Innate immune memory after brain injury drives inflammatory cardiac dysfunction.

The medical burden of stroke extends beyond the brain injury itself and is largely determined by chronic comorbidities that develop secondarily. We hypothesized that these comorbidities might share a common immunological cause, yet chronic effects post-stroke on systemic immunity are underexplored. Here, we identify myeloid innate immune memory as a cause of remote organ dysfunction after stroke. Single-cell sequencing revealed persistent pro-inflammatory changes in monocytes/macrophages in multiple organs up to 3 months after brain injury, notably in the heart, leading to cardiac fibrosis and dysfunction in both mice and stroke patients. IL-1ß was identified as a key driver of epigenetic changes in innate immune memory. These changes could be transplanted to naive mice, inducing cardiac dysfunction. By neutralizing post-stroke IL-1ß or blocking pro-inflammatory monocyte trafficking with a CCR2/5 inhibitor, we prevented post-stroke cardiac dysfunction. Such immune-targeted therapies could potentially prevent various IL-1ß-mediated comorbidities, offering a framework for secondary prevention immunotherapy.

Brain ischemia causes systemic Notch1 activity in endothelial cells to drive atherosclerosis.

Stroke leads to persistently high risk for recurrent vascular events caused by systemic atheroprogression that is driven by endothelial cell (EC) activation. However, whether and how stroke induces sustained pro-inflammatory and proatherogenic endothelial alterations in systemic vessels remain poorly understood. We showed that brain ischemia induces persistent activation, the upregulation of adhesion molecule VCAM1, and increased senescence in peripheral ECs until 4 weeks after stroke onset. This aberrant EC activity resulted from sustained Notch1 signaling, which was triggered by increased circulating Notch1 ligands DLL1 and Jagged1 after stroke in mice and humans. Consequently, this led to increased myeloid cell adhesion and atheroprogression by generating a senescent, pro-inflammatory endothelium. Notch1- or VCAM1-blocking antibodies and the genetic ablation of endothelial Notch1 reduced atheroprogression after stroke. Our findings revealed a systemic machinery that induces the persistent activation of peripheral ECs after stroke, which paves the way for therapeutic interventions or the prevention of recurrent vascular events following stroke.

Magnetically powered microwheel thrombolysis of occlusive thrombi in zebrafish.

Tissue plasminogen activator (tPA) is the only FDA-approved treatment for ischemic stroke but carries significant risks, including major hemorrhage. Additional options are needed, especially in small vessel thrombi which account for ~25% of ischemic strokes. We have previously shown that tPA-functionalized colloidal microparticles can be assembled into microwheels (µwheels) and manipulated under the control of applied magnetic fields to enable rapid thrombolysis of fibrin gels in microfluidic models of thrombosis. Transparent zebrafish larvae have a highly conserved coagulation cascade that enables studies of hemostasis and thrombosis in the context of intact vasculature, clotting factors, and blood cells. Here, we show that tPA-functionalized µwheels can perform rapid and targeted recanalization in vivo. This effect requires both tPA and µwheels, as minimal to no recanalization is achieved with tPA alone, µwheels alone, or tPA-functionalized microparticles in the absence of a magnetic field. We evaluated tPA-functionalized µwheels in CRISPR-generated plasminogen (plg) heterozygous and homozygous mutants and confirmed that tPA-functionalized µwheels are dose-dependent on plasminogen for lysis. We have found that magnetically powered µwheels as a targeted tPA delivery system are dramatically more efficient at plasmin-mediated thrombolysis than systemic delivery in vivo. Further development of this system in fish and mammalian models could enable a less invasive strategy for alleviating ischemia that is safer than directed thrombectomy or systemic infusion of tPA.

The choroid plexus maintains adult brain ventricles and subventricular zone neuroblast pool, which facilitates poststroke neurogenesis.

The brain's neuroreparative capacity after injuries such as ischemic stroke is partly contained in the brain's neurogenic niches, primarily the subventricular zone (SVZ), which lies in close contact with the cerebrospinal fluid (CSF) produced by the choroid plexus (ChP). Despite the wide range of their proposed functions, the ChP/CSF remain among the most understudied compartments of the central nervous system (CNS). Here, we report a mouse genetic tool (the ROSA26iDTR mouse line) for noninvasive, specific, and temporally controllable ablation of CSF-producing ChP epithelial cells to assess the roles of the ChP and CSF in brain homeostasis and injury. Using this model, we demonstrate that ChP ablation causes rapid and permanent CSF volume loss in both aged and young adult brains, accompanied by disruption of ependymal cilia bundles. Surprisingly, ChP ablation did not result in overt neurological deficits at 1 mo postablation. However, we observed a pronounced decrease in the pool of SVZ neuroblasts (NBs) following ChP ablation, which occurs due to their enhanced migration into the olfactory bulb. In the middle cerebral artery occlusion model of ischemic stroke, NB migration into the lesion site was also reduced in the CSF-depleted mice. Thus, our study establishes an important role of ChP/CSF in regulating the regenerative capacity of the adult brain under normal conditions and after ischemic stroke.

Endothelial peroxiredoxin-4 is indispensable for blood-brain barrier integrity and long-term functional recovery after ischemic stroke.

The endothelial lining of cerebral microvessels is damaged relatively early after cerebral ischemia/reperfusion (I/R) injury and mediates blood-brain barrier (BBB) disruption, neurovascular injury, and long-term neurological deficits. I/R induces BBB leakage within 1 h due to subtle structural alterations in endothelial cells (ECs), including reorganization of the actin cytoskeleton and subcellular redistribution of junctional proteins. Herein, we show that the protein peroxiredoxin-4 (Prx4) is an endogenous protectant against endothelial dysfunction and BBB damage in a murine I/R model. We observed a transient upregulation of Prx4 in brain ECs 6 h after I/R in wild-type (WT) mice, whereas tamoxifen-induced, selective knockout of Prx4 from endothelial cells (eKO) mice dramatically raised vulnerability to I/R. Specifically, eKO mice displayed more BBB damage than WT mice within 1 to 24 h after I/R and worse long-term neurological deficits and focal brain atrophy by 35 d. Conversely, endothelium-targeted transgenic (eTG) mice overexpressing Prx4 were resistant to I/R-induced early BBB damage and had better long-term functional outcomes. As demonstrated in cultures of human brain endothelial cells and in animal models of I/R, Prx4 suppresses actin polymerization and stress fiber formation in brain ECs, at least in part by inhibiting phosphorylation/activation of myosin light chain. The latter cascade prevents redistribution of junctional proteins and BBB leakage under conditions of Prx4 repletion. Prx4 also tempers microvascular inflammation and infiltration of destructive neutrophils and proinflammatory macrophages into the brain parenchyma after I/R. Thus, the evidence supports an indispensable role for endothelial Prx4 in safeguarding the BBB and promoting functional recovery after I/R brain injury.

Pia-FLOW: Deciphering hemodynamic maps of the pial vascular connectome and its response to arterial occlusion.

The pial vasculature is the sole source of blood supply to the neocortex. The brain is contained within the skull, a vascularized bone marrow with a unique anatomical connection to the brain meninges. Recent developments in tissue clearing have enabled detailed mapping of the entire pial and calvarial vasculature. However, what are the absolute flow rate values of those vascular networks? This information cannot accurately be retrieved with the commonly used bioimaging methods. Here, we introduce Pia-FLOW, a unique approach based on large-scale transcranial fluorescence localization microscopy, to attain hemodynamic imaging of the whole murine pial and calvarial vasculature at frame rates up to 1,000 Hz and spatial resolution reaching 5.4 µm. Using Pia-FLOW, we provide detailed maps of flow velocity, direction, and vascular diameters which can serve as ground-truth data for further studies, advancing our understanding of brain fluid dynamics. Furthermore, Pia-FLOW revealed that the pial vascular network functions as one unit for robust allocation of blood after stroke.

Stroke in the Time of Circadian Medicine.

Time-of-day significantly influences the severity and incidence of stroke. Evidence has emerged not only for circadian governance over stroke risk factors, but also for important determinants of clinical outcome. In this review, we provide a comprehensive overview of the interplay between chronobiology and cerebrovascular disease. We discuss circadian regulation of pathophysiological mechanisms underlying stroke onset or tolerance as well as in vascular dementia. This includes cell death mechanisms, metabolism, mitochondrial function, and inflammation/immunity. Furthermore, we present clinical evidence supporting the link between disrupted circadian rhythms and increased susceptibility to stroke and dementia. We propose that circadian regulation of biochemical and physiological pathways in the brain increase susceptibility to damage after stroke in sleep and attenuate treatment effectiveness during the active phase. This review underscores the importance of considering circadian biology for understanding the pathology and treatment choice for stroke and vascular dementia and speculates that considering a patient's chronotype may be an important factor in developing precision treatment following stroke.

Hypertension: Causes and Consequences of Circadian Rhythms in Blood Pressure.

Hypertension is extremely common, affecting approximately 1 in every 2 adults globally. Chronic hypertension is the leading modifiable risk factor for cardiovascular disease and premature mortality worldwide. Despite considerable efforts to define mechanisms that underlie hypertension, a potentially major component of the disease, the role of circadian biology has been relatively overlooked in both preclinical models and humans. Although the presence of daily and circadian patterns has been observed from the level of the genome to the whole organism, the functional and structural impact of biological rhythms, including mechanisms such as circadian misalignment, remains relatively poorly defined. Here, we review the impact of daily rhythms and circadian systems in regulating blood pressure and the onset, progression, and consequences of hypertension. There is an emphasis on the impact of circadian biology in relation to vascular disease and end-organ effects that, individually or in combination, contribute to complex phenotypes such as cognitive decline and the loss of cardiac and brain health. Despite effective treatment options for some individuals, control of blood pressure remains inadequate in a substantial portion of the hypertensive population. Greater insight into circadian biology may form a foundation for novel and more widely effective molecular therapies or interventions to help in the prevention, treatment, and management of hypertension and its related pathophysiology.

Mitofusin-2 Regulates Platelet Mitochondria and Function.

BACKGROUND: Single-nucleotide polymorphisms linked with the rs1474868 T allele (MFN2 [mitofusin-2] T/T) in the human mitochondrial fusion protein MFN2 gene are associated with reduced platelet MFN2 RNA expression and platelet counts. This study investigates the impact of MFN2 on megakaryocyte and platelet biology. METHODS: Mice with megakaryocyte/platelet deletion of Mfn2 (Mfn2-/- [Mfn2 conditional knockout]) were generated using Pf4-Cre crossed with floxed Mfn2 mice. Human megakaryocytes were generated from cord blood and platelets isolated from healthy subjects genotyped for rs1474868. Ex vivo approaches assessed mitochondrial morphology, function, and platelet activation responses. In vivo measurements included endogenous/transfused platelet life span, tail bleed time, transient middle cerebral artery occlusion, and pulmonary vascular permeability/hemorrhage following lipopolysaccharide-induced acute lung injury. RESULTS: Mitochondria was more fragmented in megakaryocytes derived from Mfn2-/- mice and from human cord blood with MFN2 T/T genotype compared with control megakaryocytes. Human resting platelets of MFN2 T/T genotype had reduced MFN2 protein, diminished mitochondrial membrane potential, and an increased rate of phosphatidylserine exposure during ex vivo culture. Platelet counts and platelet life span were reduced in Mfn2-/- mice accompanied by an increased rate of phosphatidylserine exposure in resting platelets, especially aged platelets, during ex vivo culture. Mfn2-/- also decreased platelet mitochondrial membrane potential (basal) and activated mitochondrial oxygen consumption rate, reactive oxygen species generation, calcium flux, platelet-neutrophil aggregate formation, and phosphatidylserine exposure following dual agonist activation. Ultimately, Mfn2-/- mice showed prolonged tail bleed times, decreased ischemic stroke infarct size after cerebral ischemia-reperfusion, and exacerbated pulmonary inflammatory hemorrhage following lipopolysaccharide-induced acute lung injury. Analysis of MFN2 SNPs in the iSPAAR study (Identification of SNPs Predisposing to Altered ALI Risk) identified a significant association between MFN2 and 28-day mortality in patients with acute respiratory distress syndrome. CONCLUSIONS: Mfn2 preserves mitochondrial phenotypes in megakaryocytes and platelets and influences platelet life span, function, and outcomes of stroke and lung injury.

Circadian Biology and the Neurovascular Unit.

Mammalian physiology and cellular function are subject to significant oscillations over the course of every 24-hour day. It is likely that these daily rhythms will affect function as well as mechanisms of disease in the central nervous system. In this review, we attempt to survey and synthesize emerging studies that investigate how circadian biology may influence the neurovascular unit. We examine how circadian clocks may operate in neural, glial, and vascular compartments, review how circadian mechanisms regulate cell-cell signaling, assess interactions with aging and vascular comorbidities, and finally ask whether and how circadian effects and disruptions in rhythms may influence the risk and progression of pathophysiology in cerebrovascular disease. Overcoming identified challenges and leveraging opportunities for future research might support the development of novel circadian-based treatments for stroke.

Biological and Procedural Predictors of Outcome in the Stroke Preclinical Assessment Network (SPAN) Trial.

BACKGROUND: The SPAN trial (Stroke Preclinical Assessment Network) is the largest preclinical study testing acute stroke interventions in experimental focal cerebral ischemia using endovascular filament middle cerebral artery occlusion (MCAo). Besides testing interventions against controls, the prospective design captured numerous biological and procedural variables, highlighting the enormous heterogeneity introduced by the multicenter structure that might influence stroke outcomes. Here, we leveraged the unprecedented sample size achieved by the SPAN trial and the prospective design to identify the biological and procedural variables that affect experimental stroke outcomes in transient endovascular filament MCAo. METHODS: The study cohort included all mice enrolled and randomized in the SPAN trial (N=1789). Mice were subjected to 60-minute MCAo and followed for a month. Thirteen biological and procedural independent variables and 4 functional (weight loss and 4-point neuroscore on days 1 and 2, corner test on days 7 and 28, and mortality) and 3 tissue (day 2, magnetic resonance imaging infarct volumes and swelling; day 30, magnetic resonance imaging tissue loss) outcome variables were prospectively captured. Multivariable regression with stepwise elimination was used to identify the predictors and their effect sizes. RESULTS: Older age, active circadian stage at MCAo, and thinner and longer filament silicone tips predicted higher mortality. Older age, larger body weight, longer anesthesia duration, and longer filament tips predicted worse neuroscores, while high-fat diet and blood flow monitoring predicted milder neuroscores. Older age and a high-fat diet predicted worse corner test performance. While shorter filament tips predicted more ipsiversive turning, longer filament tips appeared to predict contraversive turning. Age, sex, and weight interacted when predicting the infarct volume. Older age was associated with smaller infarcts on day 2 magnetic resonance imaging, especially in animals with larger body weights; this association was most conspicuous in females. High-fat diet also predicted smaller infarcts. In contrast, the use of cerebral blood flow monitoring and more severe cerebral blood flow drop during MCAo, longer anesthesia, and longer filament tips all predicted larger infarcts. Bivariate analyses among the dependent variables highlighted a disconnect between tissue and functional outcomes. CONCLUSIONS: Our analyses identified variables affecting endovascular filament MCAo outcome, an experimental stroke model used worldwide. Multiple regression refuted some commonly reported predictors and revealed previously unrecognized associations. Given the multicenter prospective design that represents a sampling of real-world conditions, the degree of heterogeneity mimicking clinical trials, the large number of predictors adjusted for in the multivariable model, and the large sample size, we think this is the most definitive analysis of the predictors of preclinical stroke outcome to date. Future multicenter experimental stroke trials should standardize or at least ensure a balanced representation of the biological and procedural variables identified herein as potential confounders.

Cardiomyocyte PANX1 Controls Glycolysis and Neutrophil Recruitment in Hypertrophy.

BACKGROUND: PANX1 (pannexin 1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation, blood pressure regulation, and myocardial infarction. However, the possible role of PANX1 in cardiomyocytes in the progression of heart failure has not yet been investigated. METHOD: We generated a novel mouse line with constitutive deletion of PANX1 in cardiomyocytes (Panx1MyHC6). RESULTS: PANX1 deletion in cardiomyocytes had no effect on unstressed heart function but increased the glycolytic metabolism and resulting glycolytic ATP production, with a concurrent decrease in oxidative phosphorylation, both in vivo and in vitro. In vitro, treatment of H9c2 (H9c2 rat myoblast cell line) cardiomyocytes with isoproterenol led to PANX1-dependent release of ATP and Yo-Pro-1 uptake, as assessed by pharmacological blockade with spironolactone and siRNA-mediated knockdown of PANX1. To investigate nonischemic heart failure and the preceding cardiac hypertrophy, we administered isoproterenol, and we demonstrated that Panx1MyHC6 mice were protected from systolic and diastolic left ventricle volume increases as a result of cardiomyocyte hypertrophy. Moreover, we found that Panx1MyHC6 mice showed decreased isoproterenol-induced recruitment of immune cells (CD45+), particularly neutrophils (CD11b+ [integrin subunit alpha M], Ly6g+ [lymphocyte antigen 6 family member G]), to the myocardium. CONCLUSIONS: Together, these data demonstrate that PANX1 deficiency in cardiomyocytes increases glycolytic metabolism and protects against cardiac hypertrophy in nonischemic heart failure at least in part by reducing immune cell recruitment. Our study implies PANX1 channel inhibition as a therapeutic approach to ameliorate cardiac dysfunction in patients with heart failure.

LOX-1 and MMP-9 Inhibition Attenuates the Detrimental Effects of Delayed rt-PA Therapy and Improves Outcomes After Acute Ischemic Stroke.

BACKGROUND: Acute ischemic stroke triggers endothelial activation that disrupts vascular integrity and increases hemorrhagic transformation leading to worsened stroke outcomes. rt-PA (recombinant tissue-type plasminogen activator) is an effective treatment; however, its use is limited due to a restricted time window and hemorrhagic transformation risk, which in part may involve activation of MMPs (matrix metalloproteinases) mediated through LOX-1 (lectin-like oxLDL [oxidized low-density lipoprotein] receptor 1). This study's overall aim was to evaluate the therapeutic potential of novel MMP-9 (matrix metalloproteinase 9) ± LOX-1 inhibitors in combination with rt-PA to improve stroke outcomes. METHODS: A rat thromboembolic stroke model was utilized to investigate the impact of rt-PA delivered 4 hours poststroke onset as well as selective MMP-9 (JNJ0966) ±LOX-1 (BI-0115) inhibitors given before rt-PA administration. Infarct size, perfusion, and hemorrhagic transformation were evaluated by 9.4-T magnetic resonance imaging, vascular and parenchymal MMP-9 activity via zymography, and neurological function was assessed using sensorimotor function testing. Human brain microvascular endothelial cells were exposed to hypoxia plus glucose deprivation/reperfusion (hypoxia plus glucose deprivation 3 hours/R 24 hours) and treated with ±tPA and ±MMP-9 ±LOX-1 inhibitors. Barrier function was assessed via transendothelial electrical resistance, MMP-9 activity was determined with zymography, and LOX-1 and barrier gene expression/levels were measured using qRT-PCR (quantitative reverse transcription PCR) and Western blot. RESULTS: Stroke and subsequent rt-PA treatment increased edema, hemorrhage, MMP-9 activity, LOX-1 expression, and worsened neurological outcomes. LOX-1 inhibition improved neurological function, reduced edema, and improved endothelial barrier integrity. Elevated MMP-9 activity correlated with increased edema, infarct volume, and decreased neurological function. MMP-9 inhibition reduced MMP-9 activity and LOX-1 expression. In human brain microvascular endothelial cells, LOX-1/MMP-9 inhibition differentially attenuated MMP-9 levels, inflammation, and activation following hypoxia plus glucose deprivation/R. CONCLUSIONS: Our findings indicate that LOX-1 inhibition and ± MMP-9 inhibition attenuate negative aspects of ischemic stroke with rt-PA therapy, thus resulting in improved neurological function. While no synergistic effect was observed with simultaneous LOX-1 and MMP-9 inhibition, a distinct interaction is evident.

Focal Brain Lesions Causing Acquired Amusia Map to a Common Brain Network.

Music is a universal human attribute. The study of amusia, a neurologic music processing deficit, has increasingly elaborated our view on the neural organization of the musical brain. However, lesions causing amusia occur in multiple brain locations and often also cause aphasia, leaving the distinct neural networks for amusia unclear. Here, we utilized lesion network mapping to identify these networks. A systematic literature search was carried out to identify all published case reports of lesion-induced amusia. The reproducibility and specificity of the identified amusia network were then tested in an independent prospective cohort of 97 stroke patients (46 female and 51 male) with repeated structural brain imaging, specifically assessed for both music perception and language abilities. Lesion locations in the case reports were heterogeneous but connected to common brain regions, including bilateral temporoparietal and insular cortices, precentral gyrus, and cingulum. In the prospective cohort, lesions causing amusia mapped to a common brain network, centering on the right superior temporal cortex and clearly distinct from the network causally associated with aphasia. Lesion-induced longitudinal structural effects in the amusia circuit were confirmed as reduction of both gray and white matter volume, which correlated with the severity of amusia. We demonstrate that despite the heterogeneity of lesion locations disrupting music processing, there is a common brain network that is distinct from the language network. These results provide evidence for the distinct neural substrate of music processing, differentiating music-related functions from language, providing a testable target for noninvasive brain stimulation to treat amusia.

Electrophysiological Correlates of Dentate Nucleus Deep Brain Stimulation for Poststroke Motor Recovery.

While ipsilesional cortical electroencephalography has been associated with poststroke recovery mechanisms and outcomes, the role of the cerebellum and its interaction with the ipsilesional cortex is still largely unknown. We have previously shown that poststroke motor control relies on increased corticocerebellar coherence (CCC) in the low beta band to maintain motor task accuracy and to compensate for decreased excitability of the ipsilesional cortex. We now extend our work to investigate corticocerebellar network changes associated with chronic stimulation of the dentato-thalamo-cortical pathway aimed at promoting poststroke motor rehabilitation. We investigated the excitability of the ipsilesional cortex, the dentate (DN), and their interaction as a function of treatment outcome measures. Relative to baseline, 10 human participants (two women) at the end of 4-8 months of DN deep brain stimulation (DBS) showed (1) significantly improved motor control indexed by computerized motor tasks; (2) significant increase in ipsilesional premotor cortex event-related desynchronization that correlated with improvements in motor function; and (3) significant decrease in CCC, including causal interactions between the DN and ipsilesional cortex, which also correlated with motor function improvements. Furthermore, we show that the functional state of the DN in the poststroke state and its connectivity with the ipsilesional cortex were predictive of motor outcomes associated with DN-DBS. The findings suggest that as participants recovered, the ipsilesional cortex became more involved in motor control, with less demand on the cerebellum to support task planning and execution. Our data provide unique mechanistic insights into the functional state of corticocerebellar-cortical network after stroke and its modulation by DN-DBS.

Severity-Dependent Interhemispheric White Matter Connectivity Predicts Poststroke Neglect Recovery.

Left-sided spatial neglect is a very common and challenging issue after right-hemispheric stroke, which strongly and negatively affects daily living behavior and recovery of stroke survivors. The mechanisms underlying recovery of spatial neglect remain controversial, particularly regarding the involvement of the intact, contralesional hemisphere, with potential contributions ranging from maladaptive to compensatory. In the present prospective, observational study, we assessed neglect severity in 54 right-hemispheric stroke patients (32 male; 22 female) at admission to and discharge from inpatient neurorehabilitation. We demonstrate that the interaction of initial neglect severity and spared white matter (dis)connectivity resulting from individual lesions (as assessed by diffusion tensor imaging, DTI) explains a significant portion of the variability of poststroke neglect recovery. In mildly impaired patients, spared structural connectivity within the lesioned hemisphere is sufficient to attain good recovery. Conversely, in patients with severe impairment, successful recovery critically depends on structural connectivity within the intact hemisphere and between hemispheres. These distinct patterns, mediated by their respective white matter connections, may help to reconcile the dichotomous perspectives regarding the role of the contralesional hemisphere as exclusively compensatory or not. Instead, they suggest a unified viewpoint wherein the contralesional hemisphere can - but must not necessarily - assume a compensatory role. This would depend on initial impairment severity and on the available, spared structural connectivity. In the future, our findings could serve as a prognostic biomarker for neglect recovery and guide patient-tailored therapeutic approaches.

iMSC exosome delivers hsa-mir-125b-5p and strengthens acidosis resilience through suppression of ASIC1 protein in cerebral ischemia-reperfusion.

Acid-sensing ion channel 1 (ASIC1) is critical in acidotoxicity and significantly contributes to neuronal death in cerebral stroke. Pharmacological inhibition of ASIC1 has been shown to reduce neuronal death. However, the potential of utilizing exosomes derived from pluripotent stem cells to achieve inhibition of Asic1 remains to be explored. Developing qualified exosome products with precise and potent active ingredients suitable for clinical application is also ongoing. Here, we adopt small RNA-seq to interrogate the miRNA contents in exosomes of pluripotent stem cell induced mesenchymal stem cell (iMSC). RNA-seq was used to compare the oxygen-glucose deprivation-damaged neurons before and after the delivery of exosomes. We used Western blot to quantify the Asic1 protein abundance in neurons before and after exosome treatment. An in vivo test on rats validated the neuroprotective effect of iMSC-derived exosome and its active potent miRNA hsa-mir-125b-5p. We demonstrate that pluripotent stem cell-derived iMSCs produce exosomes with consistent miRNA contents and sustained expression. These exosomes efficiently rescue injured neurons, alleviate the pathological burden, and restore neuron function in rats under oxygen-glucose deprivation stress. Furthermore, we identify hsa-mir-125b-5p as the active component responsible for inhibiting the Asic1a protein and protecting neurons. We validated a novel therapeutic strategy to enhance acidosis resilience in cerebral stroke by utilizing exosomes derived from pluripotent stem cells with specific miRNA content. This holds promise for cerebral stroke treatment with the potential to reduce neuronal damage and improve clinical patient outcomes.

Early Versus Late Initiation of Direct Oral Anticoagulants After Ischemic Stroke in People With Atrial Fibrillation and Hemorrhagic Transformation: Prespecified Subanalysis of the Randomized Controlled ELAN Trial.

BACKGROUND: Whether hemorrhagic transformation (HT) modifies the treatment effect of early compared with late initiation of direct oral anticoagulation in people with ischemic stroke and atrial fibrillation is unknown. METHODS: This is a post hoc analysis of the ELAN trial (Early Versus Late Initiation of Direct Oral Anticoagulants in Post-Ischaemic Stroke Patients With Atrial Fibrillation). The primary outcome was a composite of recurrent ischemic stroke, symptomatic intracranial hemorrhage, major extracranial bleeding, systemic embolism, or vascular death within 30 days. Secondary outcomes were the individual components, 30- and 90-day functional outcome. We estimated outcomes based on HT, subclassified as hemorrhagic infarction (HI) or parenchymal hemorrhage (PH) on prerandomization imaging (core laboratory rating) using adjusted risk differences between treatment arms. RESULTS: Overall, 247 of 1970 participants (12.5%) had HT (114 HI 1, 77 HI 2, 34 PH 1, 22 PH 2). For the primary outcome, the estimated adjusted risk difference (early versus late) was -2.2% (95% CI, -7.8% to 3.5%) in people with HT (HI: -4.7% [95% CI, -10.8% to 1.4%]; PH: 6.1% [95% CI, -8.5% to 20.6%]) and -0.9% (95% CI, -2.6% to 0.8%) in people without HT. Numbers of symptomatic intracranial hemorrhage were identical in people with and without HT. With early treatment, the estimated adjusted risk difference for poor 90-day functional outcome (modified Rankin Scale score, 3-6) was 11.5% (95% CI, -0.8% to 23.8%) in participants with HT (HI: 7.4% [95% CI, -6.4% to 21.2%]; PH: 25.1% [95% CI, 0.2% to 50.0%]) and -2.6% (95% CI, -7.1% to 1.8%) in people without HT. CONCLUSIONS: We found no evidence of major treatment effect heterogeneity or safety concerns with early compared with late direct oral anticoagulation initiation in people with and without HT. However, early direct oral anticoagulation initiation may worsen functional outcomes in people with PH. REGISTRATION: URL: http://www.clinicaltrials.gov; Unique identifier: NCT03148457.

Arterial Thromboembolism in Patients With Atrial Fibrillation and CHA2DS2-VASc Score 1: A Nationwide Study.

BACKGROUND: Oral anticoagulation is suggested in patients with atrial fibrillation and a CHA2DS2-VASc score ≥1 (congestive heart failure, hypertension, age ≥75 years, diabetes, stroke, vascular disease, age 65-74 years, and sex score). To assess granular differences within CHA2DS2-VASc 1, the incidence of arterial thromboembolism according to CHA2DS2-VASc 1 subgroups was examined. METHODS: The Danish National Patient Registry and the Danish Prescription Registry were linked on a nationwide level to identify patients with atrial fibrillation from 2000 to 2021 without oral anticoagulation and categorized according to CHA2DS2-VASc score: CHA2DS2-VASc 0 (male and female subjects); CHA2DS2-VASc 1 (hypertension, heart failure, diabetes, vascular disease, and age 65-74 years); or CHA2DS2-VASc 2 (age ≥75 years without other risk factors). Female sex was not considered a risk factor in any risk group. The outcome was arterial thromboembolism (ischemic stroke, embolism of extremity, or transient cerebral ischemia). Study groups were compared using Cox regression analysis. RESULTS: We included 26 701 patients with a CHA2DS2-VASc 0 score; 22 915 with CHA2DS2-VASc 1 (1483 patients with heart failure, 9066 with hypertension, 843 with diabetes, 770 with vascular disease, and 10 753 who were 65 to 74 years of age); and 14 525 patients with CHA2DS2-VASc 2 (≥75 years of age without other risk factors). With a median of 1 year of observation time, the cumulative incidence of arterial thromboembolism was 0.6% (n=154 [95% CI, 0.6%-0.8%]), 1.4% (n=16 [95% CI, 0.8%-2.2%]), 1.9% (n=141 [95% CI, 1.6%-2.2%]), 1.7% (n=12 [95% CI, 0.9%-2.9%]), 2.0% (n=13 [95% CI, 1.1%-3.4%]), 2.3% (n=187 [95% CI, 2.0%-2.7%]), and 4.4% (n=533 [95% CI, 4.1%-4.8%]) for CHA2DS2-VASc 0, heart failure, hypertension, diabetes, vascular disease, age 65 to 74 years (CHA2DS2-VASc 1), and age ≥75 years (CHA2DS2-VASc 2), respectively. No statistically significant difference was identified among subgroups of CHA2DS2-VASc 1 (P=0.15 for difference). CONCLUSIONS: For patients with atrial fibrillation, all subgroups of CHA2DS2-VASc 1 were associated with lower incidence of arterial thromboembolism compared with age ≥75 years without other risk factors (ie, CHA2DS2-VASc 2) and a higher incidence compared with CHA2DS2-VASc 0. No statistically significant difference was identified between the subgroups of CHA2DS2-VASc 1. These findings support current recommendations that patients within this intermediate risk group could be identified with a similar risk of arterial thromboembolism.

Neurofilament Light Chain and Risk of Stroke in Patients With Atrial Fibrillation.

BACKGROUND: Biomarkers reflecting brain injury are not routinely used in risk assessment of stroke in atrial fibrillation (AF). Neurofilament light chain (NFL) is a novel biomarker released into blood after cerebral insults. We investigated the association between plasma concentrations of NFL, other biomarkers, and risk of stroke and death in patients with AF not receiving oral anticoagulation. METHODS: For this observational study, baseline plasma samples were available from 3077 patients with AF randomized to aspirin in ACTIVE A (Atrial Fibrillation Clopidogrel Trial With Irbesartan for Prevention of Vascular Events; 2003 to 2008) and AVERROES (Apixaban Versus Acetylsalicylic Acid [ASA] to Prevent Stroke in Atrial Fibrillation Patients Who Have Failed or Are Unsuitable for Vitamin K Antagonist Treatment; 2007 to 2009). Median follow-up was 1.5 years. NFL was analyzed with a Single Molecule Array (Simoa). Associations with outcomes (total stroke or systemic embolism, ischemic stroke, cardiovascular death, and all-cause death) were explored with Cox regression models. RESULTS: In the combined cohort, the median NFL level was 16.9 ng/L (interquartile range, 11.1-26.5 ng/L), the median age was 71 years, 58% were men, and 13% had a history of previous stroke. NFL was associated with older age, higher creatinine, lower body mass index, previous stroke, female sex, and diabetes but not cardiac rhythm. Higher NFL was associated with a higher risk of stroke or systemic embolism (n=206) independently of clinical characteristics (hazard ratio, 1.27 [95% CI, 1.10-1.46] per doubling of NFL) and other biomarkers (hazard ratio, 1.18 [95% CI, 1.01-1.37]) and including in patients without previous stroke (hazard ratio, 1.23 [95% CI, 1.02-1.48]). NFL was also independently associated with cardiovascular (n=219) and all-cause (n=311) death. The C index for stroke using only NFL was 0.642, on par with the currently used clinical risk scores. Addition of information on NFL improved discrimination in a model also including clinical information, NT-proBNP (N-terminal pro-B-type natriuretic peptide), and high-sensitivity cardiac troponin T, yielding a C index of 0.727. CONCLUSIONS: NFL reflects overt and covert episodes of cerebral ischemia and improves risk assessment of stroke and death in patients with AF without oral anticoagulation, including in patients without previous stroke. The combination of NFL with information on age, history of stroke, and other biomarkers should be explored as a future avenue for stroke risk assessments in patients with AF.