to IgG4 anti-NF155 antibodies in the mechanism underlying IgG4 NF155+ CIDP/CCPD.

Background and purpose The impact of the paraoxonase-1 (PON1) polymorphism, Q192R, on platelet inhibition in response to clopidogrel remains controversial. We aimed to investigate the association between carrier status of PON1 Q192R and high platelet reactivity (HPR) with clopidogrel in patients undergoing elective neurointervention. Methods Post-clopidogrel platelet reactivity was measured using a VerifyNow® P2Y12 assay in P2Y12 reaction units (PRU) for consecutive patients before the treatment. Genotype testing was performed for PON1 Q192R and CYP2C19*2 and *3 (no function alleles), and *17. PRU was corrected on the basis of hematocrit. We investigated associations between factors including carrying ≥1 PON1 192R allele and HPR defined as original and corrected PRU ≥208. Results Of 475 patients (232 men, median age, 68 years), HPR by original and corrected PRU was observed in 259 and 199 patients (54.5% and 41.9%), respectively. Carriers of ≥1 PON1 192R allele more frequently had HPR by original and corrected PRU compared with non-carriers (91.5% vs 85.2%, P = 0.031 and 92.5% vs 85.9%, P = 0.026, respectively). In multivariate analyses, carrying ≥1 PON1 192R allele was associated with HPR by original (odds ratio [OR] 1.96, 95% confidence interval [CI] 1.03–3.76) and corrected PRU (OR 2.34, 95% CI 1.21–4.74) after adjustment for age, sex, treatment with antihypertensive medications, hematocrit, platelet count, total cholesterol, and carrying ≥1 CYP2C19 no function allele. Conclusions Carrying ≥1 PON1 192R allele is associated with HPR by original and corrected PRU with clopidogrel in patients undergoing elective neurointervention, although alternative results related to other genetic polymorphisms cannot be excluded.

There is a wide range of onset age in Alzheimer's disease (AD). Emerging evidence indicates variation of AD manifestations in oldest-old AD (OOAD); however, the pattern of cognitive dysfunctions remains unclear. We aimed to reveal cognitive performance characteristics and changes in brain functional connectivity in OOAD patients by a resting-state fMRI (rs-fMRI) study. We enrolled AD patients who had been referred to Kyushu University Hospital (KUH) or Sanno Hospital, and classified them into middle-old AD (MOAD) (65–79 years old) and OOAD (≥80 years old) according to the age of onset. Our subjects consisted of 19 OOAD, 17 MOAD, and 8 normal subjects. Cognitive performance was evaluated using Mini Mental State Examination-Japanese (MMSE-J) and Clinical Dementia Rating (CDR). rs-fMRI scanning and independent component analysis (ICA) were performed on Sanno Hospital patients and MOAD vs. OOAD patients were compared. The resulting significant regions were used as seeds for ROI-to-ROI analysis of the KUH dataset. Collectively, MMSE-J delayed recall sub-scores were significantly lower in OOAD patients compared with MOAD patients. ICA of the Sanno Hospital data indicated significant connectivity decrease in the default mode network (DMN) in the OOAD group compared with the MOAD group in the right superior parietal lobule (SPL). ROI-to-ROI analysis of the KUH dataset indicated significant disconnection in the OOAD group of the right SPL from the precuneus (p < 0.01). The functional connectivity from the right SPL to the precuneus was positively correlated with the MMSE-J delayed recall sub-score (p = 0.03) and negatively correlated with the CDR memory sub-scale (p = 0.04). These findings indicate that disconnection between the right SPL and the precuneus may contribute to worse memory capability in OOAD compared with MOAD.

Background and PurposeIn the acute phase of ischemia-reperfusion, hypoperfusion associated with ischemia and reperfusion in microvascular regions and disruption of the blood–brain barrier (BBB) contribute to post-ischemic brain injury. We aimed to clarify whether brain injury following transient middle cerebral artery occlusion (tMCAO) is ameliorated in Transient receptor potential vanilloid 4 knockout (Trpv4–/–) mice.MethodstMCAO was induced in wild-type (WT) and Trpv4–/– mice aged 8–10 weeks. Ischemia-induced lesion volume was evaluated by 2,3,5-triphenyltetrazolium chloride staining at 24 h post-tMCAO. Tissue water content and Evans blue leakage in the ipsilateral hemisphere and a neurological score were evaluated at 48 h post-tMCAO. Transmission electron microscopy (TEM) was performed to assess the morphological changes in microvasculature in the ischemic lesions at 6 h post-tMCAO.ResultsCompared with WT mice, Trpv4–/– mice showed reduced ischemia-induced lesion volume and reduced water content and Evans blue leakage in the ipsilateral hemisphere alongside milder neurological symptoms. The loss of zonula occludens-1 and occludin proteins in the ipsilateral hemisphere was attenuated in Trpv4–/– mice. TEM revealed that parenchymal microvessels in the ischemic lesion were compressed and narrowed by the swollen endfeet of astrocytes in WT mice, but these effects were markedly ameliorated in Trpv4–/– mice.ConclusionThe present results demonstrate that TRPV4 contributes to post-ischemic brain injury. The preserved microcirculation and BBB function shortly after reperfusion are the key neuroprotective roles of TRPV4 inhibition, which represents a promising target for the treatment of acute ischemic stroke.

The toxic conformer of amyloid β-protein (Aβ) ending at 42 (Aβ42), which contains a unique turn conformation at amino acid residue positions 22 and 23 and tends to form oligomers that are neurotoxic, was reported to play a critical role in the pathomechanisms of Alzheimer's disease (AD), in which diabetes mellitus (DM)-like mechanisms are also suggested to be operative. It remains to be established whether the attenuation of insulin signaling is involved in an increase of toxic Aβ42 conformer levels. The present study investigated the association between impaired insulin metabolism and formation of toxic Aβ42 conformers in the brains of an AD mouse model. In particular, we studied whether insulin deficiency or resistance affected the formation of toxic Aβ42 conformers in vivo. We induced insulin deficiency and resistance in 3xTg-AD mice, a mouse AD model harboring two familial AD-mutant APP (KM670/671NL) and PS1 (M146 V) genes and a mutant TAU (P301L) gene, by streptozotocin (STZ) injection and a high fructose diet (HFuD), respectively. Cognitive impairment was significantly worsened by STZ injection but not by HFuD. Dot blot analysis revealed significant increases in total Aβ42 levels and the ratio of toxic Aβ42 conformer/total Aβ42 in STZ-treated mice compared with control and HFuD-fed mice. Immunostaining showed the accumulation of toxic Aβ42 conformers and hyper-phosphorylated tau protein (p-tau), which was more prominent in the cortical and hippocampal neurons of STZ-treated mice compared with HFuD-fed and control mice. HFuD-fed mice showed only a mild-to-moderate increase of these proteins compared with controls. Toxic Aβ42 conformers were co-localized with p-tau oligomers (Pearson's correlation coefficient = 0.62) in the hippocampus, indicating their co-aggregation. Toxic Aβ42 conformer levels were inversely correlated with pancreatic insulin secretion capacity as shown by fasting immunoreactive insulin levels in STZ-treated mice (correlation coefficient = −0.5879, p = .04441), but not HFuD-fed mice, suggesting a decrease in serum insulin levels correlates with toxic Aβ42 conformer formation. Levels of p-Akt and phosphorylated glycogen synthase kinase-3β measured by a homogeneous time-resolved fluorescence assay were significantly lower in STZ-treated mice than in HFuD-fed mice, suggesting a greater inhibition of brain insulin signaling by STZ than HFuD, although both levels were significantly decreased in these groups compared with controls. Iba1-positive and NOS2-positive areas in the cortex and hippocampus were significantly increased in STZ-treated mice and to a lesser extent in HFuD-fed mice compared with controls. These findings suggest that insulin deficiency rather than insulin resistance and the resultant impairment of brain insulin signaling facilitates the formation of toxic Aβ42 conformer and its co-aggregation with p-tau oligomers, and that insulin deficiency is an important pathogenic factor in the progression of AD.

Abstract Background We compared the magnetic resonance imaging (MRI) features between Japanese and Caucasian patients with multiple sclerosis (MS), and identified the relationships between MRI features and disability. Methods From the baseline data of phase II fingolimod trials, 95 Japanese and 246 Caucasian relapsing-remitting MS patients were enrolled. The number, volume, and distribution of brain MRI lesions were evaluated using T2-weighted (T2W) images. Cross-sectional total normalized brain volume (NBV), normalized cortical gray matter volume, normalized deep gray matter volume (NDGMV), normalized white matter volume (NWMV), and normalized thalamic volume were measured. Results Japanese patients had significantly lower Expanded Disability Status Scale (EDSS) scores than Caucasian patients (mean 2.0 vs. 2.3, p = 0.008), despite a similar disease duration. Japanese patients showed a trend towards fewer T2W-lesions (median 50 vs. 65, p = 0.08) and significantly lower frequencies of cerebellar and parietal lobe lesions (p = 0.02 for both) than Caucasian patients. There were no differences in T2W-lesion volume between races, whereas Japanese patients had a significantly larger T2W-lesion volume per lesion compared with Caucasian patients (median 140 mm3 vs. 85 mm3, p

Abstract Background The first pathology observed in Parkinson’s disease (PD) is ‘dying back’ of striatal dopaminergic (DA) terminals. Connexin (Cx)30, an astrocytic gap junction protein, is upregulated in the striatum in PD, but its roles in neurodegeneration remain elusive. We investigated Cx30 function in an acute PD model by administering 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to wild-type (WT) and Cx30 knockout (KO) mice. Methods On days 1 and 7 after MPTP administration, we evaluated changes in astrocytic Cx30, Cx43, glial fibrillary acidic protein, and ionised calcium-binding adapter molecule 1 expression by immunostaining and biochemical analysis. Loss of DA neurons was evaluated by tyrosine hydroxylase immunostaining. Gene expression was analysed using A1, A2, pan-reactive astrocyte microarray gene sets, and M1, M2, and M1/M2 mixed microglial microarray gene sets. Real-time PCR and in situ hybridisation were performed to evaluate glial cell-derived neurotrophic factor (Gdnf) and S100a10 expression. Striatal GDNF protein levels were determined by enzyme-linked immunosorbent assay. Results MPTP treatment induced upregulation of Cx30 and Cx43 levels in the striatum of WT and KO mice. DA neuron loss was accelerated in Cx30 KO compared with WT mice after MPTP administration, despite no change in the striatal concentration of methyl-4-phenylpyridinium+. Astrogliosis in the striatum of Cx30 KO mice was attenuated by MPTP, whereas microglial activation was unaffected. Microarrays of the striatum showed reduced expression of pan-reactive and A2 astrocyte genes after MPTP treatment in Cx30 KO compared with WT mice, while M1, M2, and M1/M2 mixed microglial gene expression did not change. MPTP reduced the number of striatal astrocytes co-expressing Gdnf mRNA and S100β protein or S100a10 mRNA and S100β protein and also reduced the level of GDNF in the striatum of Cx30 KO compared with WT mice. Conclusions These findings indicate that Cx30 plays critical roles in astrocyte neuroprotection in an MPTP PD model.

An elevated end-diastolic (ED) ratio of the common carotid artery (CCA) is an indicator of occlusive lesions of the distal portion of the internal carotid artery. We report 2 cases of cerebral arteriovenous malformation (AVM) showing an elevated ED ratio of the CCA, which decreased after surgery. Case 1 was a 28-year-old man with chronic recurrent headache with aura, and case 2 was a 29-year-old woman with sudden-onset headache and intracerebral hemorrhage without neurologic abnormality. In both cases, digital subtraction angiography revealed a Spetzler-Martin Grade IV AVM, which was mainly fed by branches of the left middle cerebral artery with venous drainage into superficial and deep cerebral veins. Preoperative carotid ultrasonography showed an elevated CCA ED ratio (1.38 in case 1 and 1.47 in case 2; left > right) without atherosclerotic lesions. Patients’ AVMs were successfully resected. In both cases, the ED ratio was decreased after surgery (to 1.05 in case 1 and 1.20 in case 2). A decrease in vascular resistance on 1 side caused by cerebral AVM can result in an increase in the CCA ED ratio comparable to that of carotid axis occlusion. Keywords: Carotid ultrasonography, Cerebral arteriovenous malformation, End-diastolic ratio

Abstract Background Hopkins syndrome (HS) is a rare disorder presenting with acute flaccid paralysis of the limbs following an asthma attack. Neurologists encounter a diagnostic challenge if patients without a history of bronchial asthma develop neurologic features mimicking HS following acute respiratory distress. We report a case of HS occurring after a first episode of bronchial asthma associated with enterovirus D68 infection. Case presentation A 5-year-old girl developed acute respiratory distress. On the fourth hospital day, both her legs became paralyzed except for slight muscle contraction in the right lower limb. Tendon reflexes in the lower limbs were diminished and there was a positive Babinski sign on the right. Sensation was normal in all modalities, and there was no uro-rectal disturbance. Spinal magnetic resonance imaging identified T2-hyperintense lesions with spinal cord edema, mainly involving the bilateral T11 to L1 anterior horns, with left side dominance extending to the left posterior horn. The neurological and neuro-radiological findings of our case were suggestive of HS; however, she had no history of bronchial asthma. An acetylcholine inhalation challenge eventually proved the presence of reversible airway hyper-responsiveness, allowing us to diagnose HS. We identified enterovirus D68 in the patient’s intratracheal aspirates using a sensitive polymerase chain reaction assay. Intravenous immunoglobulin administrations at 2 g/kg2 for 5 consecutive days were repeated every month up to four times. After these treatments, the muscle strength of her right lower limb slightly improved while her left lower leg remained completely paralyzed. Conclusion This case emphasizes the importance of provocation tests to reveal the presence of airway hyper-responsiveness when a child shows neurological signs mimicking HS following acute respiratory distress. Furthermore, the present case suggests a possible link between HS and acute flaccid paralysis following lower respiratory tract infection by enterovirus D68.

Abstract Background The pathology of multiple system atrophy cerebellar-type (MSA-C) includes glial inflammation; however, cerebrospinal fluid (CSF) inflammatory cytokine profiles have not been investigated. In this study, we determined CSF cytokine/chemokine/growth factor profiles in MSA-C and compared them with those in hereditary spinocerebellar ataxia (SCA). Methods We collected clinical data and CSF from 20 MSA-C patients, 12 hereditary SCA patients, and 15 patients with other non-inflammatory neurological diseases (OND), and measured 27 cytokines/chemokines/growth factors using a multiplexed fluorescent bead-based immunoassay. The size of each part of the hindbrain and hot cross bun sign (HCBS) in the pons was studied by magnetic resonance imaging. Results Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-6, IL-7, IL-12, and IL-13 levels were significantly higher in MSA-C and SCA compared with OND. In MSA-C, IL-5, IL-6, IL-9, IL-12, IL-13, platelet-derived growth factor-bb, macrophage inflammatory protein (MIP)-1α, and GM-CSF levels positively correlated with anteroposterior diameters of the pontine base, vermis, or medulla oblongata. By contrast, in SCA patients, IL-12 and MIP-1α showed significant negative correlations with anteroposterior diameters of the pontine base, and unlike MSA-C, there was no cytokine with a positive correlation in SCA. IL-6 was significantly higher in MSA-C patients with the lowest grade of HCBS compared with those with the highest grade. Macrophage chemoattractant protein-1 (MCP-1) had a significant negative correlation with disease duration only in MSA-C patients. Tumor necrosis factor-alpha, IL-2, IL-15, IL-4, IL-5, IL-10, and IL-8 were all significantly lower in MSA-C and SCA compared with OND, while IL-1ra, an anti-inflammatory cytokine, was elevated only in MSA-C. IL-1β and IL-8 had positive correlations with Unified Multiple System Atrophy Rating Scale part 1 and 2, respectively, in MSA-C. Conclusions Although CSF cytokine/chemokine/growth factor profiles were similar between MSA-C and SCA, pro-inflammatory cytokines, such as IL-6, GM-CSF, and MCP-1, correlated with the disease stage in a way higher at the beginning only in MSA-C, reflecting early stronger intrathecal inflammation.

Allergic diseases are associated with central and peripheral nervous system diseases such as autism spectrum disorders and eosinophilic granulomatosis with polyangiitis, which frequently causes mononeuritis multiplex. Thus, it is possible that patients with an atopic constitution might develop multifocal inflammation in central and peripheral nervous system tissues. In a previous study in Japan, we reported a rare form of myelitis with persistent neuropathic pain (NeP) in patients with allergic disorders. However, the underlying mechanism of allergic inflammation-related NeP remains to be elucidated. First, we analyzed the effect of allergic inflammation on the nociceptive system in the spinal cord. Mice with atopy showed microglial and astroglial activation in the spinal cord and tactile allodynia. In a microarray analysis of isolated microglia from the spinal cord, endothelin receptor type B (EDNRB) was the most upregulated cell surface receptor in mice with atopy. Immunohistochemical analysis demonstrated EDNRB expression was upregulated in microglia and astroglia. The EDNRB antagonist BQ788 abolished glial activation and allodynia. These findings indicated that allergic inflammation induced widespread glial activation through the EDNRB pathway and NeP. Second, we investigated whether autoantibody-mediated pathogenesis underlies allergic inflammation-related NeP. We detected specific autoantibodies to small dorsal root ganglion (DRG) neurons and their nerve terminals in the dorsal horns of NeP patients with allergic disorders. An analysis of IgG subclasses revealed a predominance of IgG2. These autoantibodies were mostly colocalized with isolectin B4- and P2X3-positive unmyelinated C-fiber type small DRG neurons. By contrast, immunostaining for S100β, a myelinated DRG neuron marker, showed no colocalization with patient IgG. Immunoprecipitation and liquid chromatography-tandem mass spectrometry identified plexin D1 as a target autoantigen. Patients with anti-plexin D1 antibodies often present with burning pain and thermal hyperalgesia. Immunotherapies, including plasma exchange, are effective for NeP management. Therefore, anti-plexin D1 antibodies may be pathogenic for immune-mediated NeP, especially under allergic inflammation conditions. Thus, allergic inflammation may induce NeP through glial inflammation in the spinal cord and the anti-plexin D1 antibody-mediated impairment of small DRG neurons.

Background: To maximize the effect of intravenous (IV) thrombolysis and/or endovascular therapy (EVT) for acute ischemic stroke (AIS), stroke centers need to establish a parallel workflow on the basis of a code stroke (CS) protocol. At Kokura Memorial Hospital (KMH), we implemented a CS system in January 2014; however, the process of information sharing within the team has occasionally been burdensome.Objective: To solve this problem using information communication technology (ICT), we developed a novel application for smart devices, named “Task Calc. Stroke” (TCS), and aimed to investigate the impact of TCS on AIS care.Methods: TCS can visualize the real-time progress of crucial tasks for AIS on a dashboard by changing color indicators. From August 2015 to March 2017, we installed TCS at KMH and recommended its use during normal business hours (NBH). We compared the door-to-computed tomography time, the door-to-complete blood count (door-to-CBC) time, the door-to-needle for IV thrombolysis time, and the door-to-puncture for EVT time among three treatment groups, one using TCS (“TCS-based CS”), one not using TCS (“phone-based CS”), and one not based on CS (“non-CS”). A questionnaire survey regarding communication problems was conducted among the CS teams at 3 months after the implementation of TCS.Results: During the study period, 74 patients with AIS were transported to KMH within 4.5 h from onset during NBH, and 53 were treated using a CS approach (phone-based CS: 26, TSC-based CS: 27). The door-to-CBC time was significantly reduced in the TCS-based CS group compared to the phone-based CS group, from 31 to 19 min (p = 0.043). Other processing times were also reduced, albeit not significantly. The rate of IV thrombosis was higher in the TCS-based CS group (78% vs. 46%, p = 0.037). The questionnaire was correctly filled in by 34/38 (89%) respondents, and 82% of the respondents felt a reduction in communication burden by using the TCS application.Conclusions: TCS is a novel approach that uses ICT to support information sharing in a parallel CS workflow in AIS care. It shortens the processing times of critical tasks and lessens the communication burden among team members.

Background: Semi-quantitative electroencephalogram (EEG) analysis is easy to perform and has been used to differentiate dementias, as well as idiopathic and vascular Parkinson's disease.Purpose: To study whether a semi-quantitative EEG analysis can aid in distinguishing idiopathic Parkinson's disease (IPD) from atypical parkinsonian disorders (APDs), and furthermore, whether it can help to distinguish between APDs.Materials and Methods: A comprehensive retrospective review of charts was performed to include patients with parkinsonian disorders who had at least one EEG recording available. A modified grand total EEG (GTE) score evaluating the posterior background activity, and diffuse and focal slow wave activities was used in further analyses.Results: We analyzed data from 76 patients with a final diagnosis of either IPD, probable corticobasal degeneration (CBD), multiple system atrophy (MSA), or progressive supra-nuclear palsy (PSP). IPD patients had the lowest mean GTE score, followed those with CBD or MSA, while PSP patients scored the highest. However, none of these differences were statistically significant. A GTE score of ≤9 distinguished IPD patients from those with APD (p < 0.01) with a sensitivity of 100% and a specificity of 33.3%.Conclusion: The modified GTE score can distinguish patients with IPD from those with CBD, PSP or MSA at a cut-off score of 9 with excellent sensitivity but poor specificity. However, this score is not able to distinguish a particular form of APD from other forms of the disorder.

Connexin36 (Cx36) forms gap junctions between neurons, which are called electrical synapses, enabling adjacent neurons to communicate directly. The participation of chemical synapses in neurodegeneration in amyotrophic lateral sclerosis (ALS) has long been indicated, but it remains unclear whether electrical synapses are involved in the pathogenesis of ALS. We performed extensive immunopathological analyses using mutant superoxide dismutase 1 (SOD1G93A) transgenic mice and their littermates to investigate whether Cx36-made electrical synapses are affected in motor neuron diseases. We found that in the lamina IX of the lumbar spinal cord from wild type mice, about half of the Cx36 puncta existed independently of chemical synapse markers, while the rest coexisted with chemical synapse markers, such as vesicular glutamate transporter 1 (VGLUT1), which is a glutamatergic axon terminal marker, and/or glutamate decarboxylase 65 (GAD65), which is a GABAergic axon terminal marker. Cx36 single or Cx36/GAD65 double positive puncta, but not VGLUT1-containing puncta, were preferentially decreased on neuronal and dendritic surfaces of the anterior horn cells in the early stage of SOD1G93A ALS mice. Moreover, in five human autopsied sporadic ALS cases with bulbar or upper limb onset, Cx36 immunoreactivity was diminished in the proximal dendrites and neuropils of well-preserved large motor neurons in the lumbar anterior horns. These findings suggest that downregulation of neuronal and dendritic Cx36 in the spinal anterior horns commonly occurs from the early stage of hereditary and sporadic ALS. Cx36-made electrical synapses without glutamatergic signaling appear to be more vulnerable than other chemical synapses and electrical synapses with glutamatergic signaling in the early stage of motor neuron degeneration, suggesting involvement of Cx36-made electrical synapses in the pathogenesis of human ALS.

Background: Several case reports have described the concurrence of chronic inflammatory demyelinating polyneuropathy (CIDP) and membranous nephropathy (MN). The presence of autoantibodies against podocyte antigens phospholipase A2 receptor (PLA2R) and thrombospondin type 1 domain containing 7A (THSD7A) in MN suggests an autoimmune mechanism. Some CIDP patients also harbor autoantibodies against paranodal proteins such as neurofascin 155 (NF155) and contactin-1 (CNTN1). We investigated the relationship between CIDP and MN by assaying autoantibodies against paranodal and podocyte antigens in a CIDP patient with MN, and by a literature survey on the clinical features of CIDP with MN.Methods: Anti-CNTN1 and NF155 antibodies were measured by flow cytometry using HEK293 cell lines stably expressing human CNTN1 or NF155. Binding capacity of antibodies was validated by immunostaining mouse teased sciatic nerve fibers. Anti-PLA2R antibodies were measured by enzyme-linked sorbent assay and anti-THSD7A antibodies by indirect immunofluorescence assay. Clinical features between 14 CIDP with MN cases including two with anti-CNTN1 antibodies and 20 anti-CNTN1 antibody-positive CIDP cases were compared.Results: A patient whose ages was in the late 70 s complained of progressive weakness and superficial and deep sensory impairment in four extremities over 6 months. Nerve conduction studies showed prominent demyelination patterns. The patient presented with nephrotic syndrome. Renal biopsy disclosed basement membrane thickening with local subepithelial projections and glomerular deposits of IgG4, compatible with MN. Autoantibody assays revealed the presence of IgG4 and IgG1 anti-CNTN1 antibodies, but an absence of anti-NF155, anti-PLA2R, and anti-THSD7A antibodies. The patient's serum stained paranodes of teased sciatic nerves. CIDP with MN and anti-CNTN1 antibody-positive CIDP commonly showed male preponderance, relatively higher age of onset, acute to subacute onset in 35–50% of cases, distal dominant sensorimotor neuropathy, proprioceptive impairment leading to sensory ataxia, and very high cerebrospinal fluid protein levels. However, 11 of 13 CIDP patients with MN had a favorable response to mono- or combined immunotherapies whereas anti-CNTN1 antibody-positive CIDP was frequently refractory to corticosteroids and intravenous immunoglobulin administration.Conclusion: CIDP with MN and anti-CNTN1 antibody-positive CIDP show considerable overlap but are not identical. CIDP with MN is probably heterogeneous and some cases harbor anti-CNTN1 antibodies.

Glial connexins (Cxs) form gap junction channels through which a pan-glial network plays key roles in maintaining homeostasis of the central nervous system (CNS). In multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), expression of astrocytic Cx43 is lost in acute lesions but upregulated in chronic plaques, while astrocytic Cx30 is very low in normal white matter and changes in its expression have not been convincingly shown. In Cx30 or Cx43 single knockout (KO) mice and even in Cx30/Cx43 double KO mice, acute EAE is unaltered. However, the effects of Cx30/Cx43 deficiency on chronic EAE remains to be elucidated. We aimed to clarify the roles of Cx30 in chronic neuroinflammation by studying EAE induced by myelin oligodendrocyte glycoprotein peptide 35–55 in Cx30 KO mice. We found that Cx30 deficiency improved the clinical symptoms and demyelination of chronic but not acute EAE without influencing CD3+ T cell infiltration. Furthermore, increased ramified microglia in the naïve state and induced earlier and stronger microglial activation in the acute and chronic phases of EAE was observed. These activated microglia had an anti-inflammatory phenotype, as shown by the upregulation of arginase-1 and brain-derived neurotrophic factor and the downregulation of nitric oxide synthase 2. In the naïve state, Cx30 deficiency induced modest enlargement of astrocytic processes in the spinal cord gray matter and a partial reduction of Cx43 expression in the spinal cord white matter. These astrocytes in Cx30 KO mice showed earlier and stronger activation during the acute phase of EAE, with upregulated A2 astrocyte markers and a significant decrease in Cx43 in the chronic phases. Spinal cord neurons and axons were more preserved in Cx30 KO mice than in littermates in the chronic phase of EAE. These findings suggest that Cx30 deficiency increased ramified microglia in the CNS in the naïve state and improved chronic EAE through redirecting microglia toward an anti-inflammatory phenotype, suggesting a hitherto unknown critical role of astrocytic Cx30 in regulating microglial number and functional state.

We recently reported that deletion-type copy number variations of the T cell receptor (TCR) γ, α, and δ genes greatly enhanced susceptibility to multiple sclerosis (MS). However, the effect of abnormal TCR γδ gene rearrangement on MS pathogenesis remains unknown. In the present study, we aimed to clarify γδ TCR repertoire alterations and their relationship to clinical and immunological parameters in MS patients by comprehensive flow cytometric immunophenotyping. Peripheral blood mononuclear cells obtained from 30 untreated MS patients in remission and 23 age- and sex-matched healthy controls (HCs) were stained for surface markers and intracellular cytokines after stimulation with phorbol 12-myristate 13-acetate and ionomycin, and analyzed by flow cytometry. MS patients showed significantly decreased percentages of Vδ2+ and Vδ2+Vγ9+ cells in γδ T cells (pcorr = 0.0297 and pcorr = 0.0288, respectively) and elevated Vδ1/Vδ2 ratios compared with HCs (p = 0.0033). The percentages of interferon (IFN)-γ+Vδ2+ and interleukin (IL)-17A+IFN-γ+Vδ2+ cells in γδ T cells, as well as IFN-γ+ cells in Vδ2+ γδ T cells, were significantly lower in MS patients than in HCs (pcorr

Central pontine myelinolysis (CPM), which was originally considered to be the result of rapid correction of chronic hyponatremia, is not necessarily accompanied by hyponatremia or drastic changes in serum sodium level. Here, we report a case of an anorexic 55-year-old male with a history of pharyngo-laryngo-esophagogastrectomy, initially hospitalized with status epilepticus. Although his consciousness gradually recovered as we were controlling his convulsion, it deteriorated again with new onset of anisocoria, and magnetic resonance imaging (MRI) at this point revealed CPM. Rapid change of serum sodium or osmolarity, which is often associated with CPM, had not been apparent throughout his hospitalization. Instead, a review of the serum biochemistry test results showed that serum phosphate had drastically declined the day before the MRI first detected CPM. In this case, we suspect that hypophosphatemia induced by refeeding syndrome greatly contributed to the occurrence of CPM.

Background: Recent studies reported that cerebral microbleeds (CMBs), i.e. small areas of signal loss on T2*-weighted gradient-echo (GE) imaging, could develop rapidly after acute ischemic stroke. We hypothesized that CMBs rapidly emerge after carotid artery stenting (CAS). Objective: We investigated the frequency of and predisposing factors for CMBs after CAS. Methods: We retrospectively examined MRI before and after CAS in 88 consecutive patients (average age: 71.7 ± 7.2 years, average rates of carotid stenosis: 72.6 ± 12.8%) who underwent CAS for carotid artery stenosis between March 1, 2009, and September 30, 2010. We defined new CMBs as signal losses that newly appeared on the follow-up GE. We examined the association of new CMBs with demographics, risk factors, and baseline MBs. Results: Among 88 patients, 18 (20.5%) had CMBs initially, and 7 (8.0%) developed new CMBs right after CAS. New CMBs appeared on the same side of CAS in all of the 7 patients. New CMBs appeared significantly more frequently in the CMB-positive group than in the CMB-negative one (22% vs. 4%, p = 0.03) on the pre-CAS MRI. Multivariate analysis also revealed that the presence of CMBs before CAS was an independent predictor of new development of CMBs after CAS (odds ratio: 8.09, 95% confidence interval: 1.39–47.1). Conclusion: CMBs can develop rapidly after CAS, especially in patients with pre-existing CMBs. Since the existence of CMBs prior to CAS suggests a latent vascular damage which is vulnerable to hemodynamic stress following CAS, particular attention should be paid to the prevention of intracerebral hemorrhage due to hyperperfusion after CAS.

BACKGROUND: Abnormal intrathecal synthesis of IgG, reflected by cerebrospinal fluid (CSF) oligoclonal IgG bands (OBs) and increased IgG index, is much less frequently observed in Japanese multiple sclerosis (MS) cohorts compared with Western cohorts. We aimed to clarify whether genetic and common infectious backgrounds influence CSF IgG abnormality in Japanese MS patients. METHODOLOGY: We analyzed HLA-DRB1 alleles, and IgG antibodies against Chlamydia pneumoniae, Helicobacter pylori, Epstein-Barr virus nuclear antigen (EBNA), and varicella zoster virus (VZV) in 94 patients with MS and 367 unrelated healthy controls (HCs). We defined CSF IgG abnormality as the presence of CSF OBs and/or increased IgG index (>0.658). PRINCIPAL FINDINGS: CSF IgG abnormality was found in 59 of 94 (62.8%) MS patients. CSF IgG abnormality-positive patients had a significantly higher frequency of brain MRI lesions meeting the Barkhof criteria compared with abnormality-negative patients. Compared with HCs, CSF IgG abnormality-positive MS patients showed a significantly higher frequency of DRB1 1501, whereas CSF IgG abnormality-negative patients had a significantly higher frequency of DRB1 0405. CSF IgG abnormality-positive MS patients had a significantly higher frequency of anti-C. pneumoniae IgG antibodies compared with CSF IgG abnormality-negative MS patients, although there was no difference in the frequency of anti-C. pneumoniae IgG antibodies between HCs and total MS patients. Compared with HCs, anti-H. pylori IgG antibodies were detected significantly less frequently in the total MS patients, especially in CSF IgG abnormality-negative MS patients. The frequencies of antibodies against EBNA and VZV did not differ significantly among the groups. CONCLUSIONS: CSF IgG abnormality is associated with Western MS-like brain MRI features. DRB1 1501 and C. pneumoniae infection confer CSF IgG abnormality, while DRB1 0405 and H. pylori infection are positively and negatively associated with CSF IgG abnormality-negative MS, respectively, suggesting that genetic and environmental factors differentially contribute to MS susceptibility according to the CSF IgG abnormality status.

Fingolimod efficiently reduces multiple sclerosis (MS) relapse by inhibiting lymphocyte egress from lymph nodes through down-modulation of sphingosine 1-phosphate (S1P) receptors. We aimed to clarify the alterations in peripheral blood T cell subsets associated with MS relapse on fingolimod.Blood samples successively collected from 23 relapsing-remitting MS patients before and during fingolimod therapy (0.5 mg/day) for 12 months and 18 healthy controls (HCs) were analysed for T cell subsets by flow cytometry. In MS patients, the percentages of central memory T (CCR7+CD45RO+) cells (TCM) and naïve T (CCR7+CD45RO-) cells decreased significantly, while those of effector memory T (CCR7-CD45RA-) and suppressor precursor T (CD28-) cells increased in both CD4+T and CD8+T cells from 2 weeks to 12 months during fingolimod therapy. The percentages of regulatory T (CD4+CD25highCD127low) cells in CD4+T cells and CCR7-CD45RA+T cells in CD8+T cells also increased significantly. Eight relapsed patients demonstrated greater percentages of CD4+TCM than 15 non-relapsed patients at 3 and 6 months (p=0.0051 and p=0.0088, respectively). The IL17-, IL9-, and IL4-producing CD4+T cell percentages were significantly higher at pre-treatment in MS patients compared with HCs (p

BackgroundMultiple sclerosis (MS) and neuromyelitis optica (NMO) occasionally have an extremely aggressive and debilitating disease course; however, its molecular basis is unknown. This study aimed to determine a relationship between connexin (Cx) pathology and disease aggressiveness in Asian patients with MS and NMO.Methods/principal findingsSamples included 11 autopsied cases with NMO and NMO spectrum disorder (NMOSD), six with MS, and 20 with other neurological diseases (OND). Methods of analysis included immunohistochemical expression of astrocytic Cx43/Cx30, oligodendrocytic Cx47/Cx32 relative to AQP4 and other astrocytic and oligodendrocytic proteins, extent of demyelination, the vasculocentric deposition of complement and immunoglobulin, and lesion staging by CD68 staining for macrophages. Lesions were classified as actively demyelinating (n=59), chronic active (n=58) and chronic inactive (n=23). Sera from 120 subjects including 30 MS, 30 NMO, 40 OND and 20 healthy controls were examined for anti-Cx43 antibody by cell-based assay. Six NMO/NMOSD and three MS cases showed preferential loss of astrocytic Cx43 beyond the demyelinated areas in actively demyelinating and chronic active lesions, where heterotypic Cx43/Cx47 astrocyte oligodendrocyte gap junctions were extensively lost. Cx43 loss was significantly associated with a rapidly progressive disease course as six of nine cases with Cx43 loss, but none of eight cases without Cx43 loss regardless of disease phenotype, died within two years after disease onset (66.7% vs. 0%, P=0.0090). Overall, five of nine cases with Cx43 loss and none of eight cases without Cx43 loss had distal oligodendrogliopathy characterized by selective myelin associated glycoprotein loss (55.6% vs. 0.0%, P=0.0296). Loss of oligodendrocytic Cx32 and Cx47 expression was observed in most active and chronic lesions from all MS and NMO/NMOSD cases. Cx43-specific antibodies were absent in NMO/NMOSD and MS patients.ConclusionsThese findings suggest that autoantibody-independent astrocytic Cx43 loss may relate to disease aggressiveness and distal oligodendrogliopathy in both MS and NMO.

BACKGROUND: Differences in cytokine/chemokine profiles among patients with neuromyelitis optica (NMO), relapsing remitting multiple sclerosis (RRMS), and primary progressive MS (PPMS), and the relationships of these profiles with clinical and neuroimaging features are unclear. A greater understanding of these profiles may help in differential diagnosis. METHODS/PRINCIPAL FINDINGS: We measured 27 cytokines/chemokines and growth factors in CSF collected from 20 patients with NMO, 26 with RRMS, nine with PPMS, and 18 with other non-inflammatory neurological diseases (OND) by multiplexed fluorescent bead-based immunoassay. Interleukin (IL)-17A, IL-6, CXCL8 and CXCL10 levels were significantly higher in NMO patients than in OND and RRMS patients at relapse, while granulocyte-colony stimulating factor (G-CSF) and CCL4 levels were significantly higher in NMO patients than in OND patients. In NMO patients, IL-6 and CXCL8 levels were positively correlated with disability and CSF protein concentration while IL-6, CXCL8, G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN-γ were positively correlated with CSF neutrophil counts at the time of sample collection. In RRMS patients, IL-6 levels were significantly higher than in OND patients at the relapse phase while CSF cell counts were negatively correlated with the levels of CCL2. Correlation coefficients of cytokines/chemokines in the relapse phase were significantly different in three combinations, IL-6 and GM-CSF, G-CSF and GM-CSF, and GM-CSF and IFN-γ, between RRMS and NMO/NMOSD patients. In PPMS patients, CCL4 and CXCL10 levels were significantly higher than in OND patients. CONCLUSIONS: Our findings suggest distinct cytokine/chemokine alterations in CSF exist among NMO, RRMS and PPMS. In NMO, over-expression of a cluster of Th17- and Th1-related proinflammatory cytokines/chemokines is characteristic, while in PPMS, increased CCL4 and CXCL10 levels may reflect on-going low grade T cell and macrophage/microglia inflammation in the central nervous system. In RRMS, only a mild elevation of proinflammatory cytokines/chemokines was detectable at relapse.

BACKGROUND: Nationwide surveys conducted in Japan over the past thirty years have revealed a four-fold increase in the estimated number of multiple sclerosis (MS) patients, a decrease in the age at onset, and successive increases in patients with conventional MS, which shows an involvement of multiple sites in the central nervous system, including the cerebrum and cerebellum. We aimed to clarify whether genetic and infectious backgrounds correlate to distinct disease phenotypes of MS in Japanese patients. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed HLA-DRB1 and -DPB1 alleles, and IgG antibodies specific for Helicobacter pylori, Chlamydia pneumoniae, varicella zoster virus, and Epstein-Barr virus nuclear antigen (EBNA) in 145 MS patients and 367 healthy controls (HCs). Frequencies of DRB1*0405 and DPB1*0301 were significantly higher, and DRB1*0901 and DPB1*0401 significantly lower, in MS patients as compared with HCs. MS patients with DRB1*0405 had a significantly earlier age of onset and lower Progression Index than patients without this allele. The proportion and absolute number of patients with DRB1*0405 successively increased with advancing year of birth. In MS patients without DRB1*0405, the frequency of the DRB1*1501 allele was significantly higher, while the DRB1*0901 allele was significantly lower, compared with HCs. Furthermore, DRB1*0405-negative MS patients were significantly more likely to be positive for EBNA antibodies compared with HCs. CONCLUSIONS: Our study suggests that MS patients harboring DRB1*0405, a genetic risk factor for MS in the Japanese population, have a younger age at onset and a relatively benign disease course, while DRB1*0405-negative MS patients have features similar to Western-type MS in terms of association with Epstein-Barr virus infection and DRB1*1501. The recent increase of MS in young Japanese people may be caused, in part, by an increase in DRB1*0405-positive MS patients.

Although generating new neurons in the ischemic injured brain would be an ideal approach to replenish the lost neurons for repairing the damage, the adult mammalian brain retains only limited neurogenic capability. Here, we show that direct conversion of microglia/macrophages into neurons in the brain has great potential as a therapeutic strategy for ischemic brain injury. After transient middle cerebral artery occlusion in adult mice, microglia/macrophages converge at the lesion core of the striatum, where neuronal loss is prominent. Targeted expression of a neurogenic transcription factor, NeuroD1, in microglia/macrophages in the injured striatum enables their conversion into induced neuronal cells that functionally integrate into the existing neuronal circuits. Furthermore, NeuroD1-mediated induced neuronal cell generation significantly improves neurological function in the mouse stroke model, and ablation of these cells abolishes the gained functional recovery. Our findings thus demonstrate that neuronal conversion contributes directly to functional recovery after stroke. [ABSTRACT FROM AUTHOR]