Causal effects of sedentary behaviours on the risk of migraine: A univariable and multivariable Mendelian randomization study.

BACKGROUND: Migraine is a common and burdensome neurological disorder. The causal relationship between sedentary behaviours (SBs) and migraine remains instinct. We aimed to evaluate the roles of SBs including watching TV, using computer and driving in the risk of migraine. METHODS: We conducted a univariable and multivariable Mendelian randomization (MR) study based on summary datasets of large genome-wide association studies. The inverse variance weighted method was utilized as the primary analytical tool. Cochran's Q, MR-Egger intercept test, MR pleiotropy residual sum and outlier and leave-one-out were conducted as sensitivity analysis. Additionally, we performed a meta-analysis to combine the causal estimates. RESULTS: In the discovery analysis, we identified causal associations between time spent watching TV and an increased risk of migraine (p = 0.015) and migraine without aura (MO) (p = 0.002). Such causalities with increasing risk of migraine (p = 0.005), and MO (p = 0.006) were further verified using summary datasets from another study in the replication analysis. There was no significant causal association found between time spent using computer, driving and migraine or its two subtypes. The meta-analysis and multivariable MR analysis also strongly supported the causal relationships between time spent watching TV and an increased risk of migraine (p = 0.0003 and p = 0.034), as well as MO (p < 0.0001 and p = 0.0004), respectively. These findings were robust under all sensitivity analysis. CONCLUSIONS: Our study suggested that time spent watching TV may be causally associated with an increased risk of migraine, particularly MO. Large-scale and well-designed cohort studies may be warranted for further validation. SIGNIFICANCE STATEMENT: This study represents the first attempt to investigate whether a causal relationship exists between SBs and migraine. Utilizing MR analysis helps mitigate reverse causation bias and confounding factors commonly encountered in observational cohorts, thereby enhancing the robustness of derived causal associations. Our MR analysis revealed that time spent watching TV may serve as a potential risk factor for migraine, particularly MO.

Single-cell analysis of anti-BCMA CAR T cell therapy in patients with central nervous system autoimmunity.

Chimeric antigen receptor (CAR) T cell immunotherapy for the treatment of neurological autoimmune diseases is promising, but CAR T cell kinetics and immune alterations after treatment are poorly understood. Here, we performed single-cell multi-omics sequencing of paired cerebrospinal fluid (CSF) and blood samples from patients with neuromyelitis optica spectrum disorder (NMOSD) treated with anti-B cell maturation antigen (BCMA) CAR T cells. Proliferating cytotoxic-like CD8+ CAR T cell clones were identified as the main effectors in autoimmunity. Anti-BCMA CAR T cells with enhanced features of chemotaxis efficiently crossed the blood-CSF barrier, eliminated plasmablasts and plasma cells in the CSF, and suppressed neuroinflammation. The CD44-expressing early memory phenotype in infusion products was potentially associated with CAR T cell persistence in autoimmunity. Moreover, CAR T cells from patients with NMOSD displayed distinctive features of suppressed cytotoxicity compared with those from hematological malignancies. Thus, we provide mechanistic insights into CAR T cell function in patients with neurological autoimmune disease.

Individual cerebellar metabolic connectome in patients with MTLE and NTLE associated with surgical prognosis.

PURPOSE: This study aimed to comprehensively explore the different metabolic connectivity topological changes in MTLE and NTLE, as well as their association with surgical outcomes. METHODS: This study enrolled a cohort of patients with intractable MTLE and NTLE. Each individual's metabolic connectome, as determined by Kullback-Leibler divergence similarity estimation for the [18F]FDG PET image, was employed to conduct a comprehensive analysis of the cerebral metabolic network. Alterations in network connectivity were assessed by extracting and evaluating the strength of edge and weighted connectivity. By utilizing these two connectivity strength metrics with the cerebellum, we explored the network properties of connectivity and its association with prognosis in surgical patients. RESULTS: Both MTLE and NTLE patients exhibited substantial alterations in the connectivity of the metabolic network at the edge and nodal levels (p < 0.01, FDR corrected). The key disparity between MTLE and NTLE was observed in the cerebellum. In MTLE, there was a predominance of increased connectivity strength in the cerebellum. Whereas, a decrease in cerebellar connectivity was identified in NTLE. It was found that in MTLE, higher edge connectivity and weighted connectivity strength in the contralateral cerebellar hemisphere correlated with improved surgical outcomes. Conversely, in NTLE, a higher edge metabolic connectivity strength in the ipsilateral cerebellar hemisphere suggested a worse surgical prognosis. CONCLUSION: The cerebellum exhibits distinct topological characteristics in the metabolic networks between MTLE and NTLE. The hyper- or hypo-metabolic connectivity in the cerebellum may be a prognostic biomarker of surgical prognosis, which might aid in therapeutic decision-making for TLE individuals.

Contributing factors and interventions for fear of falling in stroke survivors: a systematic review.

OBJECTIVES: The purpose of this study was to provide a comprehensive overview of the prevalence, measurement tools, influencing factors, and interventions for fear of falling (FOF) in stroke survivors. METHODS: A PRISMA-guided systematic literature review was conducted. PubMed, EMBASE, Cochrane, and Web of Science were systematically searched. The search time was up to February 2023. All observational and experimental studies investigating FOF in stroke patients were included. The assessment tool of the Joanna Briggs Institute was used to assess the quality of the included studies and the risk of bias assessment. (PROSPERO: CRD42023412522). RESULT: A total of 25 observational studies and 10 experimental studies were included. The overall quality of the included studies was "low" to "good." The most common tool used to measure the FOF was the Falls Efficacy Scale-International (FES-I). The prevalence of FOF was 42%- 93.8%. Stroke survivors with physical impairments have the highest prevalence of FOF. The main risk factors for the development of FOF in stroke survivors were female gender, use of assistive devices, balance, limb dysfunction, and functional mobility. The combination of cognitive behavioral and exercise interventions is the most effective strategy. CONCLUSIONS: This review suggests that the prevalence of FOF in stroke survivors is high and that understanding the factors associated with FOF in stroke patients can help develop multifactorial prevention strategies to reduce FOF and improve quality of life. In addition, a uniform FOF measurement tool should be used to better assess the effectiveness of interventions for stroke survivors. ETHICS APPROVAL: PROSPERO registration (CRD42023412522).

Ldl-stimulated microglial activation exacerbates ischemic white matter damage.

The role of microglia in triggering the blood-brain barrier (BBB) impairment and white matter damage after chronic cerebral hypoperfusion is unclear. Here we demonstrated that the vessel-adjacent microglia were specifically activated by the leakage of plasma low-density lipoprotein (LDL), which led to BBB breakdown and ischemic demyelination. Interestingly, we found that LDL stimulation enhanced microglial phagocytosis, causing excessive engulfment of myelin debris and resulting in an overwhelming lipid burden in microglia. Surprisingly, these lipid-laden microglia exhibited a suppressed profile of inflammatory response and compromised pro-regenerative properties. Microglia-specific knockdown of LDLR or systematic medication lowering circulating LDL-C showed protective effects against ischemic demyelination. Overall, our findings demonstrated that LDL-stimulated vessel-adjacent microglia possess a disease-specific molecular signature, characterized by suppressed regenerative properties, which is associated with the propagation of demyelination during ischemic white matter damage.

Genetic causal association between physical activities and epilepsy: A Mendelian randomization study.

BACKGROUND: Despite numerous investigations into the relationship between physical activities (PA) and epilepsy, the causal effects remain contentious. Thus, we conducted a two-sample Mendelian randomization (MR) study to assess the potential causality. METHODS: Single-nucleotide polymorphisms (SNPs) predisposed to self-reported moderate and vigorous physical activities (MPA and VPA) and overall acceleration average (OAA) calculated through wrist-worn accelerometers were selected as exposure instrumental variables. Five subtypes of epilepsy, including all epilepsy, focal epilepsy and generalized epilepsy (with or without each other), focal epilepsy-strict definition and generalized epilepsy-strict definition (without overlap), were chosen as the outcomes. The MR study utilized the inverse-variance weighted (IVW) method as the primary analytical tool, supplemented by MR-Egger, simple mode, weighted mode, and weighted median methods. Cochran's Q and MR-Egger intercept tests were employed to assess heterogeneity and pleiotropy, while MR pleiotropy residual sum and outlier and leave-one-out analyses were conducted to identify potential SNP outliers. RESULTS: The study indicated that OAA was genetically linked to a decreased risk of both focal epilepsy (OR = 0.812, 95% CI: 0.687-0.960, p = .015, IVW) and focal epilepsy-strict definition (OR = 0.732, 95% CI: 0.596-0.900, p = .003, IVW; OR = 0.749, 95% CI: 0.573-0.979, p = .035, Weighted median). Genetically predicted MPA and VPA did not exhibit a causal association with all epilepsy or its subtypes (p>.05). No evidence of heterogeneity, pleiotropy, or SNP outlier was observed. CONCLUSIONS: Our findings suggested that PA with accelerometer monitoring may potentially reduce the risk of focal epilepsy, while there is no evidence supporting causal association between self-reported MPA or VPA and either focal or generalized epilepsy.

B cell lineage reconstitution underlies CAR-T cell therapeutic efficacy in patients with refractory myasthenia gravis.

B-cell maturation antigen (BCMA), expressed in plasmablasts and plasma cells, could serve as a promising therapeutic target for autoimmune diseases. We reported here chimeric antigen receptor (CAR) T cells targeting BCMA in two patients with highly relapsed and refractory myasthenia gravis (one with AChR-IgG, and one with MuSk-IgG). Both patients exhibited favorable safety profiles and persistent clinical improvements over 18 months. Reconstitution of B-cell lineages with sustained reduced pathogenic autoantibodies might underlie the therapeutic efficacy. To identify the possible mechanisms underlying the therapeutic efficacy of CAR-T cells in these patients, longitudinal single-cell RNA and TCR sequencing was conducted on serial blood samples post infusion as well as their matching infusion products. By tracking the temporal evolution of CAR-T phenotypes, we demonstrated that proliferating cytotoxic-like CD8 clones were the main effectors in autoimmunity, whereas compromised cytotoxic and proliferation signature and profound mitochondrial dysfunction in CD8+ Te cells before infusion and subsequently defect CAR-T cells after manufacture might explain their characteristics in these patients. Our findings may guide future studies to improve CAR T-cell immunotherapy in autoimmune diseases.

Causal association between the peripheral immunity and the risk and disease severity of multiple sclerosis.

Background: Growing evidence links immunological responses to Multiple sclerosis (MS), but specific immune factors are still unclear. Methods: Mendelian randomization (MR) was performed to investigate the association between peripheral hematological traits, MS risk, and its severity. Then, further subgroup analysis of immune counts and circulating cytokines and growth factors were performed. Results: MR revealed higher white blood cell count (OR [95%CI] = 1.26 [1.10,1.44], P = 1.12E-03, P adjust = 3.35E-03) and lymphocyte count (OR [95%CI] = 1.31 [1.15,1.50], P = 5.37E-05, P adjust = 3.22E-04) increased the risk of MS. In further analysis, higher T cell absolute count (OR [95%CI] = 2.04 [1.36,3.08], P = 6.37E-04, P adjust = 2.19E-02) and CD4+ T cell absolute count (OR [95%CI] = 2.11 [1.37,3.24], P = 6.37E-04, P adjust = 2.19E-02), could increase MS risk. While increasing CD25++CD4+ T cell absolute count (OR [95%CI] = 0.75 [0.66,0.86], P = 2.12E-05, P adjust = 1.72E-03), CD25++CD4+ T cell in T cell (OR [95%CI] = 0.79[0.70,0.89], P = 8.54E-05, P adjust = 5.29E-03), CD25++CD4+ T cell in CD4+ T cell (OR [95%CI] = 0.80[0.72,0.89], P = 1.85E-05, P adjust = 1.72E-03), and CD25++CD8+ T cell in T cell (OR [95%CI] = 0.68[0.57,0.81], P = 2.22E-05, P adjust = 1.72E-03), were proved to be causally defensive for MS. For the disease severity, the suggestive association between some traits related to CD4+ T cell, Tregs and MS severity were demonstrated. Moreover, elevated levels of IL-2Ra had a detrimental effect on the risk of MS (OR [95%CI] = 1.22 [1.12,1.32], P = 3.20E-06, P adjust = 1.34E-04). Conclusions: This study demonstrated a genetically predicted causal relationship between elevated peripheral immune cell counts and MS. Subgroup analysis revealed a specific contribution of peripheral immune cells, holding potential for further investigations into the underlying mechanisms of MS and its severity.

Rhizopus microsporus and Mucor racemosus coinfection following COVID-19 detected by metagenomics next-generation sequencing: A case of disseminated mucormycosis.

Mucormycosis is an invasive opportunistic fungal infection, which may be lethal and mostly affects patients with immunodeficiency or diabetes mellitus. Among Mucorales fungi, Rhizopus spp. is the most common cause of mucormycosis, followed by genera such as Mucor and Lichtheimia. Here we report a patient with severe COVID-19 infection who developed nasal pain, facial swelling, prominent black eschar on the nasal root. CT scan revealed pansinusitis along the maxillary, ethmoidal, and sphenoid sinuses. Mixed mold infection with Rhizopus microsporus and Mucor racemosus was detected by blood metagenomics next-generation sequencing (mNGS) and later nasal mucosa histological investigation confirmed mucormycosis. Severe COVID-19 infection led to the patient's thrombocytopenia and leukopenia. Later disseminated mucormycosis aggravated the infection and sepsis eventually resulted in death. It is the first case report of mucormycosis in which R. microsporus and M. racemosus as the etiologic agents were found simultaneously in one patient. COVID-19 infection combined with disseminated mucormycosisis can be fatal and mNGS is a fast, sensitive and accurate diagnostic method for fungi detection.

Serum LDL Promotes Microglial Activation and Exacerbates Demyelinating Injury in Neuromyelitis Optica Spectrum Disorder.

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune inflammatory demyelinating disease of the central nervous system (CNS) accompanied by blood-brain barrier (BBB) disruption. Dysfunction in microglial lipid metabolism is believed to be closely associated with the neuropathology of NMOSD. However, there is limited evidence on the functional relevance of circulating lipids in CNS demyelination, cellular metabolism, and microglial function. Here, we found that serum low-density lipoprotein (LDL) was positively correlated with markers of neurological damage in NMOSD patients. In addition, we demonstrated in a mouse model of NMOSD that LDL penetrates the CNS through the leaky BBB, directly activating microglia. This activation leads to excessive phagocytosis of myelin debris, inhibition of lipid metabolism, and increased glycolysis, ultimately exacerbating myelin damage. We also found that therapeutic interventions aimed at reducing circulating LDL effectively reversed the lipid metabolic dysfunction in microglia and mitigated the demyelinating injury in NMOSD. These findings shed light on the molecular and cellular mechanisms underlying the positive correlation between serum LDL and neurological damage, highlighting the potential therapeutic target for lowering circulating lipids to alleviate the acute demyelinating injury in NMOSD.

Identification of bidirectional causal links between gut microbiota and narcolepsy type 1 using Mendelian randomization.

STUDY OBJECTIVES: Narcolepsy type 1 (NT1), characterized by cataplexy and orexin deficiency, is a rare and frequently debilitating neurological disorder. It has been noted to have connections with the gut microbiota, yet the exact causal relationships remain unclear. METHODS: We conducted a comprehensive bidirectional Mendelian randomization (MR) study to rigorously investigate the causal links between the gut microbiota and NT1, utilizing genetic datasets from the MiBioGen consortium and FinnGen consortium, respectively. The inverse-variance weighted (IVW) method was employed to obtain the primary MR estimates, supplemented by several alternative methods as well as sensitivity analyses including Cochran's Q, MR-Egger, MR pleiotropy residual sum and outlier, leave-one-out, and genetic colocalization. RESULTS: Our findings indicated that an increased relative abundance of five genera including Blautia (p = 4.47E-5), Collinsella (p = 0.036), Gordonibacter (p = 0.047), Hungatella (p = 0.015), and Lachnospiraceae UCG010 (p = 0.027) may be associated with a decreased risk of NT1. Conversely, an increased relative abundance of class Betaproteobacteria (p = 0.032), genus Alloprevotella (p = 0.009), and genus Ruminiclostridium6 (p = 0.029) may potentially heighten the risk of NT1. The onset of NT1 may lead to a decrease in the relative abundance of genus Eubacterium eligens group (p = 0.022), while a increase in the family Family XI (p = 0.009), genus Hungatella (p = 0.005), genus Prevotella (p = 0.013), and unknown genus id.2001 (p = 0.019). These findings remained robust under all sensitivity analyses. CONCLUSIONS: Our results offer robust evidence for the bidirectional causal links between particular gut microbial taxa and NT1, underscoring the significance of the microbiota-gut-brain axis in the pathological process of NT1.

Single-cell analysis of refractory anti-SRP necrotizing myopathy treated with anti-BCMA CAR-T cell therapy.

Immune-mediated necrotizing myopathy (IMNM) is an autoimmune disorder associated with the presence of autoantibodies, characterized by severe clinical presentation with rapidly progressive muscular weakness and elevated levels of creatine kinase, while traditional pharmacological approaches possess varying and often limited effects. Considering the pathogenic role of autoantibodies, chimeric antigen receptor (CAR)-T cells targeting B cell maturation antigen (BCMA) have emerged as a promising therapeutic strategy. We reported here a patient with anti-signal recognition particle IMNM refractory to multiple available therapies, who was treated with BCMA-targeting CAR-T cells, exhibited favorable safety profiles, sustained reduction in pathogenic autoantibodies, and persistent clinical improvements over 18 mo. Longitudinal single-cell RNA, B cell receptor, T cell receptor sequencing analysis presented the normalization of immune microenvironment after CAR-T cell infusion, including reconstitution of B cell lineages, replacement of T cell subclusters, and suppression of overactivated immune cells. Analysis on characteristics of CAR-T cells in IMNM demonstrated a more active expansion of CD8+ CAR-T cells, with a dynamic phenotype shifting pattern similar in CD4+ and CD8+ CAR-T cells. A comparison of CD8+ CAR-T cells in patients with IMNM and those with malignancies collected at different timepoints revealed a more NK-like phenotype with enhanced tendency of cell death and neuroinflammation and inhibited proliferating ability of CD8+ CAR-T cells in IMNM while neuroinflammation might be the distinct characteristics. Further studies are warranted to define the molecular features of CAR-T cells in autoimmunity and to seek higher efficiency and longer persistence of CAR-T cells in treating autoimmune disorders.

Soluble TREM2 triggers microglial dysfunction in neuromyelitis optica spectrum disorders.

Microglia-mediated neuroinflammation contributes to acute demyelination in neuromyelitis optica spectrum disorders (NMOSD). Soluble triggering receptor expressed on myeloid cells 2 (sTREM2) in the CSF has been associated with microglial activation in several neurodegenerative diseases. However, the basis for this immune-mediated attack and the pathophysiological role of sTREM2 in NMOSD remain to be elucidated. Here, we performed Mendelian randomization analysis and identified a genetic association between increased CSF sTREM2 and NMOSD risk. CSF sTREM2 was elevated in patients with NMOSD and was positively correlated with neural injury and other neuroinflammation markers. Single-cell RNA sequencing of human macrophage/microglia-like cells in CSF, a proxy for microglia, showed that increased CSF sTREM2 was positively associated with microglial dysfunction in patients with NMOSD. Furthermore, we demonstrated that sTREM2 is a reliable biomarker of microglial activation in a mouse model of NMOSD. Using unbiased transcriptomic and lipidomic screens, we identified that excessive activation, overwhelmed phagocytosis of myelin debris, suppressed lipid metabolism and enhanced glycolysis underlie sTREM2-mediated microglial dysfunction, possibly through the nuclear factor kappa B (NF-κB) signalling pathway. These molecular and cellular findings provide a mechanistic explanation for the genetic association between CSF sTREM2 and NMOSD risk and indicate that sTREM2 could be a potential biomarker of NMOSD progression and a therapeutic target for microglia-mediated neuroinflammation.

TREM2-IGF1 Mediated Glucometabolic Enhancement Underlies Microglial Neuroprotective Properties During Ischemic Stroke.

Microglia, the major resident immune cells in the central nervous system, serve as the frontline soldiers against cerebral ischemic injuries, possibly along with metabolic alterations. However, signaling pathways involved in the regulation of microglial immunometabolism in ischemic stroke remain to be further elucidated. In this study, using single-nuclei RNA sequencing, a microglial subcluster up-regulated in ischemic brain tissues is identified, with high expression of Igf1 and Trem2, neuroprotective transcriptional signature and enhanced oxidative phosphorylation. Microglial depletion by PLX3397 exacerbates ischemic brain damage, which is reversed by repopulating the microglia with high Igf1 and Trem2 phenotype. Mechanistically, Igf1 serves as one of the major down-stream molecules of Trem2, and Trem2-Igf1 signaling axis regulates microglial functional and metabolic profiles, exerting neuroprotective effects on ischemic stroke. Overexpression of Igf1 and supplementation of cyclocreatine restore microglial glucometabolic levels and cellular functions even in the absence of Trem2. These findings suggest that Trem2-Igf1 signaling axis reprograms microglial immunometabolic profiles and shifts microglia toward a neuroprotective phenotype, which has promising therapeutic potential in treating ischemic stroke.

Trem2 deficiency attenuates microglial phagocytosis and autophagic-lysosomal activation in white matter hypoperfusion.

Chronic cerebral hypoperfusion leads to sustained demyelination and a unique response of microglia. Triggering receptor expressed on myeloid cells 2 (Trem2), which is expressed exclusively on microglia in the central nervous system (CNS), plays an essential role in microglial response in various CNS disorders. However, the specific role of Trem2 in chronic cerebral hypoperfusion has not been elucidated. In this study, we investigated the specific role of Trem2 in a mouse model of chronic cerebral hypoperfusion induced by bilateral carotid artery stenosis (BCAS). Our results showed that chronic hypoperfusion induced white matter demyelination, microglial phagocytosis, and activation of the microglial autophagic-lysosomal pathway, accompanied by an increase in Trem2 expression. After Trem2 knockout, we observed attenuation of white matter lesions and microglial response. Trem2 deficiency also suppressed microglial phagocytosis and relieved activation of the autophagic-lysosomal pathway, leading to microglial polarization towards anti-inflammatory and homeostatic phenotypes. Furthermore, Trem2 knockout inhibited lipid droplet accumulation in microglia in vitro. Collectively, these findings suggest that Trem2 deficiency ameliorated microglial phagocytosis and autophagic-lysosomal activation in hypoperfusion-induced white matter injury, and could be a promising target for the treatment of chronic cerebral hypoperfusion.

Research hotspots and trends on neuromyelitis optica spectrum disorders: insights from bibliometric analysis.

Neuromyelitis optica spectrum disorders (NMOSD) are demyelinating diseases of the central nervous system, have drawn the attention of many researchers due to the relapsing courses and cumulative disability. A first bibliometric analysis of NMOSD was conducted to identify the research hotspots and emerging trends. Articles relevant to NMOSD published in the core collection of Web of Science were retrieved and analyzed through visualized analysis using CiteSpace and VOSviewer, focusing on annual publication trends, countries, institutions, authors, journals, and keywords. The analysis showed that over the past 30 years, publications related to NMOSD had shown steady growth with slight fluctuations. The United States played an important part in this field, with the highest outputs and the greatest number of citations. Research hotspots of NMOSD had gradually shifted from the definition, biomarkers, and diagnostic criteria to diagnosis and treatment, particularly immunotherapy. This bibliometric analysis provides researchers with a theoretical basis for studying NMOSD and offers guidance for future research directions.

Risk Factors, Pathophysiologic Mechanisms, and Potential Treatment Strategies of Futile Recanalization after Endovascular Therapy in Acute Ischemic Stroke.

Endovascular therapy is the first-line treatment for acute ischemic stroke. However, studies have shown that, even with the timely opening of occluded blood vessels, nearly half of all patients treated with endovascular therapy for acute ischemic stroke still have poor functional recovery, a phenomenon called "futile recanalization.". The pathophysiology of futile recanalization is complex and may include tissue no-reflow (microcirculation reperfusion failure despite recanalization of the occluded large artery), early arterial reocclusion (reocclusion of the recanalized artery 24-48 hours post endovascular therapy), poor collateral circulation, hemorrhagic transformation (cerebral bleeding following primary ischemic stroke), impaired cerebrovascular autoregulation, and large hypoperfusion volume. Therapeutic strategies targeting these mechanisms have been attempted in preclinical research; however, translation to the bedside remains to be explored. This review summarizes the risk factors, pathophysiological mechanisms, and targeted therapy strategies of futile recanalization, focusing on the mechanisms and targeted therapy strategies of no-reflow to deepen the understanding of this phenomenon and provide new translational research ideas and potential intervention targets for improving the efficacy of endovascular therapy for acute ischemic stroke.