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.

Low-frequency stimulation enhances ensemble co-firing and dexterity after stroke.

Electrical stimulation is a promising tool for modulating brain networks. However, it is unclear how stimulation interacts with neural patterns underlying behavior. Specifically, how might external stimulation that is not sensitive to the state of ongoing neural dynamics reliably augment neural processing and improve function? Here, we tested how low-frequency epidural alternating current stimulation (ACS) in non-human primates recovering from stroke interacted with task-related activity in perilesional cortex and affected grasping. We found that ACS increased co-firing within task-related ensembles and improved dexterity. Using a neural network model, we found that simulated ACS drove ensemble co-firing and enhanced propagation of neural activity through parts of the network with impaired connectivity, suggesting a mechanism to link increased co-firing to enhanced dexterity. Together, our results demonstrate that ACS restores neural processing in impaired networks and improves dexterity following stroke. More broadly, these results demonstrate approaches to optimize stimulation to target neural dynamics.

Single Residue Variation in Skeletal Muscle Myosin Enables Direct and Selective Drug Targeting for Spasticity and Muscle Stiffness.

Muscle spasticity after nervous system injuries and painful low back spasm affect more than 10% of global population. Current medications are of limited efficacy and cause neurological and cardiovascular side effects because they target upstream regulators of muscle contraction. Direct myosin inhibition could provide optimal muscle relaxation; however, targeting skeletal myosin is particularly challenging because of its similarity to the cardiac isoform. We identified a key residue difference between these myosin isoforms, located in the communication center of the functional regions, which allowed us to design a selective inhibitor, MPH-220. Mutagenic analysis and the atomic structure of MPH-220-bound skeletal muscle myosin confirmed the mechanism of specificity. Targeting skeletal muscle myosin by MPH-220 enabled muscle relaxation, in human and model systems, without cardiovascular side effects and improved spastic gait disorders after brain injury in a disease model. MPH-220 provides a potential nervous-system-independent option to treat spasticity and muscle stiffness.

Mapping the Fine-Scale Organization and Plasticity of the Brain Vasculature.

The cerebral vasculature is a dense network of arteries, capillaries, and veins. Quantifying variations of the vascular organization across individuals, brain regions, or disease models is challenging. We used immunolabeling and tissue clearing to image the vascular network of adult mouse brains and developed a pipeline to segment terabyte-sized multichannel images from light sheet microscopy, enabling the construction, analysis, and visualization of vascular graphs composed of over 100 million vessel segments. We generated datasets from over 20 mouse brains, with labeled arteries, veins, and capillaries according to their anatomical regions. We characterized the organization of the vascular network across brain regions, highlighting local adaptations and functional correlates. We propose a classification of cortical regions based on the vascular topology. Finally, we analysed brain-wide rearrangements of the vasculature in animal models of congenital deafness and ischemic stroke, revealing that vascular plasticity and remodeling adopt diverging rules in different models.

Selenium Drives a Transcriptional Adaptive Program to Block Ferroptosis and Treat Stroke.

Ferroptosis, a non-apoptotic form of programmed cell death, is triggered by oxidative stress in cancer, heat stress in plants, and hemorrhagic stroke. A homeostatic transcriptional response to ferroptotic stimuli is unknown. We show that neurons respond to ferroptotic stimuli by induction of selenoproteins, including antioxidant glutathione peroxidase 4 (GPX4). Pharmacological selenium (Se) augments GPX4 and other genes in this transcriptional program, the selenome, via coordinated activation of the transcription factors TFAP2c and Sp1 to protect neurons. Remarkably, a single dose of Se delivered into the brain drives antioxidant GPX4 expression, protects neurons, and improves behavior in a hemorrhagic stroke model. Altogether, we show that pharmacological Se supplementation effectively inhibits GPX4-dependent ferroptotic death as well as cell death induced by excitotoxicity or ER stress, which are GPX4 independent. Systemic administration of a brain-penetrant selenopeptide activates homeostatic transcription to inhibit cell death and improves function when delivered after hemorrhagic or ischemic stroke.

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.

Single-Cell Transcriptomics and Fate Mapping of Ependymal Cells Reveals an Absence of Neural Stem Cell Function.

Ependymal cells are multi-ciliated cells that form the brain's ventricular epithelium and a niche for neural stem cells (NSCs) in the ventricular-subventricular zone (V-SVZ). In addition, ependymal cells are suggested to be latent NSCs with a capacity to acquire neurogenic function. This remains highly controversial due to a lack of prospective in vivo labeling techniques that can effectively distinguish ependymal cells from neighboring V-SVZ NSCs. We describe a transgenic system that allows for targeted labeling of ependymal cells within the V-SVZ. Single-cell RNA-seq revealed that ependymal cells are enriched for cilia-related genes and share several stem-cell-associated genes with neural stem or progenitors. Under in vivo and in vitro neural-stem- or progenitor-stimulating environments, ependymal cells failed to demonstrate any suggestion of latent neural-stem-cell function. These findings suggest remarkable stability of ependymal cell function and provide fundamental insights into the molecular signature of the V-SVZ niche.

Treg cell-derived osteopontin promotes microglia-mediated white matter repair after ischemic stroke.

The precise mechanisms underlying the beneficial effects of regulatory T (Treg) cells on long-term tissue repair remain elusive. Here, using single-cell RNA sequencing and flow cytometry, we found that Treg cells infiltrated the brain 1 to 5 weeks after experimental stroke in mice. Selective depletion of Treg cells diminished oligodendrogenesis, white matter repair, and functional recovery after stroke. Transcriptomic analyses revealed potent immunomodulatory effects of brain-infiltrating Treg cells on other immune cells, including monocyte-lineage cells. Microglia depletion, but not T cell lymphopenia, mitigated the beneficial effects of transferred Treg cells on white matter regeneration. Mechanistically, Treg cell-derived osteopontin acted through integrin receptors on microglia to enhance microglial reparative activity, consequently promoting oligodendrogenesis and white matter repair. Increasing Treg cell numbers by delivering IL-2:IL-2 antibody complexes after stroke improved white matter integrity and rescued neurological functions over the long term. These findings reveal Treg cells as a neurorestorative target for stroke recovery.

Post-injury immunosuppression and secondary infections are caused by an AIM2 inflammasome-driven signaling cascade.

Loss of lymphocytes, particularly T cell apoptosis, is a central pathological event after severe tissue injury that is associated with increased susceptibility for life-threatening infections. The precise immunological mechanisms leading to T cell death after acute injury are largely unknown. Here, we identified a monocyte-T cell interaction driving bystander cell death of T cells in ischemic stroke and burn injury. Specifically, we found that stroke induced a FasL-expressing monocyte population, which led to extrinsic T cell apoptosis. This phenomenon was driven by AIM2 inflammasome-dependent interleukin-1ß (IL-1ß) secretion after sensing cell-free DNA. Pharmacological inhibition of this pathway improved T cell survival and reduced post-stroke bacterial infections. As such, this study describes inflammasome-dependent monocyte activation as a previously unstudied cause of T cell death after injury and challenges the current paradigms of post-injury lymphopenia.

Regulation of cerebral blood flow in humans: physiology and clinical implications of autoregulation.

Brain function critically depends on a close matching between metabolic demands, appropriate delivery of oxygen and nutrients, and removal of cellular waste. This matching requires continuous regulation of cerebral blood flow (CBF), which can be categorized into four broad topics: 1) autoregulation, which describes the response of the cerebrovasculature to changes in perfusion pressure; 2) vascular reactivity to vasoactive stimuli [including carbon dioxide (CO2)]; 3) neurovascular coupling (NVC), i.e., the CBF response to local changes in neural activity (often standardized cognitive stimuli in humans); and 4) endothelium-dependent responses. This review focuses primarily on autoregulation and its clinical implications. To place autoregulation in a more precise context, and to better understand integrated approaches in the cerebral circulation, we also briefly address reactivity to CO2 and NVC. In addition to our focus on effects of perfusion pressure (or blood pressure), we describe the impact of select stimuli on regulation of CBF (i.e., arterial blood gases, cerebral metabolism, neural mechanisms, and specific vascular cells), the interrelationships between these stimuli, and implications for regulation of CBF at the level of large arteries and the microcirculation. We review clinical implications of autoregulation in aging, hypertension, stroke, mild cognitive impairment, anesthesia, and dementias. Finally, we discuss autoregulation in the context of common daily physiological challenges, including changes in posture (e.g., orthostatic hypotension, syncope) and physical activity.

Tissue-Specific Macrophage Responses to Remote Injury Impact the Outcome of Subsequent Local Immune Challenge.

Myocardial infarction, stroke, and sepsis trigger systemic inflammation and organism-wide complications that are difficult to manage. Here, we examined the contribution of macrophages residing in vital organs to the systemic response after these injuries. We generated a comprehensive catalog of changes in macrophage number, origin, and gene expression in the heart, brain, liver, kidney, and lung of mice with myocardial infarction, stroke, or sepsis. Predominantly fueled by heightened local proliferation, tissue macrophage numbers increased systemically. Macrophages in the same organ responded similarly to different injuries by altering expression of tissue-specific gene sets. Preceding myocardial infarction improved survival of subsequent pneumonia due to enhanced bacterial clearance, which was caused by IFNÉ£ priming of alveolar macrophages. Conversely, EGF receptor signaling in macrophages exacerbated inflammatory lung injury. Our data suggest that local injury activates macrophages in remote organs and that targeting macrophages could improve resilience against systemic complications following myocardial infarction, stroke, and sepsis.

ATP-induced conformational change of axonemal outer dynein arms revealed by cryo-electron tomography.

Axonemal outer dynein arm (ODA) motors generate force for ciliary beating. We analyzed three states of the ODA during the power stroke cycle using in situ cryo-electron tomography, subtomogram averaging, and classification. These states of force generation depict the prepower stroke, postpower stroke, and intermediate state conformations. Comparison of these conformations to published in vitro atomic structures of cytoplasmic dynein, ODA, and the Shulin-ODA complex revealed differences in the orientation and position of the dynein head. Our analysis shows that in the absence of ATP, all dynein linkers interact with the AAA3/AAA4 domains, indicating that interactions with the adjacent microtubule doublet B-tubule direct dynein orientation. For the prepower stroke conformation, there were changes in the tail that is anchored on the A-tubule. We built models starting with available high-resolution structures to generate a best-fitting model structure for the in situ pre- and postpower stroke ODA conformations, thereby showing that ODA in a complex with Shulin adopts a similar conformation as the active prepower stroke ODA in the axoneme.

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.

Neutrophils in aging and aging-related pathologies.

Over the past millennia, life expectancy has drastically increased. While a mere 25 years during Bronze and Iron ages, life expectancy in many European countries and in Japan is currently above 80 years. Such an increase in life expectancy is a result of improved diet, life style, and medical care. Yet, increased life span and aging also represent the most important non-modifiable risk factors for several pathologies including cardiovascular disease, neurodegenerative diseases, and cancer. In recent years, neutrophils have been implicated in all of these pathologies. Hence, this review provides an overview of how aging impacts neutrophil production and function and conversely how neutrophils drive aging-associated pathologies. Finally, we provide a perspective on how processes of neutrophil-driven pathologies in the context of aging can be targeted therapeutically.

Novel insight into the etiology of ischemic stroke gained by integrative multiome-wide association study.

Stroke, characterized by sudden neurological deficits, is the second leading cause of death worldwide. Although genome-wide association studies (GWAS) have successfully identified many genomic regions associated with ischemic stroke (IS), the genes underlying risk and their regulatory mechanisms remain elusive. Here, we integrate a large-scale GWAS (N = 1 296 908) for IS together with molecular QTLs data, including mRNA, splicing, enhancer RNA (eRNA), and protein expression data from up to 50 tissues (total N = 11 588). We identify 136 genes/eRNA/proteins associated with IS risk across 60 independent genomic regions and find IS risk is most enriched for eQTLs in arterial and brain-related tissues. Focusing on IS-relevant tissues, we prioritize 9 genes/proteins using probabilistic fine-mapping TWAS analyses. In addition, we discover that blood cell traits, particularly reticulocyte cells, have shared genetic contributions with IS using TWAS-based pheWAS and genetic correlation analysis. Lastly, we integrate our findings with a large-scale pharmacological database and identify a secondary bile acid, deoxycholic acid, as a potential therapeutic component. Our work highlights IS risk genes/splicing-sites/enhancer activity/proteins with their phenotypic consequences using relevant tissues as well as identify potential therapeutic candidates for IS.

Structural Plasticity in Adulthood with Motor Learning and Stroke Rehabilitation.

The development of advanced noninvasive techniques to image the human brain has enabled the demonstration of structural plasticity during adulthood in response to motor learning. Understanding the basic mechanisms of structural plasticity in the context of motor learning is essential to improve motor rehabilitation in stroke patients. Here, we review and discuss the emerging evidence for motor-learning-related structural plasticity and the implications for stroke rehabilitation. In the clinical context, a few studies have started to assess the effects of rehabilitation on structural measures to understand recovery poststroke and additionally to predict intervention outcomes. Structural imaging will likely have a role in the future in providing measures that inform patient stratification for optimal outcomes.

Predicting functional outcome in ischemic stroke patients using genetic, environmental, and clinical factors: a machine learning analysis of population-based prospective cohort study.

Ischemic stroke (IS) is a leading cause of adult disability that can severely compromise the quality of life for patients. Accurately predicting the IS functional outcome is crucial for precise risk stratification and effective therapeutic interventions. We developed a predictive model integrating genetic, environmental, and clinical factors using data from 7819 IS patients in the Third China National Stroke Registry. Employing an 80:20 split, we randomly divided the dataset into development and internal validation cohorts. The discrimination and calibration performance of models were evaluated using the area under the receiver operating characteristic curves (AUC) for discrimination and Brier score with calibration curve in the internal validation cohort. We conducted genome-wide association studies (GWAS) in the development cohort, identifying rs11109607 (ANKS1B) as the most significant variant associated with IS functional outcome. We employed principal component analysis to reduce dimensionality on the top 100 significant variants identified by the GWAS, incorporating them as genetic factors in the predictive model. We employed a machine learning algorithm capable of identifying nonlinear relationships to establish predictive models for IS patient functional outcome. The optimal model was the XGBoost model, which outperformed the logistic regression model (AUC 0.818 versus 0.756, P < .05) and significantly improved reclassification efficiency. Our study innovatively incorporated genetic, environmental, and clinical factors for predicting the IS functional outcome in East Asian populations, thereby offering novel insights into IS functional outcome.