中性粒细胞在缺血性卒中急性期，停留在血管中的作用

BACKGROUND: Obesity-induced hyperglycemia is a significant risk factor for stroke. Integrin α9β1 is expressed on neutrophils and stabilizes adhesion to the endothelium via ligands, including Fn-EDA (fibronectin containing extra domain A) and tenascin C. Although myeloid deletion of α9 reduces susceptibility to ischemic stroke, it is unclear whether this is mediated by neutrophil-derived α9. We determined the role of neutrophil-specific α9 in stroke outcomes in a mice model with obesity-induced hyperglycemia. METHODS: α9Neu-KO (α9fl/flMRP8Cre+) and littermate control α9WT (α9fl/flMRP8Cre−) mice were fed on a 60% high-fat diet for 20 weeks to induce obesity-induced hyperglycemia. Functional outcomes were evaluated up to 28 days after stroke onset in mice of both sexes using a transient (30 minutes) middle cerebral artery ischemia. Infarct volume (magnetic resonance imaging) and postreperfusion thrombo-inflammation (thrombi, fibrin, neutrophil, phospho-nuclear factor kappa B [p-NFκB], TNF [tumor necrosis factor]-α, and IL [interleukin]-1β levels, markers of neutrophil extracellular traps) were measured post 6 or 48 hours of reperfusion. In addition, functional outcomes (modified Neurological Severity Score, rota-rod, corner, and wire-hanging test) were measured for up to 4 weeks. RESULTS: Stroke upregulated neutrophil α9 expression more in obese mice (P<0.05 versus lean mice). Irrespective of sex, deletion of neutrophil α9 improved functional outcomes up to 4 weeks, concomitant with reduced infarct, improved cerebral blood flow, decreased postreperfusion thrombo-inflammation, and neutrophil extracellular traps formation (NETosis) (P<0.05 versus α9WT obese mice). Obese α9Neu-KO mice were less susceptible to thrombosis in FeCl3 injury-induced carotid thrombosis model. Mechanistically, we found that α9/cellular fibronectin axis contributes to NETosis via ERK (extracellular signal-regulated kinase) and PAD4 (peptidyl arginine deiminase 4), and neutrophil α9 worsens stroke outcomes via cellular fibronectin-EDA but not tenascin C. Obese wild-type mice infused with anti–integrin α9 exhibited improved functional outcomes up to 4 weeks (P<0.05 versus vehicle). CONCLUSIONS: Genetic ablation of neutrophil-specific α9 or pharmacological inhibition improves long-term functional outcomes after stroke in mice with obesity-induced hyperglycemia, most likely by limiting thrombo-inflammation.

Abstract Background Thrombosis is the key driver for ischemic events, including stroke, which is the leading cause of global mortality. However, current antithrombotic therapies carry substantial bleeding risks. Targeting protease-activated receptor-1 (PAR-1), a thrombin-activated receptor central to thrombus growth, represents a promising antithrombotic strategy for safer intervention. Material and Methods we mined the venom gland transcriptome of Conus spp., applied in silico Furin cleavage prediction, and synthesized and screened the anti-PAR-1 activity of a series of conotoxin-derived peptides. Results Cb-26 exhibited the strongest but reversible activity in inhibiting PAR-1-mediated platelet activation and aggregation in vitro. Crucially, Cb-26 inhibited platelet adhesion, neutrophil extracellular traps formation, and thrombus growth under shear conditions in whole blood from healthy donors. In murine models, Cb-26 significantly delayed carotid occlusion and reduced cerebral infarct size in photochemical-induced ischemic stroke, without affecting blood coagulation and bleeding time. Conclusion These results suggest that Cb-26 is a selective and reversible PAR-1 antagonist and represents a promising antithrombotic candidate without apparent bleeding side effects.

Ischemic stroke induces sustained microvascular dysfunction and inflammation, contributing to futile recanalization and poor tissue recovery despite successful large-vessel reopening. Neutrophil adhesion and capillary stalling are key drivers of this microvascular failure. While Ibudilast, a small-molecule phosphodiesterase (PDE3/4) inhibitor with vasodilatory and anti-inflammatory properties, has shown promise in modulating neuroinflammation, its impact on post-stroke microvascular flow and neutrophil-mediated obstruction remains poorly understood. In this study, we investigated the effects of Ibudilast on cerebral perfusion, neutrophil dynamics, and infarct size following transient middle cerebral artery occlusion (MCAO) in young (4-6 months) and aged (18-20 months) male and female C57BL/6 mice, a design intended to reflect clinically relevant variability in stroke outcomes. Ibudilast (30 mg/kg, intraperitoneally) was administered at 30 min and 6 h post-occlusion, and in vivo imaging was performed at 3hPR and 24hPR to assess hemodynamic parameters and neutrophil behavior. Treatment preserved vessel diameter, maintained flow velocity, restored red blood cell flux, and enhanced perfusion in both surface and capillary compartments, with improvements most pronounced in young and female cohorts. Neutrophil adhesion and capillary stalling were reduced. In vehicle-treated animals, neutrophil stalls correlated strongly with impaired vascular flow, whereas this relationship was absent in the treated group. These microvascular improvements were accompanied by a significant reduction in infarct volume (>50 %) at 24hPR compared to vehicle-treated controls. Together, these findings highlight Ibudilast's therapeutic potential for preserving microvascular integrity and guiding adjunctive interventions to mitigate futile recanalization in acute ischemic stroke.

No abstract available

Introduction: Acute cerebral blood flow occlusion by thrombus is a major cause of ischemic stroke. The composition of the resulting thrombus varies depending on the underlying medical condition. Notably, neutrophils, the most abundant white blood cells, play a crucial role in acute ischemic stroke and can form neutrophil extracellular traps (NETs). NETs are web-like structures consisting of DNA, granule proteins, and histones. NETs contribute to thrombo-inflammation and stabilize the thrombus, potentially leading to worse outcomes. While long non-coding RNA (lncRNA) do not encode proteins, they are involved in essential biological processes. The link between lncRNAs and cardiovascular disease is increasingly studied, and their role in ischemic stroke has recently come to attention. We, therefore, hypothesize that lncRNAs within NETs play a critical role in the formation and regulation of thrombi. Method: Human thrombi obtained from endovascular thrombectomy were categorized based on their underlying medical conditions, such as dyslipidemia, hypertension, or a combination of two or more conditions. Frozen sections of the thrombi were immunostained with markers including Citrullinated H3 (CitH3), CD45, neutrophil elastase (NE), and DAPI, and then analyzed using holotomography, confocal microscopy, and spatial transcriptomics. In spatial transcriptomics analysis, NETs and other leukocytes were selected as distal and proximal ROIs for detailed gene expression profiling. The gene expression of HDAC class 1 genes (HDAC1, HDAC2, HDAC3, and HDAC8) was further examined by qPCR. Results: Holotomography analysis revealed that cells in the thrombi of patients with underlying conditions exhibited a high mean refractive index. Immunofluorescence imaging also identified a high percentage of NETs in these samples. Spatial transcriptomics analysis confirmed that NETs in patients with hypertension showed upregulated expression of lncRNAs. Additionally, qPCR results demonstrated increased expression of HDAC class 1 genes, which are associated with NET formation, in patients with underlying conditions. Conclusion: LncRNAs play a crucial role in the formation and regulation of thrombi. Therefore, NETs-related lncRNAs could serve as valuable therapeutic targets and diagnostic markers for acute ischemic stroke patients with underlying diseases.

Introduction: Both histological and transcriptomic analyses of acute ischemic stroke (AIS) clots have identified features associated with mechanical thrombectomy (MT) outcome. However, few studies have explored how fluorescence histology and mRNA sequencing from the same clot can pinpoint specific biological phenomena associated with MT failure. Hypothesis: Joint analysis of paired clot immunofluorescence histology and mRNA sequencing will identify Neutrophil Extracellular Trap (NET) enrichment as a biomarker of MT first pass (FP) outcome. Methods: We performed paired histological and transcriptomic analysis of 32 stroke clots retrieved by MT (n=16 each of FP success and failure). Immunofluorescence histology was completed by co-staining 4µm clot tissue sections with antibodies against NET components (neutrophil elastase [ELANE], citrullinated histone [CitHis]) and super resolution imaging with instant structured illumination microscopy (ISIM) at 100X magnification. Differentially expressed genes (DEGs) were identified between transcriptomes of FP success and failure using the criteria of logFC≥1.5 and q<0.05. Principal component analysis (PCA) and Ingenuity Pathway Analysis (IPA) were completed to evaluate how well DEGs separated clots based on FP outcome and to identify enriched biological processes. Results: 44 DEGs were identified, most of which were downregulated in clots with successful FP. Expression of DEGs was able to separate clots by FP outcome. The NET signaling pathway was identified as the most enriched biological process in the FP failure group, with related genes downregulated in the FP success group. NETs were detected in clots with FP failure. Conclusion: Our results suggest that future multi-omic models, upon integration with pre-MT CT radiomics, can be developed to inform thrombolytic therapy and device selection, as well as predict FP outcome.

BACKGROUND Respiratory viruses, such as influenza viruses and SARS-CoV-2, cause severe infections of the respiratory system. Cohort studies and clinical observations indicate that patients with severe influenza A virus (IAV) infections are at an increased risk of developing an ischemic stroke event. However, the underlying mechanisms remain elusive. To this end, we investigated the consequences of IAV infection on cerebral damage in a mouse model of ischemic stroke. METHODS We intranasally inoculated male C57BL6/N mice with the mouse-adapted IAV strain A/Puerto Rico 8/34 or PBS as a vehicle control. At 3, 7, and 10 days post-infection, mice were subjected to transient middle cerebral artery occlusion, followed by sacrifice 24 hours after reperfusion for subsequent analysis. The anticoagulant drug acetylsalicylic acid was administered as treatment 1 day before transient middle cerebral artery occlusion. RESULTS Our research demonstrated a time-dependent deterioration of cerebral ischemia after transient middle cerebral artery occlusion, resulting in increased infarct volume and a worsened neurological outcome at the propagation and inflammation phases of infection. Our observations revealed an elevation in procoagulant activity and an increase in thrombosis within the microvasculature after infection and stroke. This effect was attributed to an infection-mediated inflammatory milieu and accelerated neutrophil response. Upon infection, the release of increased neutrophil extracellular traps by neutrophils had detrimental consequences for transient middle cerebral artery occlusion development. Administration of acetylsalicylic acid or control antiviral therapy prevented the IAV-induced exacerbation of stroke and reduced brain damage by reducing NETosis and coagulation. CONCLUSIONS These findings suggest that IAV infections enhance the systemic propensity for NETosis and foster a procoagulant state, thereby increasing the risk of cerebral damage and thrombosis following stroke. Targeting a combination of neutrophils and coagulation molecules simultaneously represents a promising treatment approach for clinical stroke.

Neutrophils are readily activated immune cells after ischemic stroke in mice and patients. Still, the impact of gut microbiota on neutrophil activation and its influence on inflammatory brain injury remain undefined. We report that natural microbiota colonization of germ-free (GF) mice induces substantial neutrophil activation and deteriorates stroke pathology. The colonized Ex-GF stroke mice had considerably larger infarct sizes and higher sensorimotor deficits than GF littermates. Furthermore, employing an antibiotic-based mouse model of microbiota deficiency, we demonstrate that gut microbiota depletion induces a juvenile neutrophil phenotype characterized by the upregulation of resting state surface receptors, reduced inflammatory proteins, and levels of circulating NETs. This disarming of neutrophil responses was associated with decreased expression of brain inflammatory genes, vascular thrombus formation, reduced infarct size, and alleviated behavioral deficits. We conclude that gut microbes strongly influence neutrophil activation after stroke and thus directly contribute to stroke severity. • Cohousing transfers gut microbiota to germ-free mice, increasing neutrophil activation. • Antibiotics-induced microbiota deficiency reduces neutrophil activation and protects the stroke brain. • Microbiota deficiency reduces circulating NETs, brain vascular thrombus formation and neuroinflammation after stroke.

Neutrophil extracellular traps (NETs) have been implicated in thrombotic diseases. There is no definitive explanation for how NETs form during acute ischemic strokes (AIS). The purpose of our study was to investigate the potential mechanism and role of NETs formation in the AIS process. As well as 45 healthy subjects, 45 patients with AIS had ELISA tests performed to detect NET markers. Expression of high-mobility group box 1 (HMGB1) on platelet microvesicles (PMVs) was analyzed by flow cytometry in healthy subjects and AIS patients’ blood samples. We established middle cerebral artery occlusion (MCAO) mice model to elucidate the interaction between PMPs and NETs. A significant elevation in NET markers was found in patient plasma in AIS patients, and neutrophils generated more NETs from patients’ neutrophils. HMGB1 expression was upregulated on PMVs from AIS patients and induced NET formation. NETs enhanced Procoagulant activity (PCA) through tissue factor and via platelet activation. Targeting lactadherin in genetical and in pharmacology could regulate the formation of NETs in MCAO model. NETs mediated by PMVs derived HMGB1 exacerbate thrombosis and brain injury in AIS. 59eUbqf-2Yb-L2dvdE9ZeT Video Abstract Video Abstract

Introduction: Stroke is a leading cause of death and disability worldwide, with diabetes being a significant risk factor that exacerbates stroke outcomes. While previous studies have identified various cell populations characteristic of stroke and other neurological diseases through transcriptomic analysis of brain and blood cells, the interaction between leukocytes and brain endothelial cells during stroke remains inadequately understood. This study aimed to investigate these intercellular interactions in the context of diabetes and stroke. Methods: We utilized a permanent ischemic stroke model in leptin receptor-deficient db/db mice (a model for type 2 diabetes) and their non-diabetic counterparts, db/+ mice. Three days post-stroke, brain cells and circulating leukocytes were isolated and subjected to single-cell RNA sequencing (scRNAseq). The CellChat tool was employed to analyze the intercellular communication between these cells. Additionally, in vivo two-photon microscopy was used to observe real-time interactions between leukocytes and brain endothelial cells in cortical veins. Results: scRNAseq analysis revealed that diabetic mice had a higher proportion of myeloid cells in the blood compared to controls. Post-stroke, both diabetic and non-diabetic mice showed an increased presence of microglia and macrophages in the brain. CellChat analysis indicated that both diabetes and stroke significantly elevated the number and strength of cell-cell interactions, with the highest levels observed in the diabetic stroke group. Notably, interactions involving the integrin family and ICAM/VCAM, known for their roles in cell adhesion between blood leukocytes and brain endothelial cells, were particularly prominent in the diabetic mice. In vivo imaging confirmed significant leukocyte accumulation in the cortical veins immediately after stroke in both genotypes, with delayed clearance observed in the diabetic group. Conclusion: The findings suggest that diabetes exacerbates the inflammatory response in stroke by enhancing leukocyte-endothelial interactions, leading to prolonged leukocyte retention in the cerebral vasculature, which likely contributes to increased inflammation and worsened stroke outcomes in diabetic conditions.

No abstract available

Ischemic stroke continues to be a leading cause of mortality and long‐term disability, affecting millions globally each year. Although nanoenzymes are explored as therapeutic candidates, conventional nanoenzymes predominantly target antioxidative mechanisms, which are insufficient to address the complex pathophysiology of ischemic stroke. In response to this challenge, this work introduces Transition Metal Nanocluster catalysts (TMNCs), which are widely recognized for their antioxidative and enzyme‐mimicking properties. However, this research reveals these nanoclusters, specifically molybdenum (Mo), vanadium (V) and tungsten (W), exhibit a novel and critical Deoxyribonuclease I (DNase1)‐like activity, previously unrecognized in the context of stroke therapy. Among these, Mo nanoclusters (Mo NCs) emerged as the most potent DNase1 mimics, efficiently degrading neutrophil extracellular traps (NETs) and disrupting the detrimental NET‐free radical cycle that exacerbates ischemic damage. This unique mechanism not only addresses oxidative stress but also mitigates NET‐associated inflammation, offering a dual‐action therapeutic approach. Behavioral studies in animal models demonstrated that Mo NCs significantly accelerated motor function recovery while providing robust neuroprotection.

Excessive production of neutrophil extracellular traps (NETs) contributes to immunothrombosis activation in ischemic stroke pathogenesis. The metabolic and mechanistic regulators of NET formation in relation to platelet activation during ischemic stroke remain poorly understood. In the present study, using multiple animal and clinical models of stroke, we examined the role of homocysteine and its biochemical modifications on NET formation and concomitant neuronal damage, platelet aggregation, motor and gait functions. Phosphoproteomics analysis of neutrophils in response to homocysteine revealed an enrichment of kinases and phosphoproteins associated with thrombosis. Homocysteinylated albumin induced significant NET formation via Erk1/2, Akt, ATM, and PAD4‐dependent pathways, independent of reactive oxygen species. Hyperhomocysteinemic mice fed a methionine‐rich diet exhibited elevated NETs components (neutrophil elastase, citrullinated histones, cell‐free DNA (cfDNA)), platelet activation, neuronal damage, and impaired motor, balance, and learning functions. UCCAO‐induced ischemia exacerbated neuronal damage, motor dysfunction, and platelet activation in hyperhomocysteinemic mice, which were reversed by disrupting NETs with N‐acetyl cysteine and DNase. In stroke patients, homocysteine, neutrophil elastase, and cfDNA levels were significantly elevated, independent of comorbidities (e.g., hypertension, type 2 diabetes), etiology (TOAST classification), or stroke severity. Additionally, stroke patients generated autoantibodies against homocysteinylated albumin, which positively correlated with neutrophil elastase levels. This study identifies homocysteine and its modifications as key metabolic regulators of NETosis in stroke, linking NETs formation to platelet activation and neuronal damage. These findings highlight potential therapeutic targets for mitigating stroke pathogenesis through the modulation of NETs.

Background Patients with cancer exhibit an increased risk of acute ischemic stroke (AIS), and neutrophil extracellular traposis (NETosis) has been proposed as a mechanism underlying cancer-associated hypercoagulability. However, studies validating these findings in independent cohorts are limited. Objective We sought to explore whether NETosis-associated markers (plasma DNA and nucleosomes) are increased in patients with active cancer and AIS, and whether these increases correlate with coagulopathy markers in cancer patients. Methods We analyzed NETosis-associated markers in cancer patients with and without AIS and healthy controls, and assessed the correlation between these markers and coagulopathy markers. Additionally, we compared the levels of Netosis-associated markers between cancer patients with conventional stroke mechanisms (CSM) and those with embolic stroke of undetermined source (ESUS). Results Plasma DNA and nucleosome levels were significantly higher in cancer patients with AIS than in cancer controls and healthy controls (p < 0.001, respectively). Both markers correlated with D-dimer levels in cancer patients with AIS. In a sub-analysis, cancer patients with ESUS showed higher levels of NETosis-associated markers compared to those with CSM, whereas vascular risk factors were more frequently observed in cancer patients with CSM. Conclusion These findings suggest that NETosis may contribute to hypercoagulability in patients with active cancer and AIS, particularly in those with ESUS. These results provide additional evidence supporting the establishment of pathophysiology-based therapeutic approaches.

Objective To investigate the correlation between Neutrophil Extracellular Traps (NETs) content and macrophages in thrombi of acute ischemic stroke (AIS) patients, as well as the differential degradation and clearance capacities of macrophages polarized into distinct functional states. Methods 60 AIS patients treated with endovascular mechanical thrombectomy at Bozhou People’s Hospital were enrolled. Thrombus samples from 30 patients underwent immunohistochemical staining for citrullinated histone 3 (CitH3), CD16, and CD163. CitH3-positive area percentage was quantified to evaluate NETs content. Pearson’s correlation analysis was applied to assess associations between M1(CD16⁺) and M2(CD163⁺) macrophage densities and the CitH3-positive area in thrombus. For the remaining 30 thrombi, co-culture experiments with polarized macrophages were conducted. CitH3 concentrations before and after co-culture were measured via enzyme-linked immunosorbent assay (ELISA), with a blank control group as a reference. Statistical comparisons between groups were performed using Student’s t-tests. Results All 30 thrombi exhibited positive expression of CitH3, CD16, and CD163. CD16+ and CD163+ macrophage densities significantly correlated with CitH3-positive area percentage (r = 0.538 and 0.641, P < 0.05). Co-culture with M1 or M2 macrophages significantly reduced CitH3 concentrations compared to the blank control (P < 0.05). Notably, M1 macrophages demonstrated superior NETs degradation efficacy compared to M2, as evidenced by lower post-co-culture CitH3 levels (P = 0.038). Conclusion NETs are contained in the thrombus of patients with acute ischemic stroke. The numbers of M1 macrophages and M2 macrophages in thrombus are positively correlated with the content of NETs. M1 and M2 macrophages derived from human monocytes have the ability to degrade and clear NETs, and the effect of M1 macrophages in degrading and clearing NETs may be stronger than M2 macrophages.

In stroke patients, local sampling of pial blood within the occluded vasculature before recanalization by mechanical thrombectomy emerged as powerful tool enabling insights into ultra-early stroke pathophysiology. Thereby, a strong intravascular inflammatory response hallmarked by hyper-acute neutrophil recruitment, altered lymphocyte composition and platelet activation could be observed. These human findings mirror experimental stroke. Here, neutrophil and T-cell activation are driven by platelets involving engagement of platelet glycoprotein receptor (GP)Ib, GPVI and CD84 as well as α-granule release orchestrating infarct progression. Thus, targeting of early intravascular inflammation may evolve as a new therapeutic strategy to augment the effects of recanalization.

Neovascularization and vascular remodeling are functionally important for brain repair after stroke. We show that neutrophils accumulate in the peri-infarct cortex during all stages of ischemic stroke. Neutrophils producing intravascular and intraparenchymal neutrophil extracellular traps (NETs) peak at 3–5 days. Neutrophil depletion reduces blood-brain barrier (BBB) breakdown and enhances neovascularization at 14 days. Peptidylarginine deiminase 4 (PAD4), an enzyme essential for NET formation, is upregulated in peri-ischemic brains. Overexpression of PAD4 induces an increase in NET formation that is accompanied by reduced neovascularization and increased BBB damage. Disruption of NETs by DNase 1 and inhibition of NET formation by genetic ablation or pharmacologic inhibition of PAD increases neovascularization and vascular repair and improves functional recovery. Furthermore, PAD inhibition reduces stroke-induced STING-mediated production of IFN-β, and STING knockdown and IFN receptor-neutralizing antibody treatment reduces BBB breakdown and increases vascular plasticity. Collectively, our results indicate that NET release impairs vascular remodeling during stroke recovery. Following ischemic insult, neovascularization and vascular remodelling play an essential part in the repair of brain damage. Here the authors show that neutrophil extracellular traps serve a detrimental role in the regeneration process, limiting the functional recovery of the brain post injury.

Following cerebral ischemia, neutrophil extracellular traps (NETs) contribute significantly to brain damage by exacerbating delayed immune cell infiltration and vascular injury. They are detected both in brain tissue and within blood vessels. Danger-associated molecular pattern (DAMP) molecules have been implicated in inducing NETosis after cerebral ischemia. This study investigated the role of High mobility group box 1 (HMGB1), a prototype DAMP molecule, in NETosis induction following photothrombotic stroke (PTS), with a particular focus on neutrophil-platelet interactions. In PTS, thrombi consist primarily of aggregated platelets and neutrophils, lacking significant fibrin content. Triphenyltetrazolium chloride (TTC) staining revealed rapid but progressive expansion of the infarct area in the PTS model, commencing within 1 h and continuing until 24 h. Concomitant with this, peripheral neutrophils isolated following PTS exhibited progressive NETosis, particularly intravascular NETosis. This was evidenced by significant increase in citrullinated histone H3 (CitH3), a marker of NETosis, as early as 1 h post-PTS. Furthermore, serum levels of free DNA gradually and significantly increased, further supporting the induction of NETosis following PTS. Intranasal administration of BBCA, a peptidylarginine deiminase (PAD) inhibitor, effectively suppressed the induction of intravascular NETosis. Importantly, BBCA administration, both 30 min before and 4 h after PTS surgery, significantly reduced infarct volumes at 24 h and improved neurological outcomes. These findings underscore the crucial role of NETosis in both the initiation and progression of ischemic brain damage in this model. Following PTS, HMGB1 rapidly accumulated in serum, detectable as early as 1 h. Immunofluorescence staining revealed initial localization of HMGB1 in neurons, followed by its accumulation within activated neutrophils and platelets within blood vessels. Functional inhibition of HMGB1 by intranasal administration of an HMGB1 A box 4 h post-PTS significantly suppressed NETosis induction, reduced infarct volume, and improved neurological deficits, confirming the pivotal role of HMGB1 in NETosis induction. Notably, we observed a rapid platelet activation and concomitant HMGB1 induction within activated platelets after PTS. Co-culture experiments using naïve PMNs-platelets isolated following PTS demonstrate that extracellular HMGB1, particularly one derived from platelets, plays a critical role in activating neutrophils and inducing intravascular NETosis via a TLR4-dependent manner. Collectively, these findings highlight the critical role of NETosis not only in the initial stages of thrombus formation but also in the subsequent progression of ischemic brain damage in the PTS animal model. HMGB1, particularly platelet-derived HMGB1, emerges as a key mediator to this process. Therefore, targeting NETosis through modulation of HMGB1 presents a promising multipotent therapeutic strategy for mitigating ischemic brain damage.

The recruitment and formation of neutrophil extracellular traps (NETs) by neutrophils play an important role in reperfusion injury in ischemic stroke. Current nanosystem‐based therapeutic strategies are mainly confined within the blood‐brain barrier (BBB), ignoring the constant intrusion from external challenges. Here, considering the unique vascular localization of NETs, a DNase I‐mediated NETs‐targeting nanoparticle is developed to integrate the catalytic and chemotactic functions of DNase I and achieve the synergistic regulation of the internal and external microenvironment of the neurovascular unit (NVU). DNase I navigates the nanoparticles to the lesion, enabling the accumulation in the brain through damaged BBB. The removal of intravascular NETs mitigates the ongoing destruction of the endothelium and reduces the recruitment of immunothrombosis. The main nanoparticles with dual antioxidant activity rescue neuronal apoptosis by scavenging reactive oxygen species (ROS) and protecting mitochondria. Reduced infarct size and remodeling of microenvironment homeostasis shown in the middle cerebral artery occlusion/reperfusion (MCAO) mouse model. This strategy provides new insights into the vascular side treatment of ischemic stroke. Targeting mediated by enzyme chemotaxis is first validated and showed the potential of a universal chemotactic targeted delivery strategy.

Background: Neutrophil extracellular trap (NET) production has been implicated in the pathogenesis of thromboinflammatory conditions such as Sickle Cell Disease (SCD), contributing to heightened risk for ischemic stroke. NETs are catalyzed by the enzyme Peptidyl Arginine Deiminase 4 (PAD4) and neutrophil derived reactive oxygen species (ROS), especially NADPH oxidase (NOX) which interacts with PAD4 and is therefore critical for neutrophil function. However, the role that NOX-dependent ROS and NETs play in the accelerated cerebral microvascular thrombosis associated with thromboinflammatory conditions, such as SCD, has not been fully elucidated and is the aim of this study. Methods: The in-vitro effects of targeting PAD4 and NOX were examined using physiologically relevant NET assays with neutrophils isolated from healthy volunteers (control) and SCD patients. In addition, in-vivo intravascular effects of targeting PAD4 and NOX in the cerebral microcirculation of C57BL/6 and sickle transgenic mice (STM) were assessed using a photoactivation thrombosis model (light/dye) coupled with real-time fluorescence intravital microscopy. Results: We found that targeting PAD4 and NOX in human neutrophils significantly inhibited ionomycin dependent H3cit+ neutrophils. Targeting PAD4 and NOX in-vivo resulted in prolonged blood flow cessation in cerebrovascular arterioles as well as venules. Moreover, we were able to replicate the effects of PAD4 and NOX targeting in a clinical model of accelerated thromboinflammation by increasing blood flow cessation times in cerebral microvessels in STM. These findings concurred with the clinical setting i.e. neutrophils isolated from SCD patients, which possessed an attenuation of H3cit+ neutrophil production on targeting PAD4 and NOX. Conclusions: Taken together, our compelling data suggests that PAD4 and NOX play a significant role in neutrophil driven thromboinflammation. Targeting PAD4 and NOX limits pathological H3cit+ neutrophils, which may further explain attenuation of cerebral thrombosis. Overall, this study presents a viable pre-clinical model of prevention and management of thromboinflammatory complications such as ischemic stroke.

Neutrophils are reported to be critical mediators of to poor outcome after subarachnoid hemorrhage (SAH). Following ischemic stroke, neutrophils cause vascular occlusion via neutrophil extracellular traps (NETs). In this study, our hypothesis was that NETs cause vascular occlusion leading to delayed cerebral ischemia (DCI) and worse outcome after SAH. SAH mice (male and female, young and aged) were administered one of the following: a neutrophil depleting antibody, a PAD4 inhibitor (to prevent NETosis), DNAse-I (to degrade NETs), or a vehicle (control). Mice had neurological assessment daily until day 7 and then were euthanized to quantify the amount of brain vascular NETs. Various subcohorts of mice were used to assess neutrophil propensity to NETose, in vivo formation of NETs via intravital microscopy, and MRI assessment of cerebral perfusion and infarct development. We also examined if NETs were present after SAH in 128 humans by measuring NETs markers via ELISA at 1, 2, 4, 7, and 10 days post-SAH. In mice, SAH induced a pro-NETosis phenotype selectively in skull neutrophils and caused intravascular NETs to be present by day 1 which persisted until at least day 7. Neutrophil depletion reduced NETs, improved cerebral perfusion and neurological outcome, and attenuated the incidence of DCI. Similar findings were observed for PAD4 inhibition. However, degrading NETs only marginally improved outcomes. Aneurysmal SAH patients who developed DCI had elevated markers of NETs compared to non-DCI patients. In conclusion, after SAH, skull-derived neutrophils are primed for NETosis, and neutrophils and NETosis are therapeutic targets to reduce DCI by preventing vascular occlusion by NETs.

Rationale: Large vessel recanalization in ischemic stroke does not always go along with tissue reperfusion, a phenomenon called “no-reflow”. However, knowledge of the mechanism of no-reflow is limited because identifying microvascular obstruction across the cortex and subcortex both in clinical and experimental models is challenging. In this study, we developed a smart three-dimensional recognition pipeline for microvascular obstruction during post-ischemia reperfusion to examine the underlying mechanism of no-reflow. Methods: Transient (60 min) occlusion of the middle cerebral artery (tMCAo) in mice was induced using a filament. Two different fluorophore-conjugated tomato lectins were injected into mice via the tail vein before and after ischemia/reperfusion (I/R), respectively, one to label all blood vessels and the other to label functional blood vessels. Post-I/R microvascular obstruction was visualized using combined iDISCO+-based tissue clearing and optical imaging. Arterioles and capillaries were distinguished using whole-mount immunolabeling with an anti-αSMA antibody. Circulating neutrophils were depleted utilizing an anti-Ly6G antibody. Brain slices were immunostained with the anti-Ly6G antibody to identify co-localized blockage points and neutrophils. MATLAB software was used to quantify the capillary diameters in the ipsilateral brain from the normal and tMCAo mice. Results: Microcirculatory reperfusion deficit worsened over time after I/R. Microvascular obstruction occurred not only in arterioles but also in capillaries, with capillary obstruction associated with local capillary lumen narrowing. In addition, the depletion of circulating neutrophils mitigated reperfusion deficit to a large extent after I/R. The co-localization of blockage points and neutrophils revealed that some neutrophils plugged capillaries with coexisting capillary lumen narrowing and that no neutrophil was trapped in heaps of blockage points. Quantification of the capillary diameter showed that capillary lumen shrunk after I/R but returned to typical measurements when intravascular neutrophils were depleted. Conclusions: According to our findings, both vascular lumen narrowing and neutrophil trapping in cerebral microcirculation are the key causes of microvascular obstruction after I/R. Also, the primary contribution by neutrophils to microvascular obstruction does not occur through microemboli plugging but rather via the exacerbation of capillary lumen narrowing. Our proposed method will help monitor microcirculatory reperfusion deficit, explore the mechanism of no-reflow, and evaluate the curative effect of drugs targeting no-reflow.

Significance Ischemic stroke is a leading cause of death and disability with limited therapies. Neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8) is a ubiquitin-like protein that is involved in protein neddylation. The first-in-class anticancer agent called MLN4924 plays a crucial role in suppressing tumorigenesis and attenuating inflammatory responses due to specifically inhibiting NEDD8-activating enzyme. Here, we investigated the potential protective role of MLN4924 after experimental stroke. We showed that the neddylation pathway is overactivated in the brain following cerebral ischemia. Inhibition of neddylation by MLN4924 protects the brain against ischemic injury by attenuating neutrophil extravasation and maintaining blood–brain barrier integrity. Our findings provide insights into the promising treatment with neddylation inhibition for ischemic brain injury. Blood–brain barrier (BBB) breakdown and inflammation occurring at the BBB have a key, mainly a deleterious role in the pathophysiology of ischemic stroke. Neddylation is a ubiquitylation-like pathway that is critical in various cellular functions by conjugating neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) to target proteins. However, the roles of neddylation pathway in ischemic stroke remain elusive. Here, we report that NEDD8 conjugation increased during acute phase after ischemic stroke and was present in intravascular and intraparenchymal neutrophils. Inhibition of neddylation by MLN4924, also known as pevonedistat, inactivated cullin-RING E3 ligase (CRL), and reduced brain infarction and improved functional outcomes. MLN4924 treatment induced the accumulation of the CRL substrate neurofibromatosis 1 (NF1). By using virus-mediated NF1 silencing, we show that NF1 knockdown abolished MLN4924-dependent inhibition of neutrophil trafficking. These effects were mediated through activation of endothelial P-selectin and intercellular adhesion molecule-1 (ICAM-1), and blocking antibodies against P-selectin or anti–ICAM-1 antibodies reversed NF1 silencing-induced increase in neutrophil infiltration in MLN4924-treated mice. Furthermore, we found that NF1 silencing blocked MLN4924-afforded BBB protection and neuroprotection through activation of protein kinase C δ (PKCδ), myristoylated alanine-rich C-kinase substrate (MARCKS), and myosin light chain (MLC) in cerebral microvessels after ischemic stroke, and treatment of mice with the PKCδ inhibitor rottlerin reduced this increased BBB permeability. Our study demonstrated that increased neddylation promoted neutrophil trafficking and thus exacerbated injury of the BBB and stroke outcomes. We suggest that the neddylation inhibition may be beneficial in ischemic stroke.

Flow stagnation of peri-ischemic capillaries due to dynamic leukocyte stalls has been described to be a contributor to ongoing penumbral injury in transient brain ischemia, but has not been investigated in permanent experimental stroke so far. Moreover, it is discussed that obstructing neutrophils are involved in this process; however, their contribution has not yet been proven. Here, we characterize the dynamics of neutrophil granulocytes in two models of permanent stroke (photothrombosis and permanent middle cerebral artery occlusion) using intravital two-photon fluorescence microscopy. Different to previous studies on LysM-eGFP+ cells we additionally apply a transgenic mouse model with tdTomato-expressing neutrophils to avoid interference from additional immune cell subsets. We identify repetitively occurring capillary stalls of varying duration promoted by neutrophils in both models of permanent cerebral ischemia, validating the suitability of our new transgenic mouse model in determining neutrophil occlusion formation in vivo. Flow cytometric analysis of peripheral blood (PB) and brain tissue from mice subjected to photothrombosis reveal an increase in the total proportion of neutrophils, with selective upregulation of endothelial adherence markers in the PB. In conclusion, the dynamic microcirculatory stall phenomenon that is described after transient ischemia followed by reperfusion also occurs after permanent small- or large-vessel stroke and is clearly attributable to neutrophils.

Introduction: Mechanical thrombectomy (MT) effectively restores large-vessel patency in acute ischemic stroke (AIS), yet incomplete reperfusion and blood–brain barrier (BBB) disruption with hemorrhagic transformation often limit its clinical benefit. Growing evidence implicates thrombo-inflammation, particularly neutrophil extracellular trap (NET)-driven microvascular obstruction, as a key barrier to microcirculatory reperfusion. Ischemia–reperfusion rapidly activates circulating neutrophils, triggering NET release and brain infiltration that promote microvascular thrombosis, impede capillary flow, and disrupt the BBB. Colchicine, an FDA-approved microtubule inhibitor, suppresses neutrophil effector functions in inflammatory diseases. We hypothesized that colchicine administration at reperfusion would attenuate neutrophil-driven thrombo-inflammation and improve MT outcomes. Methods: A transient middle cerebral artery occlusion (tMCAO) model was established in male C57BL/6J mice to mimic MT. Colchicine (0.4, 0.8, or 1.6 mg/kg, i.p.) was administered immediately before reperfusion; the optimal dose was determined by infarct volume reduction on 2,3,5-triphenyltetrazolium chloride (TTC) staining at day 3. Using this dose, we assessed leukocyte counts and phenotypes, neutrophil F-actin polymerization (flow cytometry, phalloidin staining), and cerebral blood flow (laser speckle imaging) at 4–24 h post-tMCAO. Brain tissue was analyzed for leukocyte infiltration, neutrophil activation, NET formation, fibrin deposition, and BBB integrity (flow cytometry, immunohistochemistry). Sensorimotor and cognitive outcomes were evaluated up to 14 days. Results: Colchicine significantly reduced infarct volume at day 3 and inhibited neutrophil F-actin polymerization. At 4 h post-tMCAO, colchicine reduced circulating neutrophil recruitment and activation. At 24 h, colchicine markedly decreased NET formation and fibrin deposition in cerebral microvessels, improved regional cerebral blood flow, and attenuated leukocyte brain infiltration, neutrophil pro-inflammatory activation, and BBB disruption. Colchicine-treated mice exhibited better sensorimotor and cognitive performance and reduced brain tissue loss up to 14 days. Conclusions: Colchicine administered at reperfusion attenuates neutrophil-driven thrombo-inflammation, improving neurovascular outcomes in a murine MT-mimicking stroke model. These findings support colchicine as a potential adjunct to reperfusion therapy for AIS.

Pericyte-mediated capillary constriction decreases cerebral blood flow in stroke after an occluded artery is unblocked. The determinants of pericyte tone are poorly understood. We show that a small rise in cytoplasmic Ca2+ concentration ([Ca2+]i) in pericytes activated chloride efflux through the Ca2+-gated anion channel TMEM16A, thus depolarizing the cell and opening voltage-gated calcium channels. This mechanism strongly amplified the pericyte [Ca2+]i rise and capillary constriction evoked by contractile agonists and ischemia. In a rodent stroke model, TMEM16A inhibition slowed the ischemia-evoked pericyte [Ca2+]i rise, capillary constriction, and pericyte death; reduced neutrophil stalling; and improved cerebrovascular reperfusion. Genetic analysis implicated altered TMEM16A expression in poor patient recovery from ischemic stroke. Thus, pericyte TMEM16A is a crucial regulator of cerebral capillary function and a potential therapeutic target for stroke and possibly other disorders of impaired microvascular flow, such as Alzheimer’s disease and vascular dementia.

Despite successful clot retrieval in large vessel occlusion stroke, ∼50% of patients have an unfavorable clinical outcome. The mechanisms underlying this functional reperfusion failure remain unknown, and therapeutic options are lacking. In the thrombin-model of middle cerebral artery (MCA) stroke in mice, we show that, despite successful thrombolytic recanalization of the proximal MCA, cortical blood flow does not fully recover. Using in vivo two-photon imaging, we demonstrate that this is due to microvascular obstruction of ∼20%-30% of capillaries in the infarct core and penumbra by neutrophils adhering to distal capillary segments. Depletion of circulating neutrophils using an anti-Ly6G antibody restores microvascular perfusion without increasing the rate of hemorrhagic complications. Strikingly, infarct size and functional deficits are smaller in mice treated with anti-Ly6G. Thus, we propose neutrophil stalling of brain capillaries to contribute to reperfusion failure, which offers promising therapeutic avenues for ischemic stroke.

Introduction: Futile reperfusion remains a major challenge in acute ischemic stroke (AIS), where restoration of large vessel patency fails to translate into microvascular reflow. The mechanisms underlying microcirculatory failure after reperfusion and effective therapeutic targets remain elusive. Methods: We combined in vivo two-photon microscopy, immunostaining, electron microscopy, bulk and single-cell RNA sequencing, in vitro cell experiments and translational studies in both murine stroke models and AIS patients. Platelet GPVI activation was assessed in blood samples from AIS patients and mice. Functional consequences of GPVI deficiency were examined in Gp6 knockout ( Gp6 -/- ) mice subjected to transient middle cerebral artery occlusion (tMCAO). Mechanistic pathways were validated in vitro, and a novel small-molecule GPVI inhibitor (ND886) was identified and evaluated in vitro and in vivo. Results: Early and robust platelet GPVI activation was observed in AIS patients and experimental stroke models. Two-photon imaging demonstrated that neutrophil adhesion, capillary plugging, and reduced cortical microvascular flow, which were markedly attenuated after reperfusion in Gp6 -/- mice. These mice exhibited improved cerebral blood flow, reduced BBB leakage, and smaller infarct volumes. Electron microscopy revealed reduced α-granule release in Gp6 -/- platelets, consistent with attenuated activation. Bulk RNA-seq of neutrophils and single-cell transcriptomics of brain tissue showed significant downregulation of neutrophil adhesion, migration, and immune activation pathways, as well as suppressed endothelial immune activation in Gp6 -/- mice. Mechanistically, GPVI-mediated platelet activation promoted neutrophil activation via the CD14/TLR2 pathway. ND886 selectively inhibited GPVI signaling, reduced neutrophil adhesion, preserved microvascular flow, and decreased infarct size in vivo. Conclusions: In conclusion, platelet GPVI activation is a central driver of neutrophil-mediated microvascular dysfunction following reperfusion in AIS. Targeting GPVI activation and thromboinflammation with a novel small-molecule inhibitor represents a promising strategy to overcome futile reperfusion. A Phase II randomized trial (NCT 05836753) is underway to evaluate the safety and efficacy of ND886 in AIS patients receiving reperfusion therapy, with results to be presented in the Late-Breaking session.

Ever since the introduction of thrombolysis and the subsequent expansion of endovascular treatments for acute ischemic stroke, it remains to be identified why the actual outcomes are less favorable despite recanalization. Here, by high spatio-temporal resolution imaging of capillary circulation in mice, we introduce the pathological phenomenon of dynamic flow stalls in cerebral capillaries, occurring persistently in salvageable penumbra after reperfusion. These stalls, which are different from permanent cellular plugs of no-reflow, were temporarily and repetitively occurring in the capillary network, impairing the overall circulation like small focal traffic jams. In vivo microscopy in the ischemic penumbra revealed leukocytes traveling slowly through capillary lumen or getting stuck, while red blood cell flow was being disturbed in the neighboring segments under reperfused conditions. Stall dynamics could be modulated, by injection of an anti-Ly6G antibody specifically targeting neutrophils. Decreased number and duration of stalls were associated with improvement in penumbral blood flow within 2–24 h after reperfusion along with increased capillary oxygenation, decreased cellular damage and improved functional outcome. Thereby, dynamic microcirculatory stall phenomenon can be a contributing factor to ongoing penumbral injury and is a potential hyperacute mechanism adding on previous observations of detrimental effects of activated neutrophils in ischemic stroke.

Platelet–endothelial interactions have a critical role in microcirculatory function, which maintains tissue homeostasis. The subtle equilibrium between platelets and the vessel wall is disturbed by the coronavirus disease 2019 (COVID-19), which affects all three components of Virchow’s triad (endothelial injury, stasis and a hypercoagulable state). Endotheliitis, vasculitis, glycocalyx degradation, alterations in blood flow and viscosity, neutrophil extracellular trap formation and microparticle shedding are only few pathomechanisms contributing to endothelial damage and microthrombosis resulting in capillary plugging and tissue ischemia. In the following opinion paper, we discuss major pathological processes leading to microvascular endothelial activation and thrombosis formation as a possible major adverse factor driving the deterioration of patient disease course in severe COVID-19.

BACKGROUND Alzheimer's disease (AD) is characterized by progressive, irreversible neurodegeneration, leading to memory loss and cognitive decline. In mouse models of AD, global decreases in cerebral blood flow (CBF) are brought on by the plugging of capillaries by arrested neutrophils, and the administration of the neutrophil-specific antibody against Ly6G (anti-Ly6G) reduces these capillary stalls in minutes and improves cognitive function within hours. This suggests that at least some aspects of neural activity impairment are reversible, but the mechanism of this recovery - and what specific neural activity is normalized - is not yet known. In agreement with prior studies, we found orientation tuning selectivity to drifting gratings in primary visual cortex neurons to be broadened in mouse models of AD. Here, we hypothesized that the impaired neural response can be modified by blood flow improvement with anti-Ly6G treatment (4 mg/kg) in the APP/PS1 mouse model of AD. METHOD We transfected neurons in layer 2/3 of the primary visual cortex (V1) of mice with a fluorescent calcium indicator using AAV9 vectors (pAAV.Syn.GCaMP6s.WPRE.SV40, 10^12 vg/mL). We also injected the fluorescent labels methoxy-X04 to detect amyloid plaques and Texas-red-dextran in the vasculature to detect blocked capillaries. Drifting grating visual stimuli were presented to anesthetized mice with using MouseGoggles during recording with two-photon microscopy before and one day after anti-Ly6G or isotype control antibody administration (Fig. 1). RESULT One day after anti-Ly6G administration to reduce stalls and increase CBF, we observed a significant sharpening of orientation tuning relative to baseline in the same animals, as well as a trend toward increased spontaneous activity in V1 neurons (Fig. 2). Such normalization of neural activity patterns likely underlies the improved cognitive function that is seen within hours of blood flow increase in AD mouse models. CONCLUSION Our data suggests some aspects of the neural and behavioral deficits in AD are acutely recoverable by increasing CBF. Such recovery demonstrates a promising avenue for future therapeutic targets to combat the symptoms of AD in humans.

Growing evidence supports the suggestion that the peripheral immune system plays a role in different pathologies associated with cognitive impairment, such as vascular dementia (VD) or Alzheimer's disease (AD). The aim of this review is to summarize, within the peripheral immune system, the implications of different types of myeloid cells in AD and VD, with a special focus on post‐stroke cognitive impairment and dementia (PSCID). We will review the contributions of the myeloid lineage, from peripheral cells (neutrophils, platelets, monocytes and monocyte‐derived macrophages) to central nervous system (CNS)‐associated cells (perivascular macrophages and microglia). Finally, we will evaluate different potential strategies for pharmacological modulation of pathological processes mediated by myeloid cell subsets, with an emphasis on neutrophils, their interaction with platelets and the process of immunothrombosis that triggers neutrophil‐dependent capillary stall and hypoperfusion, as possible effector mechanisms that may pave the way to novel therapeutic avenues to stop dementia, the epidemic of our time.

Following ischemic stroke, polymorphonuclear neutrophils (PMNs) are rapidly recruited to the ischemic brain tissue and exacerbate stroke injury by release of reactive oxygen species (ROS), proteases and proinflammatory cytokines. PMNs may aggravate post-ischemic microvascular injury by obstruction of brain capillaries, contributing to reperfusion deficits in the stroke recovery phase. Thus, experimental studies which specifically depleted PMNs by delivery of anti-Ly6G antibodies or inhibited PMN brain entry, e.g., by CXC chemokine receptor 2 (CXCR2) or very late antigen-4 (VLA-4) blockade in the acute stroke phase consistently reduced neurological deficits and infarct volume. Although elevated PMN responses in peripheral blood are similarly predictive for large infarcts and poor stroke outcome in human stroke patients, randomized controlled clinical studies targeting PMN brain infiltration did not improve stroke outcome or even worsened outcome due to serious complications. More recent studies showed that PMNs have decisive roles in post-ischemic angiogenesis and brain remodeling, most likely by promoting extracellular matrix degradation, thereby amplifying recovery processes in the ischemic brain. In this minireview, recent findings regarding the roles of PMNs in ischemic brain injury and post-ischemic brain remodeling are summarized.

Background Neutrophil activation and neutrophil extracellular traps (NETs) participate in downstream microvascular thromboinflammation (DMT) and blood brain barrier disruption in acute ischemic stroke (AIS). Objectives The aim of this study was to test whether deoxyribonuclease 1 (DNase 1) infusion, which cleaves extracellular DNA, could reduce DMT in a transient middle cerebral artery (MCA) occlusion stroke model in rats. Methods Eighteen rats were subjected to 120-minute transient MCA occlusion. DNase 1 (3 mg/kg, 20% intravenous, and 80% intraperitoneal injection) or vehicle were randomly infused 30 minutes after MCA occlusion. Main outcome criteria were the infarct volume assessed with magnetic resonance imaging, neurological disability, and the rate of hemorrhagic transformation measured at 24 hours. Brain DMT was assessed with biomarkers of platelet, coagulation, and neutrophil activation quantified in brain homogenates. Results The infusion of DNase 1 significantly reduced the infarct volume (P = .024) and improved 24-hour neurological outcome (P = .031) compared with vehicle. Staining for fibrin(ogen) and citrullinated histones H3 colocalized with extracellular DNA in the occluded microvessels. Brain thrombin–antithrombin complexes and fibrinogen deposits were significantly reduced in DNase 1-treated rats compared with vehicle (P = .027 and P = .036, respectively). The blood brain barrier disruption assessed with brain immunoglobulin G measurement and brain edema were also reduced in DNase 1-treated rats (P = .015 and P = .031, respectively). Conclusion Our results confirm that NETs contribute to DMT during AIS and that early NET-targeting therapy may represent a new strategy to improve the clinical benefit of large vessel recanalization in AIS.

Neutrophils are key effector cells in the pathogenesis of stroke. Using in vivo murine models, Dhanesha and colleagues identify the multifunctional protein pyruvate kinase muscle 2 (PKM2) in neutrophils as a key modulator of outcome. Induction of stroke results in nuclear translocation of PKM2 in neutrophils driving a thromboinflammatory reaction through STAT3 signaling that exacerbates the severity of cerebral ischemia-reperfusion injuries, a process that is potentially amenable to timely, targeted pharmacological inhibition.

Background Thromboinflammatory processes modulate the complex pathophysiology of cerebral ischemia-reperfusion (I/R) injury in ischemic stroke, but the exact underlying mechanisms remain poorly understood. Emerging evidence indicates that neutrophil extracellular traps (NETs) might play an important role in the thromboinflammatory cascade. In addition, the link between von Willebrand factor (VWF) and neutrophil recruitment in the ischemic brain might promote thromboinflammation, possibly by the formation of NETs. Objectives To study NET formation in a murine model of cerebral I/R injury in ischemic stroke. Methods The filament–induced transient middle cerebral artery occlusion model was used to induce 60 minutes of focal cerebral ischemia after which reperfusion was allowed. At different time points postischemia, NETs were identified in the ischemic mouse brain using quantitative immunofluorescence microscopy. Results NETs could be identified in the ipsilateral brain hemisphere. Interestingly, NETs could already be detected at 6 hours poststroke. Their presence increased at 12 hours, was highest at 24 hours, and decreased again 48 hours postischemia. Remarkably, NETs were predominantly localized within the brain vasculature postischemia, suggesting that NETs play a role in secondary microthrombosis. Strikingly, NET formation was significantly decreased in VWF–deficient mice compared to littermate wild-type mice 24 hours postischemia, indicating a possible role for VWF in promoting NETosis in the ischemic brain. Conclusion This study identified the spatiotemporal profile of NET formation in a mouse model of cerebral I/R injury in ischemic stroke. NETs, potentially in combination with VWF, might be attractive targets for the development of novel therapeutic strategies in ischemic stroke treatment.

Endovascular treatment (EVT) for large vessel occlusion in acute ischemic stroke patients (AIS) improves reperfusion rates, yet a considerable number of patients do not recover despite successful recanalization. Microvascular occlusions may underlie these reperfusion failures after EVT and periprocedural antithrombotic agents - unfractionated heparin (UFH) and/or acetylsalicylic acid (ASA)- may be administered to mitigate these complications. However, the MR CLEAN MED Trial, investigating the safety and efficacy of periprocedural UFH, ASA or UFH+ASA in AIS patients undergoing EVT, found an association between administration of these antithrombic agents and a higher rate of symptomatic intracranial hemorrhages. Our recent work showed that EVT elicited activation of the contact system and microthrombi formation in a large animal stroke model. Neutrophil activation and NETosis may also impact secondary thromboinflammation, potentiating thrombogenesis even in the presence of UFH and ASA, while exacerbating bleeding risk. Thus, safer and more effective antithrombotic drugs are needed. Prior studies implicate neutrophil extracellular traps (NETs) in AIS through direct cellular activation, coagulation stimulation, and inflammation. Heparin can dismantle NETs, potentially improving outcomes; however, links between NETs and coagulation activity in AIS patients remain unclear. To better characterize the thrombo-inflammatory mechanisms in humans we carried out a prespecified substudy of the MR CLEAN MED Trial, addressing plasma biomarkers, exploring the full coagulation cascade alongside markers of neutrophil activation/NETosis. The clinical outcomes included function (in)dependence (mRS), mortality, stroke severity (NIHSS) and follow-up infarct volume (FIV). Plasma samples collected at three time points (T0 = baseline; T1 = 1 hour post-EVT; T2 = 24 hours post-EVT) of 216 patients from the MR CLEAN MED Trial, were included. Coagulation activity was assessed by measuring activated coagulation factor (F) XII (FXIIa), FXIa, FIXa, FXa, FVIIa and thrombin (T) in complex with their natural inhibitors (antithrombin (AT), C1-esterase inhibitor (C1Inh) or alpha-1-antitrypsin (a1AT)) using ELISA. NETs markers (Histone-DNA, MPO-DNA complex, citrullinated Histone H3) were measured with ELISA. Correlations between coagulation activity and NETs were analyzed via Pearson correlation; related-Samples Friedman's two-way ANOVA by ranks and multivariable regression was used for analyzing differences in coagulation and associations with outcomes, including interaction terms for coagulation activity and NETs. Among patients receiving EVT only (n=53), FXIIa:AT and FXIIa:C1Inh showed no significant changes over time. In the UFH (n=60) and UFH+ASA (n=55) groups, FXIIa:AT significantly increased at T1 compared to baseline (UFH: 35.8 pM [17.2–111.2] vs. 116.5 pM [52.9–284.5], p=0.002; UFH+ASA: 31.2 pM [20.8–76.1] vs. 94.8 pM [45.2–182.0], n.s.), returning to baseline by T2 (31.2 pM [16.2–51.5], p=0.000; 28.5 pM [12.6–57.3], p=0.016, respectively). ASA-treated patients (n=48) showed a significant increase in FXIIa:C1Inh at T1 (1210.2 pM [990.1–1368.0] vs. 1478.4 pM [1224.3–1590.6], p=0.031). FXIa:C1Inh showed a similar pattern in the ASA group, but not in UFH-treated patients. In the UFH+ASA group, FXIa:a1AT decreased at T1 versus baseline (29.5 pM [13.7–65.1] vs. 18.1 pM [3.6–37.4], p=0.071), returning near baseline at T2 (33.5 pM [5.4–47.0], n.s.). Standard care showed no change in FXIIa or FXIa, but FVIIa:AT increased at T1 (153.3 pM [80.2–386.7] vs. 302.3 pM [190.0–761.3], n.s.). Coagulation activity showed low to moderate correlation with NETs markers in all groups. In the UFH+ASA group, there was a moderate to strong correlation between FXIIa:AT and FXIa:a1AT, FXIa:α1AT and FXa:AT, and FXIa:a1AT and histone-DNA. No significant associations were found between coagulation activity and outcomes. In summary, periprocedural antithrombotic treatment in EVT-treated stroke patients increased FXII activation compared to EVT only. However, coagulation activity did not associate with outcomes, nor were interactions found between NETs presence and coagulation activity on outcome measures. Based on these and previous animal data, FXIIa or downstream FXIa ± NETosis inhibition may hold promise to improve periprocedural antithrombotic management, balancing efficacy and bleeding risk.

Background Accumulating evidence indicates that neutrophil activation (NA) contributes to microvascular thromboinflammation in acute ischemic stroke (AIS) due to a large vessel occlusion. Preclinical data have suggested that intravenous thrombolysis (IVT) before endovascular therapy (EVT) could dampen microvascular thromboinflammation. In this study we investigated the association between NA dynamics and stroke outcome, and the impact of IVT on NA in patients with AIS treated with EVT. Methods A single-center prospective study was carried out, including patients treated with EVT for whom three blood samples (before, within 1 hour, 24 hours post-EVT) were drawn to measure plasma myeloperoxidase (MPO) concentration as a marker of NA. Unfavorable outcome was defined as a modified Rankin score of 3–6 at 3 months. Results Between 2016 and 2020, 179 patients were included. The plasma MPO concentration peaked significantly 1 hour post-EVT (median increase 21.0 ng/mL (IQR −2.1–150)) and returned to pre-EVT baseline values 24 hours after EVT (median change from baseline −0.8 ng/mL (IQR −7.6–6.7)). This peak was strongly associated with unfavorable outcomes at 3 months (aOR 0.53 (95% CI 0.34 to 0.84), P=0.007). IVT before EVT abolished this 1 hour post-EVT MPO peak. Changes in plasma MPO concentration (baseline to 1 hour post-EVT) were associated with unfavorable outcomes only in patients not treated with IVT before EVT (aOR 0.54 (95% CI 0.33 to 0.88, P=0.013). However, we found no significant heterogeneity in the associations between changes in plasma MPO concentration and outcomes. Conclusions A peak in plasma MPO concentration occurs early after EVT and is associated with unfavorable outcomes. IVT abolished the post-EVT MPO peak and may modulate the association between NA and outcomes.

Thrombosis is viewed as a multi‐step, multi‐cellular process driven by inflammatory stimuli and leukocyte (e.g. neutrophil) activation, and is a major pathophysiological contributor of stroke. Accumulating data supports the hypothesis that anti‐inflammatory therapies may limit thrombosis and anti‐thrombotic therapies may reduce vascular inflammation. One such target is the anti‐inflammatory and pro‐resolving endogenous mediator Annexin A1 (AnxA1). The development of therapies that temper inflammation and enhance resolution offer potential therapeutic strategies for the treatment and management of stroke and other thromboinflammatory diseases. Here we sought to comprehensively elucidate the functional significance of targeting the AnxA1/Fpr2/ALX pathway in thrombosis.

Introduction: In stroke clots, neutrophil extracellular traps (NETs) promote thrombosis, enhance clot stability, and decrease clot amenability to thrombectomy and thrombolysis. Objective: We examine the relationship between NET enrichment and clot mechanical characteristics, radiomics, and histological microstructure. Methods: White blood cells (WBCs) were concentrated from human donor whole blood. Platelet-rich plasma and red blood cells (RBCs) were isolated by centrifugation and mixed with WBCs to produce 0%, 20%, and 40% RBC clot analogs. Lipopolysaccharide (LPS) was added (0μL, 2μL, 6μL into 1mL dH2O) to enrich clots with varying NET amounts. CaCl2 induced clotting. Clots were incubated while rotating at 37°C. Clot analogs were assessed mechanically by uniaxial stretch tester and stress-strain curves, radiomically through high-resolution microCT and radiomic feature analysis, and histologically through whole-slide imaging of H&E-stained tissue and immunofluorescence (Citrullinated Histone Antibody and DAPI counterstain) to quantify NETs. Statistical analysis assessed what mechanical, radiomic, and histological features were significantly different among clots of various NET enrichment. unsupervised clustering was performed on radiomic and histological features to identify signatures unique to NET-enriched clots. Results: LPS addition produced clots with increasing NET enrichment and microstructural complexity. For fibrin-platelet rich clots (0% RBC), NET enrichment produced significant increase in mechanical stiffness, measured by breaking strength (max load) and Young’s Modulus. For each percent composition, radiomic and histomic profiling clustered clot analogs well by NET-enrichment, with NET-enriched clots demonstrating distinct radiomic and histological feature trends. See Figure 1. Conclusion: NET enrichment produces mechanically stiffer clot analogs with distinct microstructure, radiomic, and histological profiles.

Traumatic brain injuries (TBI) and stroke are major causes of morbidity and mortality in both developing and developed countries. The complex and heterogeneous pathophysiology of TBI and cerebral ischemia‐reperfusion injury (CIRI), in addition to the blood‐brain barrier (BBB) resistance, is a major barrier to the advancement of diagnostics and therapeutics. Clinical data showed that the severity of TBI and stroke is positively correlated with the number of neutrophils in peripheral blood and brain injury sites. Furthermore, neutrophil extracellular traps (NETs) released by neutrophils correlate with worse TBI and stroke outcomes by impairing revascularization and vascular remodeling. Therefore, targeting neutrophils to deliver NETs inhibitors to brain injury sites and reduce the formation of NETs can be an optimal strategy for TBI and stroke therapy. Herein, the study designs and synthesizes a reactive oxygen species (ROS)‐responsive neutrophil‐targeting delivery system loaded with peptidyl arginine deiminase 4 (PAD4) inhibitor, GSK484, to prevent the formation of NETs in brain injury sites, which significantly inhibited neuroinflammation and improved neurological deficits, and improved the survival rate of TBI and CIRI. This strategy may provide a groundwork for the development of targeted theranostics of TBI and stroke.

Background: Neutrophil Extracellular Traps (NETs) are web-like structures of DNA, histones, and granule proteins that trap pathogens as part of innate immunity but also scaffold red blood cells, fibrin, and platelets in pathological thrombosis. Thrombus NETs have been associated with failed thrombolysis and thrombectomy for acute ischemic stroke (AIS). We previously used mass spectrometry to describe a novel association between NET proteins and atrial fibrillation (AF). This association has not been validated by traditional immunohistochemical methods. Aims/Purpose: We used immunohistochemistry to quantify NETs in AIS thrombus taken from patients with and without comorbid AF. Methods: We selected markers for each of the components of NETS: (1) a granule protein, myeloperoxidase (MPO), (2) citrullinated histone 3 (CitH3), and cellular nucleic acid (NA, using Hoechst 33342). This was a single-blind study using cryopreserved thrombi from 30 patients, 15 with AF. Stroke thrombi were collected over a two-year span, and immediately frozen to -80*C post-thrombectomy. To capture the coverage of NET markers more comprehensively for each patient, we used cryostat to sample four sections (five-micron thickness) from each thrombus. We then fixed sections with 4% formaldehyde in phosphate buffered saline (PBS), performed immunohistological staining, and quantified extent of staining using ImageJ. Data for each NET marker from a single thrombus is represented as the mean percentage of thrombus area staining positively across the four corresponding sections. After performing Shapiro Wilks test for normality, statistical comparisons of NET marker staining coverage between AF and non-AF thrombi were performed with either unpaired T test or Mann-Whitney U test, depending on whether a parametric or non-parametric test was appropriate. Results: Thrombi from patients with AF had greater MPO coverage(17.9 +/- 1.47% versus 11.6 +/- 1.29% in non-AF, p<0.01) and CitH3 coverage(12.6 +/- 1.46% versus 7.4 +/- 1.11% in non-AF, p<0.01). Staining for nucleic acid with Hoechst 33342 trended toward higher coverage in AF but did not reach significance (27.8 +/- 3.35% versus 22.6 +/- 3.20% in non-AF, p=0.30). Conclusion: The present study provides immunohistochemical validation, supporting novel observations in prior proteomic investigations, of increased NETs in stroke thrombi of patients with AF. This can help to identify patient cohorts for future studies of therapies directed at NETs.

Early breach of the blood–brain barrier (BBB) and consequently extravasation of blood-borne substances into the brain parenchyma is a common hallmark of ischemic stroke. Although BBB breakdown is associated with an increased risk of cerebral hemorrhage and poor clinical prognosis, the cause and mechanism of this process are largely unknown. The aim of this study was to establish an imaging and analysis protocol which enables investigation of the dynamics of BBB breach in relation to hemodynamic properties along the arteriovenous axis. Using longitudinal intravital two-photon imaging following photothrombotic induction of ischemic stroke through a cranial window, we were able to study the response of the cerebral vasculature to ischemia, from the early critical hours to the days/weeks after the infarct. We demonstrate that disruption of the BBB and hemodynamic parameters, including perturbed blood flow, can be studied at single-vessel resolution in the three-dimensional space as early as 30 min after vessel occlusion. Further, we show that this protocol permits longitudinal studies on the response of individual blood vessels to ischemia over time, thus enabling detection of (maladaptive) vascular remodeling such as intussusception, angiogenic sprouting and entanglement of vessel networks. Taken together, this in vivo two-photon imaging and analysis protocol will be useful in future studies investigating the molecular and cellular mechanisms, and the spatial contribution, of BBB breach to disease progression which might ultimately aid the development of new and more precise treatment strategies for ischemic stroke.

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No abstract available

Purpose This study aimed to investigate the diagnostic potential of neutrophil extracellular traps (NETs)-related genes in acute ischemic stroke (AIS) through comprehensive bioinformatics analysis. Methods Two GEO datasets (GSE37587 and GSE16561) were integrated to identify differentially expressed genes (DEGs) between AIS patients and healthy controls. Gene Set Enrichment Analysis (GSEA) was performed to explore functional pathways, while single-sample GSEA (ssGSEA) was used to evaluate immune cell infiltration patterns. NETs-related DEGs (NDEGs) were identified by intersecting the DEGs with previously reported NETS-related genes. Functional enrichment of NDEGs was performed using Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Key genes were identified via machine learning algorithms, including least absolute shrinkage and selection operator (LASSO) and random forest (RF). A diagnostic model was constructed based on the identified hub genes and validated using an independent dataset (GSE58294). Potential regulatory miRNAs and candidate therapeutic compounds were predicted using the TargetScan and DSigDB databases, respectively. Results The discovery dataset included 73 AIS patients and 24 healthy controls, revealed 551 DEGs (225 upregulated, 326 downregulated). The analysis of ssGSEA revealed notable immune dysregulation in AIS patients, characterized by increased neutrophil infiltration and decreased level of Th17, Th1, and TFH cells. GSEA indicated that DEGs were enriched in neutrophil degranulation and innate immune system. NDEGs were significantly enriched in immune regulation and leukocyte apoptosis (GO) and NETs formation pathway (KEGG). Four hub genes—SRC, TLR8, FCAR, and HIF1A—were identified using LASSO and RF algorithms. A diagnostic model based on these genes yielded area under the curve (AUC) values of 0.880 in the training dataset and 0.936 in the validation dataset. Furthermore, three regulatory miRNAs (miR-146a-5p, miR-155-5p, and miR-21-5p) and 23 candidate therapeutic drugs were predicted. Conclusion To our knowledge, this represents the first comprehensive investigation of NETs-related gene signatures in AIS patients compared with healthy controls. These findings deepen our understanding of immune cell infiltration and the underlying molecular mechanisms involved in stroke, offering novel insights that may enhance diagnostic accuracy and therapeutic strategies for AIS.

Background: Radiomic and transcriptomic analyses have independently identified features linked to mechanical thrombectomy (MT) outcomes in acute ischemic stroke (AIS). In this study, we integrate paired radiomic and transcriptomic profiling of AIS clots to identify Neutrophil Extracellular Trap (NET) enrichment as a predictor of first-pass MT success. We further assess the potential to non-invasively detect NET enrichment using pre-thrombectomy CT imaging. Methods: We performed radiomic and transcriptomic analysis of 32 stroke clots retrieved by MT (n=16 each of modified first pass [mFP] success and failure). Clots were segmented from pre-MT CTA and nCCT scans and radiomic features (RFs) were extracted using pyRadiomics. Normality, equal variance, and two-sample testing were completed to identify which RFs were significantly different between mFP outcomes. Differentially expressed genes (DEGs) were identified between transcriptomes of mFP success and failure using the criteria of logFC≥1.5 and q<0.05. A NET enrichment score was computed from expression data and correlated with RFs to identify a RF signature predictive of NET enrichment. Immunofluorescence (IF) staining was completed on retrieved clot tissue to provide ground truth labeling of NETs. Results: 44 DEGs were identified between mFP outcomes. From ontology analysis, NET Formation, Neutrophil Degranulation, and the NET Signaling Pathway were among the most enriched terms in the mFP failure group, with related genes downregulated in the mFP success group. 40 RFs were significantly different between mFP outcomes. Of these, 6 were found to be correlated with and predictive of clot NET enrichment. IF quantification validated that transcriptomic NET signatures accurately reflected NET presence within clot tissues. Conclusion: Our findings indicate that NET enrichment within thrombi is associated with reduced mFP success, and that radiomic features extracted from pre-thrombectomy CT imaging can serve as non-invasive biomarkers of clot NET content. This radiomic signature may aid in pre-procedural decision-making, including thrombolytic therapy planning and thrombectomy device selection.

BACKGROUND Ischemic stroke (IS) accounts for 71 % of all strokes, whose diagnosis and prognosis require further exploration. Neutrophil extracellular traps (NETs) are produced by neutrophils, and there is already evidence that NETs play a role in IS, but further studies about crosstalk between immune cells, pathways and NETs are still needed. MATERIALS AND METHODS To assess the expression of neutrophil extracellular traps (NETs), we utilized single sample Gene Set Enrichment Analysis. Stroke-associated NETs genes (SN genes) were identified through differential expression analysis combined with Weighted Correlation Network Analysis. Based on these SN genes, we developed a sophisticated diagnostic model incorporating machine learning techniques. Furthermore, we constructed a single-cell atlas of neutrophil transitions in post-stroke mice. Validation of our findings was conducted both in vitro and in vivo. In vitro, we employed oxygen-glucose deprivation (OGD) experiments to simulate ischemic conditions, facilitating the assessment of NETs formation and monitoring alterations in SN genes expression within neutrophils. In vivo, validation involved tracking changes in peripheral blood levels of these genes in a mouse model of transient middle cerebral artery occlusion (tMCAO) post-cerebral ischemia. RESULTS A detailed single-cell landscape depicting the dynamic transitions of neutrophils within the cerebral microenvironment post-stroke has been elaborately constructed. NETs displayed significant differential expression between IS and control groups in peripheral blood, correlating strongly with the activities of neutrophils and macrophages. Pathways pertinent to IS and NETs were delineated. A diagnostic model incorporating two SN genes was developed, demonstrating an AUC greater than 0.98, effectively pinpointing the hyperacute phase of IS. Additionally, the ceRNA networks concerning IS and NETs were mapped out. In vitro validation with oxygen-glucose deprivation (OGD) experiments revealed marked changes in NET formation and SN genes expression in neutrophils, corroborating our computational predictions. In vivo validation using a mouse transient middle cerebral artery occlusion (tMCAO) model confirmed significant changes in peripheral blood levels of F12 and PLXDC2 after cerebral ischemia, proving the excellent predictive value of these markers for IS. CONCLUSION This study elucidates the complex roles and dynamic changes of neutrophils within the cerebral microenvironment of mice from 3 h to 3 days following stroke onset. We have identified key genes, immune cells, signaling pathways, and ceRNA networks implicated in the formation of NETs in IS. Our study constructed a robust diagnostic model capable of detecting the hyperacute phase of IS, with an AUC value greater than 0.98. The inclusion of experimental validation for the SN genes F12 and PLXDC2 not only corroborates our model's predictive accuracy but also underscores its potential utility in clinical settings. These findings offer promising avenues for improving early diagnosis and potentially guiding therapeutic strategies in IS.

Background Ischemic stroke (IS) is the second leading cause of mortality worldwide, continuing to be a serious health concern. It is well known that oxidative stress and neutrophil response play vital roles in the pathophysiology of early IS. However, the complex interactions and critical genes associated with them have not been fully understood. Methods Two datasets (GSE37587 and GSE16561) from the Gene Expression Omnibus database were extracted and integrated as the discovery dataset. Subsequent GSVA and WGCNA approaches were used to investigate IS-specific oxidative stress-related genes (ISOSGS). Then, we explored IS-specific neutrophil-associated genes (ISNGS) using CIBERSORT analysis. Next, the protein-protein interaction network was established to ascertain candidate critical genes related with oxidative stress and neutrophil response. Furthermore, these candidate genes were validated using GSE58294 dataset and our clinical samples by RT-qPCR method. Finally, functional annotation, diagnostic capability evaluation and drug-gene interactions were performed by using GSEA analysis, ROC curves and DGIDB database. Result In our analysis of discovery dataset, 155 genes were determined as ISOSGS and 559 genes were defined as ISNGS. Afterward, 9 candidate genes were identified through the intersection of ISOSGS and ISNGS, PPI network construction, and filtration by degree algorithm. Then, six real critical genes, including STAT3, MMP9, AQP9, SELL, FPR1, and IRAK3, passed the validation using the GSE58294 dataset and our clinical samples. Further functional annotation analysis indicated these critical genes were associated with neutrophil response, especially neutrophil extracellular trap. Meanwhile, they had a good diagnostic performance. Lastly, 53 potential drugs targeting these genes were predicted by DGIDB database. Conclusion We identified 6 critical genes, STAT3, FPR1, AQP9, SELL, MMP9 and IRAK3, related to oxidative stress and neutrophil response in early IS, which may provide new insights into understanding the pathophysiological mechanism of IS. We hope our analysis could help develop novel diagnostic biomarkers and therapeutic strategies for IS.

Background Our previous work has shown that inflammatory processes play a detrimental role in the pathophysiology of acute ischemic stroke (AIS). Neutrophil extracellular traps (NETs) have been recognized as a key contributor to the proinflammatory response in AIS and could aggravate blood-brain barrier (BBB) damage. Recently, experimental and clinical researches showed that Edaravone Dexborneol (Eda.B), which is comprised of two active ingredients, Edaravone and (+)-Borneol, was effective in treatment of AIS. However, it is not clear whether the effects of Eda.B against AIS are related to NETs and BBB permeability. Methods Experiment 1 was to detect the effects of Eda.B in AIS patients. Serum samples of volunteers and AIS patients were collected before and 3 days after Edaravone Dexborneol treatment. Markers of NETs and occludin were detected by ELISA kit. Experiment 2 was to explore the effects of Eda.B on experimental stroke mice. Male C57BL/6 mice were subjected to distal middle cerebral artery occlusion (MCAO) and treated with vehicle, Eda.B, or DeoxyribonueleaseI (DNase I). After stroke, the neurobehavioral tests, infarct volume, and cerebral blood flow evaluation were determined. Leakage of Evans blue was to assess the integrity of BBB. Western blot, real-time quantitative polymerase chain reaction (RT-qPCR), and immunofluorescence were used to examine the expression of NETs and tight junction- (TJ-) associated proteins. Results Eda.B significantly improved neurological function and cerebral blood flow but reduced infarct volume after experimental stroke. Eda.B downregulated level of NETs in serum samples of AIS patients and tissue samples of MCAO mouse cortex. Eda.B and DNase I alleviated BBB permeability by upregulating TJ-associated proteins. Conclusion NETs are related to the early stage of AIS. Eda.B exerted neuroprotective effects and ameliorated BBB permeability after AIS.

Type 2 diabetes mellitus (T2DM) is associated with impaired leptomeningeal collateral compensation and poor stroke outcome. Neutrophils tethering and rolling on endothelium after stroke can also independently reduce flow velocity. However, the chronology and topological changes in collateral circulation in T2DM is not yet defined. Here, we describe the spatial and temporal blood flow dynamics and vessel diameter changes in pial arteries and veins and leukocyte-endothelial adhesion following middle cerebral artery (MCA) stroke using two-photon microscopy in awake control and T2DM mice. Relative to control mice, T2DM mice already exhibited smaller pial vessels with reduced flow velocity prior to stroke. Following stroke, T2DM mice displayed persistently reduced blood flow in pial arteries and veins, resulting in a poor recovery of downstream penetrating arterial flow and a sustained deficit in microvascular flow. There was also persistent increase of leukocyte adhesion to the endothelium of veins, coincided with elevated neutrophils infiltration into brain parenchyma in T2DM mice compared to control mice after stroke. Our data suggest that T2DM-induced increase in inflammation and chronic remodeling of leptomeningeal vessels may contribute to the observed hemodynamics deficiency after stroke and subsequent poor stroke outcome.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with high morbidity and mortality. Excessive neutrophil infiltration into the pulmonary airspace is the main cause for the acute inflammation and lung injury. Platelets have been implicated in the pathogenesis of ALI/ARDS, but the underlying mechanisms are not fully understood. We here show that the immunoreceptor tyrosine-based activation motif (ITAM)-coupled Ig-like platelet receptor glycoprotein (GP)VI plays a key role in the early phase of pulmonary thrombo-inflammation in a model of lipopolysaccharide (LPS)-induced ALI in mice. In WT control mice, intranasal LPS application triggered severe pulmonary and blood neutrophilia, hypothermia, and increased blood lactate levels. In contrast, Gp6-/- mice as well as anti-GPVI-treated WT mice were markedly protected from pulmonary and systemic compromises and showed no increased pulmonary bleeding. High resolution multicolor microscopy of lung sections and intravital confocal microcopy of the ventilated lung revealed that anti-GPVI treatment resulted in less stable platelet interaction with neutrophils and overall reduced platelet-neutrophil complex (PNC)-formation. Anti-GPVI treatment also reduced neutrophil crawling and adhesion on endothelial cells resulting in reduced neutrophil transmigration and alveolar infiltrates. Remarkably, also neutrophil activation was diminished in anti-GPVI treated animals, associated with strongly reduced formation of platelet/neutrophil clusters and neutrophil extracellular traps (NETs) compared to control. These results establish GPVI as a key mediator of neutrophil recruitment, PNC-formation, and NETosis in experimental ALI. Thus, GPVI inhibition might be a promising strategy to reduce the acute pulmonary inflammation causing ALI/ARDS.

Inhibiting the platelet glycoprotein VI (GPVI) receptor is a promising strategy for reducing cerebral ischemia-reperfusion injury (CIRI) without severe compromise of hemostasis, while targeting glycoprotein IIb/IIIa (integrin αIIbβ3) causes bleeding. The underlying cellular mechanism remains unclear. This study shows that megakaryocyte-platelet-specific deficiency of the autophagic protein Rubicon (Run domain protein as Beclin-1 interacting and cysteine-rich containing) accelerates stroke development and exacerbates cerebral hemorrhage. Rubicon interacts with Bruton's tyrosine kinase (Btk) to inhibit GPVI-mediated thrombus formation, while it prevents αIIbβ3-mediated selective autophagy and degradation of Btk to stabilize platelet thrombi. The expression of Rubicon in platelets is decreased in patients with acute ischemic-reperfusion injury. A cell-permeable peptide mimicking the Rubicon-Btk interaction significantly reduces cerebral infarction volume in a mouse model. As Rubicon is dispensable for hemostasis but crucial in the reperfusion stage of CIRI, peptides mimicking its effects may offer a selective and safe therapeutic strategy.

Experimental evidence has emerged that local platelet activation contributes to inflammation and infarct formation in acute ischemic stroke (AIS) which awaits confirmation in human studies. We conducted a prospective observational study on 258 consecutive patients undergoing mechanical thrombectomy (MT) due to large-vessel-occlusion stroke of the anterior circulation (08/2018–05/2020). Intraprocedural microcatheter aspiration of 1 ml of local (occlusion condition) and systemic arterial blood samples (self-control) was performed according to a prespecified protocol. The samples were analyzed for differential leukocyte counts, platelet counts, and plasma levels of the platelet-derived neutrophil-activating chemokine C-X-C-motif ligand (CXCL) 4 (PF-4), the neutrophil attractant CXCL7 (NAP-2), and myeloperoxidase (MPO). The clinical-biological relevance of these variables was corroborated by specific associations with molecular-cellular, structural-radiological, hemodynamic, and clinical-functional parameters. Seventy consecutive patients fulfilling all predefined criteria entered analysis. Mean local CXCL4 (+ 39%: 571 vs 410 ng/ml, P = .0095) and CXCL7 (+ 9%: 693 vs 636 ng/ml, P = .013) concentrations were higher compared with self-controls. Local platelet counts were lower (− 10%: 347,582 vs 383,284/µl, P = .0052), whereas neutrophil counts were elevated (+ 10%: 6022 vs 5485/µl, P = 0.0027). Correlation analyses revealed associations between local platelet and neutrophil counts (r = 0.27, P = .034), and between CXCL7 and MPO (r = 0.24, P = .048). Local CXCL4 was associated with the angiographic degree of reperfusion following recanalization (r =  − 0.2523, P = .0479). Functional outcome at discharge correlated with local MPO concentrations (r = 0.3832, P = .0014) and platelet counts (r = 0.288, P = .0181). This study provides human evidence of cerebral platelet activation and platelet-neutrophil interactions during AIS and points to the relevance of per-ischemic thrombo-inflammatory mechanisms to impaired reperfusion and worse functional outcome following recanalization.

COVID-19 progression often involves severe lung injury, inflammation, coagulopathy, and leukocyte infiltration into pulmonary tissues. The pathogenesis of these complications is unknown. Because vascular endothelium and neutrophils express angiotensin-converting enzyme-2 and spike (S)-proteins, which are present in bodily fluids and tissues of SARS-CoV-2-infected patients, we investigated the effect of S-proteins and cell–cell communication on human lung microvascular endothelial cells and neutrophils expression of P-selectin, markers of coagulopathy, NETosis, and inflammation. Exposure of endothelial cells or neutrophils to S-proteins and endothelial–neutrophils co-culture induced P-selectin transcription and expression, significantly increased expression/secretion of IL-6, von Willebrand factor (vWF, pro-coagulant), and citrullinated histone H3 (cit-H3, NETosis marker). Compared to the SARS-CoV-2 Wuhan variant, Delta variant S-proteins induced 1.4–15-fold higher P-selectin and higher IL-6 and vWF. Recombinant tissue factor pathway inhibitor (rTFPI), 5,5′-dithio-bis-(2-nitrobenzoic acid) (thiol blocker), and thrombomodulin (anticoagulant) blocked S-protein-induced vWF, IL-6, and cit-H3. This suggests that following SARS-CoV-2 contact with the pulmonary endothelium or neutrophils and endothelial–neutrophil interactions, S-proteins increase adhesion molecules, induce endothelial injury, inflammation, NETosis and coagulopathy via the tissue factor pathway, mechanisms involving functional thiol groups, and/or the fibrinolysis system. Using rTFPI, effectors of the fibrinolysis system and/or thiol-based drugs could be viable therapeutic strategies against SARS-CoV-2-induced endothelial injury, inflammation, NETosis, and coagulopathy.

BACKGROUND Endovascular treatment (EVT) alone was not demonstrated to be non-inferior to intravenous alteplase bridging EVT in acute large vessel occlusion (LVO) stroke. Using cerebral ischemia/reperfusion (I/R) injury model, intravenous tenecteplase (TNK) was administrated after ischemia followed by reperfusion at various time points. OBJECTIVES To investigate whether intravenous TNK bridging EVT vs EVT alone could improve I/R injury, and this effect may be associated with the time from TNK to reperfusion. METHODS Rats received intravenous TNK (1.4 mg/kg) or vehicle (sterile water) 1.0 hour after middle cerebral artery occlusion (MCAO), followed by reperfusion after 0.5 or 1.0 hour following TNK. Neurological deficit scores, infarct volume, and brain edema were measured at 24 hours after MCAO. Microthrombi were determined by immunofluorescence staining for CD31+/fibrinogen+ and CD31+/thrombocyte+. Inflammatory cell infiltration in the ischemic brain region was determined by flow cytometry. RESULTS Compared with vehicle, TNK significantly reduced neurological deficit scores, brain infarction, neuro-inflammation, and blood-brain barrier (BBB) disruption, and significantly reduced intravascular fibrin and platelet deposition, and brain inflammatory cell infiltration in penumbra of I/R rats. Furthermore, a better beneficial trend was found in TNK bridging reperfusion at 0.5 hour after TNK compared with TNK bridging reperfusion at 1.0 hour after TNK. CONCLUSIONS Our results demonstrate that intravenous TNK bridging reperfusion produced neuroprotective action through dissolving microvascular thrombus and alleviating inflammatory cell infiltration to improve microcirculation, with the result of maintaining BBB integrity and inhibiting neuroinflammation, and the neuroprotective benefit may be associated with the time from TNK to reperfusion.

Neutrophil elastase (NE) is a protease released by activated neutrophils in the brain parenchyma after cerebral ischemia, which plays a pivotal role in the regulation of neutrophil extracellular traps (NETs) formation. The excess NETs could lead to blood-brain barrier (BBB) breakdown, overwhelming neuroinflammation, and neuronal injury. While the potential of targeting neutrophils and inhibiting NE activity to mitigate ischemic stroke (IS) pathology has been recognized, effective strategies that inhibit NETs formation remain under-explored. Herein, a biomimic multifunctional nanoplatform (HM@ST/TeTeLipos) was developed for active NE targeting and IS treatment. The core of the HM@ST/TeTeLipos consisted of sivelestat-loaded ditelluride-containing liposomes with ROS-responsive and NE-inhibiting properties. The outer shell was composed of platelet-neutrophil hybrid membrane vesicles (HMVs), which acted to hijack neutrophils and neutralize proinflammatory cytokines. Our studies revealed that HM@ST/TeTeLipos could effectively inhibit NE activity, thereby suppressing the release of NETs, impeding the activation of the AIM2 inflammasome, and consequently redirecting the immune response away from a pro-inflammatory M1 microglia phenotype. This resulted in enhanced neurovascular remodeling, reduced BBB disruption, and diminished neuroinflammation, ultimately promoting neuron survival. We believe that this innovative approach holds significant potential for improving the treatment of IS and various NE-mediated inflammatory diseases.

Cerebral reperfusion injury in stroke, stemming from interconnected thrombotic and inflammatory signatures, often involves platelet activation, aggregation and its interaction with various immune cells, contributing to microvascular dysfunction. However, the regulatory mechanisms behind this platelet activation and the resulting inflammation are not well understood, complicating the development of effective stroke therapies. Utilizing animal models and platelets from hemorrhagic stroke patients, our research demonstrates that human cerebral dopamine neurotrophic factor (CDNF) acts as an endogenous antagonist, mitigating platelet aggregation and associated neuroinflammation. CDNF moderates mitochondrial membrane potential, reactive oxygen species production, and intracellular calcium in activated platelets by interfering with GTP binding to Rap1b, thereby reducing Rap1b activation and downregulating the Rap1b-MAPK-PLA2 signaling pathway, which decreases release of the pro-inflammatory mediator thromboxane A2. In addition, CDNF reduces the inflammatory response in BV2 microglial cells co-cultured with activated platelets. Consistent with ex vivo findings, subcutaneous administration of CDNF in a rat model of ischemic stroke significantly reduces platelet activation, aggregation, lipid mediator production, infarct volume, and neurological deficits. In summary, our study highlights CDNF as a promising therapeutic target for mitigating platelet-induced inflammation and enhancing recovery in stroke. Harnessing the CDNF pathway may offer a novel therapeutic strategy for stroke intervention.

Phosphoinositide 3-kinase beta (PI3Kβ) plays an important role in platelet activation and thrombosis, but its role in stroke pathology remains unknown. In this study, we investigated whether inhibition of PI3Kβ protects against cerebral ischemia/reperfusion (I/R) injury by preventing circulating platelet activation and downstream microvascular thrombosis. We used a rat intraluminal filament model of transient middle cerebral artery occlusion (tMCAO) because the rapid restoration of cerebral blood flow to the ischemic area in both tMCAO and endovascular thrombectomy provides clinical relevance for this model. The results showed that TGX221, a selective PI3Kβ inhibitor, treatment immediately before the onset of reperfusion dose-dependently reduced infarct volume and improved neurological function. The protective effects were associated with blocking platelet activation and thrombotic response, thereby reducing downstream microvascular thrombosis, and maintaining reperfusion efficiency. These results suggest that PI3Kβ might be a promising target for treating downstream microvascular thrombosis induced by cerebral I/R injury and offer a novel adjunctive treatment to improve reperfusion therapy for acute ischemic stroke.

The angiogenic response after stroke correlates with mild injury and an improved recovery. Stimulation of post‐stroke angiogenesis using vascular endothelial growth factor (VEGF)‐A is associated with an increased risk of vascular destabilization, leading to life‐threatening complications. The non‐mammalian VEGF‐A homolog, VEGF‐E, stimulates stable cutaneous vascularization and promotes wound healing. Herein, we posit that VEGF‐E represents a potential mediator of reparative revascularization after ischemic stroke. C57BL6/J wildtype mice were subjected to experimental stroke, and VEGF‐E or VEGF‐A were intranasally delivered during the subacute phase. Our results indicate that VEGF‐E improves neurological recovery and increases vascular density without compromising permeability, more efficiently than VEGF‐A. We show that VEGF‐E‐mediated revascularization correlates with normal restoration of brain perfusion, whereas VEGF‐A induces cerebral hyperperfusion, indicative of vascular dysfunction. Furthermore, VEGF‐E reduces microvascular stalls, increases the density of angiogenic vasculature, and improves the interaction of brain endothelial cell with pericytes, which is critical for vascular stabilization. Using cell‐based assays, we demonstrate that stimulation of brain endothelial cells with VEGF‐E, but not with VEGF‐A, increases the expression of platelet‐derived growth factor (PDGF)‐D, a potent ligand of PDGFRβ that plays critical roles in regulating the survival and functions of perivascular cells, including pericytes. These effects are associated with activation of extracellular signal‐regulated kinase (ERK)1/2 and P38 mitogen‐activated protein kinase (MAPK). Finally, we confirm that the secretome of VEGF‐E‐stimulated brain endothelial cells ameliorates pericyte migration required for vascular recruitment. Our study indicates that VEGF‐E promotes a stable and functional revascularization after ischemic stroke, outlining its promises for therapeutic purposes.

ETHNOPHARMACOLOGICAL RELEVANCE No-reflow post-recanalization is a challenge in ischemic stroke. Our prior study demonstrated that Chinese medicine Tongxinluo capsule (TXL) improves no-reflow in stroke by suppressing leukocyte-endothelium interactions. However, it remains unclear which leukocyte subpopulation plays a major role. AIM OF THE STUDY This study aimed to further explore the mechanisms of TXL improving no-reflow in stroke. MATERIALS AND METHODS The mouse model of stroke with recanalization treatment was induced by transient middle cerebral artery occlusion (tMCAO). Neutrophil depletion was performed by injecting Ly6G-specific monoclonal antibody (anti-Ly6G). The mice were orally administered with TXL suspension. We examined the following parameters: cerebral blood flow (CBF) determined by laser speckle perfusion imaging, neutrophil percentages and apoptosis examined by flow cytometry, and granulocyte colony-stimulating factor (G-CSF) measured by cytometric bead array. RESULTS We confirmed our prior conclusion that TXL could significantly reduce blood neutrophil percentages (by up to 35%) as well as improving no-reflow in stroke (increasing CBF in the ischemic area by 9%). Interestingly, neutrophil depletion also suppressed no-reflow (increasing CBF by 15%). These results suggest a causal role for neutrophil reduction in TXL's improvement of no-reflow. Subsequently, we found that tMCAO led to a 15% increase in the percentage of bone marrow neutrophils but caused a 30% decrease in the percentage of spleen neutrophils. However, TXL did not significantly affect the percentage of bone marrow neutrophils but interestingly increased the percentage of spleen neutrophils by up to 27%. Importantly, tMCAO induced the increased granulocyte colony-stimulating factor (G-CSF) in plasma and ischemic brain tissue, while both TXL and anti-Ly6G decreased the cytokine level. Moreover, TXL didn't affect neutrophil apoptosis in blood and bone marrow, but increased spleen neutrophil apoptosis. CONCLUSIONS Our study has elucidated an important mechanism of post-stroke no-reflow: the overproduction of G-CSF by ischemic brain triggers a massive release of neutrophils from the bone marrow and spleen into the circulation, consequently obstructing the cerebral microvasculature. Interestingly, TXL counteracts post-stroke no-reflow by targeting the pathogenic surge of circulating neutrophils via a dual mechanism: inhibiting cerebral G-CSF production to reduce splenic neutrophil release; and facilitating splenic neutrophil homing to promote their clearance.

No abstract available

Dysfunction of the neurovascular unit significantly impacts the prognostic outcomes of ischemic stroke. However, effective strategies to comprehensively modulate the neurovascular unit have yet to be developed. In this work, we introduce a brain-targeting biomimetic nanozyme, A@HPB@THSA, designed to mitigate neurovascular unit dysfunction following ischemia/reperfusion. Specifically, aspirin is encapsulated within hollow Prussian blue nanozyme, which is subsequently modified with brain-targeting T7 peptide-conjugated human serum albumin, ultimately forming the composite A@HPB@THSA. The overexpression of transferrin receptors on cerebral vascular endothelial cells, along with compromised blood-brain barrier (BBB) permeability, facilitates the accumulation of A@HPB@THSA at cerebral ischemic lesions. The hollow Prussian blue nanozyme component effectively scavenges reactive oxygen species in ischemia/reperfusion-affected brain cells. While the aspirin component inhibits platelets aggregation and neutrophils infiltration, thereby preventing microvascular "no-reflow" and preserving the integrity of the BBB. In rat models of transient middle cerebral artery occlusion, A@HPB@THSA demonstrated comprehensive modulation of the neurovascular unit, including reduced BBB permeability, promotion of microglia polarization toward an anti-inflammatory phenotype, and enhanced neuronal survival. This work provides a promising platform to reverse dysfunctional neurovascular unit for ischemic stroke treatment.

Good leptomeningeal collaterals (LMCs) after large vessel occlusion (LVO) extend the time window for endovascular therapy. The mechanisms regulating LMC activation are not fully understood. The aim of this study was to investigate the potential role of two vasoactive molecules endothelin-1 (ET-1)—a vasoconstrictor agent—and nitric oxide (NO)—a vasodilator agent—in the regulation of post-stroke LMCs. Ischemic stroke patients within 6 h of LVO were included. Collateral status was assessed using the Menon scoring system based on computed tomography angiography scans. Patients were accordingly divided into three groups: poor, intermediate, and good LMCs. Recanalization was evaluated using the modified thrombolysis in cerebral infarction (mTICI) score. Serum levels of ET-1 and NO were measured at three time points: T0 (<6 h), T1 (24 h), and T2 (48 h). A total of 105 patients were enrolled (mean age 76 ± 12.8 years): 44 with good (46.2%), 36 with intermediate (37.8%), and 22 with poor LMCs (23.1%). NO values decreased, whereas ET-1 values increased from T0 to T1 in all groups of patients. No significant association was found between serum ET-1 levels and collateral status. Higher ET-1 levels at T1 correlated with poor outcome regardless of the LMC status or the degree of recanalization (p = 0.030). A significant linear positive correlation was revealed at T0 between high levels of ET-1 and the neutrophil count (Spearman’s rho = 0.236, p = 0.035). Subgroup analysis showed a significant inverse correlation at T1 between NO and the collateral score (Spearman’s rho = −0.251, p = 0.021). Although we observed no significant association between LMC score and serum ET-1 concentrations, at 24 h higher ET-1 serum levels were predictive of poor outcome and higher NO levels were correlated with poor collateral status. These findings may indicate an inadequate microvascular reperfusion, possibly due to ET-1-mediated vasoconstriction, neutrophil activation, and NO-mediated oxidative stress, suggesting their potential role in the no-reflow phenomenon.

Neutrophil extracellular traps (NETs) have been shown to promote thrombus formation. Little is known about the exact composition of thrombi that cause ischemic stroke. In particular, no information is yet available on the presence of NETs in cerebral occlusions. Such information is, however, essential to improve current thrombolytic therapy with tissue plasminogen activator (t-PA). This study aimed at investigating the presence of neutrophils and more specifically NETs in ischemic stroke thrombi. Sixty-eight thrombi retrieved from ischemic stroke patients undergoing endovascular treatment were characterized by immunostaining using neutrophil markers (CD66b and neutrophil elastase) and NET markers (citrullinated histone H3 [H3Cit] and extracellular DNA). Neutrophils and NETs were quantified. In addition, extracellular DNA was targeted by performing ex vivo lysis of retrieved thrombi with DNase 1 and t-PA. Neutrophils were detected extensively throughout all thrombi. H3Cit, a hallmark of NETs, was observed in almost all thrombi. H3Cit-positive area varied up to 13.45% of total thrombus area. Colocalization of H3Cit with extracellular DNA released from neutrophils confirmed the specific presence of NETs. H3Cit was more abundant in thrombi of cardioembolic origin compared to other etiologies. Older thrombi contained significantly more neutrophils and H3Cit compared to fresh thrombi. Interestingly, ex vivo lysis of patient thrombi was more successful when adding DNase 1 to standard t-PA. Neutrophils and NETs form important constituents of cerebral thrombi. Targeting of NETs with DNase 1 might have prothrombolytic potential in treatment of acute ischemic stroke. Ann Neurol 2017;82:223-232.

Ca Myeloid-specific Cracr2a conditional knockout mice and intravital microscopy were used to investigate the physiologic role of neutrophil Cracr2a in neutrophil recruitment in vascular inflammation. Cracr2a-deficient neutrophils or dHL-60 (differentiated human promyelocytic leukemia) cells and Cracr2a-derived peptides were used in flow cytometry, immunoprecipitation, cytosolic Ca Compared with wild-type control mice, Cracr2a conditional knockout mice exhibited significantly reduced adhesion, crawling, and transmigration of neutrophils on ear and cremaster venules in tumor necrosis factor-α-induced sterile inflammation. Neutrophil Cracr2a rapidly interacts with Stim1 (stromal interaction molecule 1) after agonist stimulation and facilitates Ca Our results demonstrate that neutrophil CRACR2A promotes neutrophil recruitment to sites of sterile inflammation, such as ischemic stroke. Blocking the STIM1-CRACR2A interaction may be a novel therapeutic strategy to mitigate inflammation and consequent tissue injury.

To better understand the functional state of circulating neutrophils in patients with ischemic stroke (IS) for planning future clinical trials. We analyzed by flow cytometry activation state of circulating neutrophils and the distribution of neutrophil peripheral subsets in 41 patients with acute IS less than 6 hours before admission and compared them with 22 age-matched healthy controls. Our results demonstrated continuous basal hyperactivation of circulating neutrophils during acute IS, characterized by lower l-selectin expression and higher CD11b expression at the cell surface, increased ROS production by neutrophils, and greater circulating levels of neutrophil elastase. Neutrophil hyperactivation was associated with deregulation of the equilibrium between apoptotic and necrotic. Patients also had higher percentages than controls of the overactive senescent (CXCR4 Altogether, our results indicate that during acute IS, the inflammatory properties of circulating neutrophils rise, associated with the expansion of harmful neutrophil subsets. These changes in neutrophil homeostasis, associated with disease severity, may play an instrumental role by contributing to systemic inflammation and to the blood-brain barrier breakdown. Our findings highlight new potential therapeutic approaches of stroke by rebalancing the ratio of senescent to immunosuppressive neutrophils or decreasing reverse neutrophil transmigration or both.

Ischemic stroke is an acute and severe neurological disease, resulting in disability and death. Reperfusion to an ischemic brain is a means to reverse brain damage after stroke; however, this causes secondary tissue damage induced by inflammation responses, called ischemia/reperfusion (I/R) injury. Adhesion of neutrophils to endothelial cells underlies the initiation of inflammation in I/R. Inspired by this interaction, we report a drug delivery system comprised of neutrophil membrane-derived nanovesicles loaded with Resolvin D2 (RvD2) that can enhance resolution of inflammation, thus protecting brain damage during ischemic stroke. In the study, the middle cerebral artery occlusion (MCAO) mouse model was developed to mimic ischemic stroke. Using intravital microscopy of a live mouse brain, we visualized the binding of nanovesicles to inflamed brain vasculature for delivery of therapeutics to ischemic stroke lesions in real-time. We also observed that RvD2-loaded nanovesicles dramatically decreased inflammation in ischemic stroke and improved mouse neurological functions. Our study provides a strategy to inhibit neuroinflammation using neutrophil-derived nanovesicles for ischemic stroke therapy.

Ischemic stroke is a leading cause of disability. Inflammation of the vessel wall following neutrophil adhesion to vascular endothelium may contribute to ischemic damage. We studied the effect of a platelet inhibitor and an angiotensin II receptor antagonist: alone or in combination, on the adhesion of neutrophils to endothelial cell line in stroke patients. Neutrophils were collected from 12 patients with ischemic stroke within 48 h. Six patients with previous stroke and six healthy volunteers served as control. Neutrophils were incubated with dipyridamole, candesartan or both and allowed to adhere to human endothelial cell line (ECV-304). Adhesion and expression of adhesion molecules (AM) were determined using fluorescence-activated cell-sorting (FACS). Dipyridamole and the combination of dipyridamole and candesartan inhibited significantly the adhesion of neutrophils from ischemic stroke patients as compared to controls with a prominent additive effect. No inhibition was seen in the control groups. These drugs also reduced significantly the expression of the AM Mac-1. Both candesartan and dipyridamole inhibited the adhesion of neutrophils to vascular endothelium in ischemic stroke patients but not in chronic stroke patients or healthy persons. This effect may be related to specific downregulation of Mac-1 by these drugs or other intracellular events.

Hemorrhagic complications represent a major limitation of intravenous thrombolysis using tPA (tissue-type plasminogen activator) in patients with ischemic stroke. The expression of tPA receptors on immune cells raises the question of what effects tPA exerts on these cells and whether these effects contribute to thrombolysis-related hemorrhagic transformation. We aim to determine the impact of tPA on immune cells and investigate the association between observed immune alteration with hemorrhagic transformation in ischemic stroke patients and in a rat model of embolic stroke. Paired blood samples were collected before and 1 hour after tPA infusion from 71 patients with ischemic stroke. Control blood samples were collected from 27 ischemic stroke patients without tPA treatment. A rat embolic middle cerebral artery occlusion model was adopted to investigate the underlying mechanisms of hemorrhagic transformation. We report that tPA induces a swift surge of circulating neutrophils and T cells with profoundly altered molecular features in ischemic stroke patients and a rat model of focal embolic stroke. tPA exacerbates endothelial injury, increases adhesion and migration of neutrophils and T cells, which are associated with brain hemorrhage in rats subjected to embolic stroke. Genetic ablation of annexin A2 in neutrophils and T cells diminishes the effect of tPA on these cells. Decoupling the interaction between mobilized neutrophils/T cells and the neurovascular unit, achieved via a S1PR (sphingosine-1-phosphate receptor) 1 modulator RP101075 and a CCL2 (C-C motif chemokine ligand 2) synthesis inhibitor bindarit, which block lymphocyte egress and myeloid cell recruitment, respectively, attenuates hemorrhagic transformation and improves neurological function after tPA thrombolysis. Our findings suggest that immune invasion of the neurovascular unit represents a previously unrecognized mechanism underlying tPA-mediated brain hemorrhage, which can be overcome by precise immune modulation during thrombolytic therapy.

Leukocyte recruitment and inflammatory response play an important patho-physiologic role after cerebral ischemia. This study aimed to evaluate whether leukocyte adhesion molecules can predict clinical outcome in patients after ischemic stroke. We prospectively examined serial changes in p-selectin glycoprotein ligand-1 (PSGL-1), macrophage antigen-1 (Mac-1), and lymphocyte function-associated antigen-1 (LFA-1) expression by leukocyte subsets using flow cytometry at various time points in 65 acute ischemic stroke patients and 60 controls. PSGL-1 expression on neutrophils and monocytes was significantly higher from day 1 to 90 after stroke as compared with control subjects (p < 0.05). The expression of monocyte Mac-1, LFA-1, and neutrophil Mac-1 were also significantly increased on days 1 and 7 after stroke than in control subjects (p < 0.05). Neutrophil PSGL-1 expression on day 1 was significantly higher in patients with early neurologic deterioration (END) (p < 0.01). Monocyte Mac-1 expression positively correlated with National Institutes of Health Stroke Scale (NIHSS) scores on admission (p = 0.013, gamma = 0.318). Underlying disease of diabetes mellitus and NIHSS score on admission were independently associated with 3-month outcome. The expressions of leukocyte adhesion molecules on admission are significantly increased in patients with acute ischemic stroke. This study shows that higher neutrophil PSGL-1 expression on admission may imply a higher risk for END and that monocyte Mac-1 expression on admission reflects the severity of ischemic stroke on admission.

We tested the hypothesis that both chronic and acute inflammatory processes contribute to worse reperfusion injury and stroke outcome in an experimental model of T2DM. Twelve- to thirteen-week-old male Zucker Diabetic Fatty (ZDF) rats vs. Zucker Lean Controls (ZLC) rats were tested at baseline and after middle cerebral artery occlusion (ischemia) and reperfusion (I-R). Neutrophil adhesion to the cerebral microcirculation, neutrophil expression of CD11b, infarction size, edema, neurologic function, sICAM, and cerebral expression of neutrophil-endothelial inflammatory genes were measured. At baseline, CD11b and sICAM were significantly increased in ZDF vs. ZLC animals (p < 0.05). After I-R, significantly more neutrophil adhesion and cell aggregates were observed in ZDF vs. ZLC (p < 0.05); infarction size, edema, and neurologic function were significantly worse in ZDF vs. ZLC (p < 0.05). CD11b and sICAM-1 remained significantly increased in ZDFs (p < 0.05), and cerebral expression of IL-1β, GRO/KC, E-selectin, and sICAM were significantly induced in ZDF, but not ZLC groups (p < 0.05) after 2.5 hours of reperfusion. Both sides of the neutrophil-endothelial interface appear to be primed prior to I-R, and remain significantly more activated during I-R in an experimental model of T2DM. Consequently, reperfusion injury appears to play a significant role in poor stroke outcome in T2DM.

Type 2 diabetes mellitus (T2DM) is associated with impaired leptomeningeal collateral compensation and poor stroke outcome. Neutrophils tethering and rolling on endothelium after stroke can also independently reduce flow velocity. However, the chronology and topological changes in collateral circulation in T2DM is not yet defined. Here, we describe the spatial and temporal blood flow dynamics and vessel remodeling in pial arteries and veins and leukocyte-endothelial adhesion following middle cerebral artery (MCA) stroke using two-photon microscopy in awake control and T2DM mice. Relative to control mice prior to stroke, T2DM mice already exhibited smaller pial vessels with reduced flow velocity. Following stroke, T2DM mice displayed persistently reduced blood flow in pial arteries and veins, resulting in a poor recovery of downstream penetrating arterial flow and a sustained deficit in microvascular flow. There was also persistent increase of leukocyte adhesion to the endothelium of veins, coincided with elevated neutrophils infiltration into brain parenchyma in T2DM mice compared to control mice after stroke. Our data suggest that T2DM-induced increase in chronic inflammation may contribute to the remodeling of leptomeningeal collateral circulation and the observed hemodynamics deficiency that potentiates poor stroke outcome.

Rovelizumab is a humanized monoclonal leukointegrin antibody under development by ICOS as a potential treatment for multiple sclerosis (MS), hemorrhagic shock, myocardial infarction (MI) and stroke. ICOS announced the commencement of phase II studies in MS patients experiencing acute exacerbations in January 1997; a randomized, double-blind, placebo-controlled phase III trial for acute ischemic stroke, to involve 800 patients, was initiated in January 1999 [312467,313014]. The compound is also undergoing preclinical investigation for cerebral vasospasm, head trauma, kidney transplantation and restenosis [346437]. In September 1999, results from a phase II clinical trial in 45 patients suffering from acute exacerbations of MS were presented at the Warburg Dillion Read Global Life Sciences Conference (New York). The study was designed to evaluate the safety and efficacy of four weekly doses of rovelizumab, as compared to placebo. Rovelizumab was shown to be safe, but demonstrated no clinical benefit for the recovery of neurological functioning [341638]. In February 1997, ICOS announced the initiation of a phase II trial in MI. The placebo-controlled trial is being coordinated by the Mayo Physician Alliance for Cardiovascular Trials and will evaluate safety, pharmacokinetics and infarct size in 60 patients [234046,264363]. Patient enrollment for this, and an open label phase II trial in trauma-induced hemorrhagic shock, was completed in September 1997 [264363]. An expanded shock trial in 150 trauma patients, is expected to complete enrollment by the end of 1998 [296831]. An expanded trial for MI was also planned [264363]. The company is to evaluate rovelizumab in patients with ischemic stroke, and a double-blind, dose-escalating, placebo-controlled phase II trial has been initiated at several centers in the US [264363]. A patient population of 48 was tested, with patient dosing occurring within 12 h of stroke onset symptoms. There was no significant difference in SAEs between rovelizumab and placebo treatment, and no immunogenicity was observed [315799]. Neuroprotection was observed in a rabbit model of focal ischemia, with greatest reduction in infarct noted in the cortical areas of the brain. Neutrophil infiltration to ischemic brain parenchyma was reduced by 90% [315799]. Rovelizumab is a monoclonal antibody directed against the CD11/CD18 cell adhesion proteins. By binding to these receptors, rovelizumab prevents the migration and adhesion of neutrophils in the central nervous system, which may cause brain inflammation and neuronal loss [167725]. Rovelizumab binds to all four known leukointegrin receptors, blocking neutrophil adhesion and binding to ICAMs [307344]. ICOS collaborated with the University of Washington on the preclinical development of this compound [175193].

No abstract

The no-reflow phenomenon refers to the observation that when an organ is made ischemic by occlusion of a large artery supplying it, restoration of patency in that artery does not restore perfusion to the microvasculature supplying the parenchyma of that organ. This has been observed after prolonged arterial occlusions in the heart (30-90 min), brain, skin, and kidney. In experimental models, zones of no reflow in the heart are characterized by ultrastructural microvascular damage, including focal endothelial swelling obstructing the lumen of small vessels. Blood elements such as neutrophil plugs, platelets, and stacking of erythrocytes have also been implicated. No reflow is associated with poor healing of the myocardial infarction. In patients, no reflow is associated with a poor clinical outcome independent of infarct size, suggesting that therapy for no reflow may be an important approach to improving outcome for ST elevation myocardial infarction. No reflow occurs after reperfusion of experimental cerebral ischemia and may be observed after only 5-min episodes of ischemia. Aggregation of blood elements may play a greater role than in cardiac no reflow. No reflow in the brain may involve cortical spreading depression with disturbed local vascular control and high, vasculotonic levels of extracellular K

Activated neutrophils appear to be directly involved in potentiating central nervous system ischemic injury. After initial endothelial adherence, neutrophils can produce capillary plugging with subsequent parenchymal infiltration and resulting cytotoxic neuronal injury. We used an in vitro leukocyte adherence assay to determine if adhesion is increased in acute stroke (within 72 h) or in patients at high risk for stroke (two or more risk factors) compared to matched controls. Neutrophils were isolated using density gradient centrifugation, and adherence to laminin or fibronectin was determined using a myeloperoxidase assay. The adhesion to laminin was significantly higher (p < 0.05) in the stroke group (23.6 ± 4.3; n = 14) compared to controls (9.7 ± 2.3; n = 12), with the risk group being intermediate (16.3 ± 4.3; n = 14). Total WBC counts were significantly higher in the stroke 8.0 ± 0.72 and risk 7.8 ± 0.41 groups (p < 0.05), compared to controls 5.3 ± 0.27. These data indicate that neutrophil adherence is increased in acute stroke and suggests that the total number of potentially adherent cells (total neutrophils times percent adherent cells) is greatly increased.

Results of recent studies reveal vascular and neuroprotective effects of matrix metalloproteinase-9 (MMP-9) inhibition and MMP-9 gene deletion in experimental stroke. However, the cellular source of MMP-9 produced in the ischemic brain and the mechanistic basis of MMP-9-mediated brain injury require elucidation. In the present study, we used MMP-9-/- mice and chimeric knockouts lacking either MMP-9 in leukocytes or in resident brain cells to test the hypothesis that MMP-9 released from leukocytes recruited to the brain during postischemic reperfusion contributes to this injury phenotype. We also tested the hypothesis that MMP-9 promotes leukocyte recruitment to the ischemic brain and thus is proinflammatory. The extent of blood-brain barrier (BBB) breakdown, the neurological deficit, and the volume of infarction resulting from transient focal stroke were abrogated to a similar extent in MMP-9-/- mice and in chimeras lacking leukocytic MMP-9 but not in chimeras with MMP-9-containing leukocytes. Zymography and Western blot analysis from these chimeras confirmed that the elevated MMP-9 expression in the brain at 24 h of reperfusion is derived largely from leukocytes. MMP-9-/- mice exhibited a reduction in leukocyte-endothelial adherence and a reduction in the number of neutrophils plugging capillaries and infiltrating the ischemic brain during reperfusion; microvessel immunopositivity for collagen IV was also preserved in these animals. These latter results document proinflammatory actions of MMP-9 in the ischemic brain. Overall, our findings implicate leukocytes, most likely neutrophils, as a key cellular source of MMP-9, which, in turn, promotes leukocyte recruitment, causes BBB breakdown secondary to microvascular basal lamina proteolysis, and ultimately contributes to neuronal injury after transient focal stroke.

Blood stasis-heat syndrome is one of the common syndromes of ischemic stroke, which is manifested as syndromes of blood stasis and heat during the pathological progression of patients with ischemic stroke, but there is a lack of systematic research on its biological essence. Thromboinflammation reaction is a newly proposed pathological mechanism highly associated with thrombosis and inflammatory reaction, and it refers to the fact that under the mediation of von Willebrand factor(vWF) and the kallikrein-kinin system, thrombosis and inflammatory reaction interact with each other. Activation of T cells and neutrophils further aggravates thrombosis and worsens the pathological progression of ischemic stroke. Therefore, thromboinflammation reaction has the characteristics of the interaction between blood stasis and heat in blood stasis-heat syndrome in traditional Chinese medicine(TCM). Based on the research progress related to thromboinflammation reaction and the clinical syndrome characteristics and biomarkers of blood stasis-heat syndrome in ischemic stroke, this paper put forward the view that thromboinflammation reaction may be the biological foundation of blood stasis-heat syndrome in ischemic stroke. Besides, this paper systematically organized the current applications and research on the mechanism of TCM monomers or compound formulas with the effects of promoting blood circulation to remove blood stasis and clearing heat and detoxifying. It is found that the pharmacological mechanisms are intimately linked to the regulation of biomarkers related to thromboinflammation reaction, verifying that the biological foundation of the blood stasis-heat syndrome in ischemic stroke is thromboinflammation reaction. This paper aims to provide a scientific basis for TCM prevention and treatment strategies for ischemic stroke targeting thromboinflammation reaction.

Immediate activated factor (F)X (FXa) inhibition exerts direct antiplatelet effects in the context of arterial thrombosis but little is known about the impact of long-term therapy on platelet function in ischemic cardiovascular diseases. Therefore, we analyzed platelet-derived effects of long-term FXa inhibition in the setting of acute myocardial infarction (AMI) and stroke. We evaluated the effect of acute versus chronic FXa inhibition on thromboinflammation following AMI and stroke in mice in vivo. Mechanistically, we identified changes in platelet gene expression and proteome under chronic FXa nonvitamin K antagonist oral anticoagulant treatment and characterized its functional consequence on platelet physiology. In a prospectively recruited cohort of patients with AMI, we determined cardiovascular magnetic resonance based cardiac endpoints under FXa nonvitamin K antagonist oral anticoagulant effects on clinical endpoints in a cohort of patients with AMI. Chronic but not acute FXa inhibition reduced cerebral and myocardial infarct size and improved cardiac function 24 hours after AMI in mice. Mechanistically, we identified an attenuated thromboinflammatory response with reduced neutrophil extracellular trap formation in mice and patient samples. Proteome and RNA expression analysis of FXa inhibitor treated patients revealed a reduction of key regulators within the membrane trafficking and secretion machinery hampering platelet α and dense granule release. Subsequent, thromboinflammatory neutrophil extracellular trap density in thrombi isolated from stroke and myocardial infarction patients was reduced. Patients with AMI treated with FXa inhibitors showed decreased infarct size after myocardial infarction compared to patients without anticoagulation treatment. Long-term FXa inhibition induces antithromboinflammatory proteome signatures in platelets, improving infarct size after myocardial infarction and stroke.

Inflammation including immunothrombosis by neutrophil extracellular traps (NETs) has important implications in acute ischemic stroke and can affect reperfusion status, susceptibility to stroke associated infections (SAI) as well as functional clinical outcome. NETs were shown to be prevalent in stroke thrombi and NET associated markers were found in stroke patients' blood. However, little is known whether blood derived NET markers reflect the amount of NETs in thrombi. Conclusions from blood derived markers to thrombus composition might open avenues for novel strategies in diagnostic and therapeutic approaches. We prospectively recruited 166 patients with acute ischemic stroke undergoing mechanical thrombectomy between March 2018 and May 2021. Available thrombi (n = 106) were stained for NET markers DNA-histone-1 complexes and myeloperoxidase (MPO). Cell free DNA (cfDNA), deoxyribonuclease (DNase) activity, MPO-histone complexes and a cytokine-panel were measured before thrombectomy and after seven days. Clinical data, including stroke etiology, reperfusion status, SAI and functional outcome after rehabilitation, were collected of all patients. NET markers were present in all thrombi. At onset the median concentration of cfDNA in blood was 0.19 µg/ml increasing to 0.30 µg/ml at 7 days. Median DNase activity at onset was 4.33 pmol/min/ml increasing to 4.96 pmol/min/ml at 7 days. Within thrombi DNA-histone-1 complexes and MPO correlated with each other (ρ = 0.792; p < 0.001). Moreover, our study provides evidence for an association between the amount of NETs and endogenous DNase activity in blood with amounts of NETs in cerebral thrombi. However, these associations need to be confirmed in larger cohorts, to investigate the potential clinical implications for individualized therapeutic and diagnostic approaches in acute ischemic stroke.

A hypercoagulable state, chronic inflammation, and increased risk of venous thrombosis and stroke are prominent features in patients with sickle cell disease (SCD). Coagulation factor XII (FXII) triggers activation of the contact system that is known to be involved in both thrombosis and inflammation, but not in physiological hemostasis. Therefore, we investigated whether FXII contributes to the prothrombotic and inflammatory complications associated with SCD. We found that when compared with healthy controls, patients with SCD exhibit increased circulating biomarkers of FXII activation that are associated with increased activation of the contact pathway. We also found that FXII, but not tissue factor, contributes to enhanced thrombin generation and systemic inflammation observed in sickle cell mice challenged with tumor necrosis factor α. In addition, FXII inhibition significantly reduced experimental venous thrombosis, congestion, and microvascular stasis in a mouse model of SCD. Moreover, inhibition of FXII attenuated brain damage and reduced neutrophil adhesion to the brain vasculature of sickle cell mice after ischemia/reperfusion induced by transient middle cerebral artery occlusion. Finally, we found higher FXII, urokinase plasminogen activator receptor, and αMβ2 integrin expression in neutrophils of patients with SCD compared with healthy controls. Our data indicate that targeting FXII effectively reduces experimental thromboinflammation and vascular complications in a mouse model of SCD, suggesting that FXII inhibition may provide a safe approach for interference with inflammation, thrombotic complications, and vaso-occlusion in patients with SCD.

Ischemia reperfusion injury (I/RI) is a common complication of cardiovascular diseases. Resolution of detrimental I/RI-generated prothrombotic and proinflammatory responses is essential to restore homeostasis. Platelets play a crucial part in the integration of thrombosis and inflammation. Their role as participants in the resolution of thromboinflammation is underappreciated; therefore we used pharmacological and genetic approaches, coupled with murine and clinical samples, to uncover key concepts underlying this role. Middle cerebral artery occlusion with reperfusion was performed in wild-type or annexin A1 (AnxA1) knockout (AnxA1 Intravital microscopy revealed heightened platelet adherence and aggregate formation post I/RI, which were further exacerbated in AnxA1

von Willebrand factor (VWF) plays an important role in ischemic stroke. However, the exact mechanism by which VWF mediates progression of ischemic stroke brain damage is not completely understood. Using flow cytometric analysis of single cell suspensions prepared from brain tissue and immunohistochemistry, we investigated the potential inflammatory mechanisms by which VWF contributes to ischemic stroke brain damage in a mouse model of cerebral ischemia/reperfusion injury. Twenty-four hours after stroke, flow cytometric analysis of brain tissue revealed that overall white blood cell recruitment in the ipsilesional brain hemisphere of VWF KO mice was 2 times lower than WT mice. More detailed analysis showed a specific reduction of proinflammatory monocytes, neutrophils and T-cells in the ischemic brain of VWF KO mice compared to WT mice. Interestingly, histological analysis revealed a substantial number of neutrophils and T-cells still within the microcirculation of the stroke brain, potentially contributing to the no-reflow phenomenon. Specific therapeutic targeting of the VWF A1 domain in WT mice resulted in reduced immune cell numbers in the affected brain and protected mice from ischemic stroke brain damage. More specifically, recruitment of proinflammatory monocytes was reduced two-fold, neutrophil recruitment was reduced five-fold and T-cell recruitment was reduced two-fold in mice treated with a VWF A1-targeting nanobody compared to mice receiving a control nanobody. In conclusion, our data identify a potential role for VWF in the recruitment of proinflammatory monocytes, neutrophils and T-cells to the ischemic brain via a mechanism that is mediated by its A1 domain.

Although post-stroke neutrophil recruitment is known to be deleterious to neural tissues in the peri-infarct area, the precise behavior of recruited neutrophils remains elusive. In this study, potential therapeutic agents for modifying neutrophil behavior in the peri-infarct area were explored through intravital imaging of an experimental stroke mouse model. By applying in vivo 2-photon imaging to study a tightly controlled photothrombotic stroke mouse model, we established a highly sensitive and reproducible method for investigating the temporal dynamics of ischemic brain lesions. Taking advantage of this system, we revealed that neutrophil depletion by a neutrophil-specific antibody ameliorated the expansion of the infarct area, confirming the deleterious effect of neutrophils in the peri-infarct cortex. To identify neutrophil-targeted therapeutic approaches, we screened various agents and found that colchicine and an anti-P-selectin antibody were the most effective in inhibiting neutrophil attachment to the vessel wall in the early phase (6 h post-infarction). Interestingly, further investigation in the later phase (16 h post-infarction) revealed that colchicine potently inhibited neutrophil infiltration into the peri-infarct cortex; however, the anti-P-selectin antibody did not. Subsequent analysis revealed that the effect of the anti-P-selectin antibody against neutrophil attachment to the vessel wall was transient and thus insufficient for mitigating neutrophil infiltration. Finally, we revealed that colchicine treatment effectively ameliorated infarct expansion. In conclusion, we have established an intravital strategy to directly investigate pathophysiology in the ischemic border zone, and found that colchicine administration in the acute phase of ischemic stroke is a potential novel therapeutic strategy.

Polymorphonuclear neutrophil granulocytes (PMNs) are part of the early post-ischemic immune response that orchestrates the removal of infarcted brain tissue. PMNs contribute to secondary brain injury in experimental stroke models. In human patients, high PMN-to-lymphocyte ratios in peripheral blood are predictive of poor stroke outcome. Following earlier studies indicating that the cerebral microvasculature forms an efficient barrier that impedes PMN brain entry even under conditions of ischemia, more recent studies combining intravital two-photon microscopy and

In acute ischemic stroke due to large vessel occlusion (LVO) infarcts rapidly grow into the penumbra, which represents dysfunctional, but still viable brain tissue amenable to rescue by vessel recanalization. However, infarct progression and/or delayed patient presentation are serious and frequent limitations of this so far only acute therapy. Thus, a major goal of translational research is to "freeze" the penumbra already during LVO (before opening the vessel) and thereby extend individual time windows for non-futile recanalization. We used the filament occlusion model of the middle cerebral artery (MCAO) in mice and assessed progressive infarction under occlusion at 2, 3, and 4 h after onset. We show that blocking the activatory platelet receptor glycoprotein (GP)VI substantially delayed progressive neocortical infarction compared to isotype control antibody treated mice. Moreover, the local vascular recruitment of infiltrating neutrophils and T-cells was mitigated. In conclusion, our experimental data support ongoing clinical trials blocking platelet GPVI in acute ischemic stroke.

Stroke remains a leading cause of mortality and disability, driven by complex, time-dependent mechanisms that aggravate ischemic injury. Collateral perfusion dictates infarct size, expansion rate, and penumbral preservation, yet its regulation is poorly understood. Beyond structural/genetic factors such as aging or cardiovascular risk, functional influences like circadian immune activity may also affect vascular patency. Neutrophils, key mediators of ischemic injury, exhibit circadian oscillations in phenotype and function that could modulate collateral flow and stroke outcome. We combined permanent and transient middle cerebral artery occlusion models in mice with flow cytometry, single-cell RNA sequencing, confocal microscopy, and laser speckle imaging to investigate time-of-day-dependent neutrophil mechanisms in stroke. Pharmacological (chloramidine, DNase-I) and genetic (Pad4 [peptidyl arginine deiminase 4] Infarct volume and neurological deficits exhibited clear circadian oscillations, with worse outcomes when stroke occurred during the murine inactive phase (Zeitgeber time 5) versus the active phase (Zeitgeber time 13). These fluctuations disappeared after neutrophil depletion or clock disruption. During the inactive phase, neutrophils displayed an activated, NET-prone phenotype, causing microvascular stalling and reduced collateral perfusion. Inhibiting NET formation pharmacologically or through Pad4 deletion restored perfusion and abolished time-of-day effects. In patients, neutrophil and NET-related biomarkers (MPO [myeloperoxidase], elastase, sCD40L [soluble CD40 ligand]) showed diurnal oscillations, peaking during the human inactive phase (evening/night), coinciding with reduced collateral flow and poorer outcomes. Time-of-day regulation of neutrophil function critically determines collateral perfusion and stroke severity. Neutrophil-driven NETosis during the inactive phase promotes microvascular obstruction and worsens outcomes. Targeting NET formation or timing therapy could enhance collateral efficacy and offer novel chronotherapeutic opportunities for stroke treatment.

Ischemic stroke (IS) is a significant global health burden, contributing to disability and imposing financial strain on societies. Taohong Siwu decoction (THSWD), a classical TCM formula, has shown neuroprotective effects in IS therapy, particularly in mitigating inflammation, but its precise mechanisms remain unclear. Neutrophil extracellular traps (NETs) have been implicated in post-stroke neuroinflammation and brain damage, making NETs a potential therapeutic target for IS. This study focuses on the protective effect and molecular mechanism of THSWD on IS injury, focuses on its inhibitory effect on the formation of NETs, and explores the potential molecular mechanism of THSWD regulating the formation of NETs. Network pharmacology was employed to predict the interactions between THSWD and disease-associated target genes. In vivo experiments were conducted using a rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) to evaluate the effects of THSWD on infarct volume, neurological deficits, cerebral microvascular injury, inflammatory cytokine secretion, and NETs formation. In vitro experiments were performed to investigate the effects of THSWD serum on NETs formation in rat neutrophils stimulated by phorbol myristate acetate (PMA). The underlying mechanism of THSWD in regulating NETs was further investigated via transcriptome sequencing (RNA-seq). Utilizing network pharmacology, 264 overlapping targets of THSWD and IS were identified, with key pathways including neutrophil-mediated immunity and NETs formation. Experimental validation in a rat MCAO/R model demonstrated that THSWD significantly reduced cerebral infarct volume, improved neurological deficits, and attenuated blood-brain barrier (BBB) disruption by restoring vascular integrity (ZO-1, Occludin) and enhancing hemodynamic recovery. Furthermore, THSWD downregulated the expression of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, IL-2, IL-5, IL-18, and TNF-β) and neutrophil infiltration while downregulating NETs markers Myeloperoxidase (MPO) and Citrullinated histone H3(CitH3) in serum and ischemic brain tissue. In vitro, THSWD-containing serum inhibited PMA-induced NETosis in neutrophils by reducing extracellular DNA release and ROS production. RNA-seq of peripheral blood neutrophils identified 326 differentially expressed genes (DEGs) commonly altered by MCAO/R injury and rescued by THSWD treatment. KEGG enrichment revealed significant involvement of the NOD-like receptor signaling pathway, with bioinformatic analysis identifying STAT1 as a core hub gene. Subsequent mechanistic validation confirmed that THSWD suppressed the activation of the STAT1/NLRP3/GSDMD signaling axis in neutrophils. THSWD ameliorates IS by inhibiting NETs formation via the STAT1/NLRP3/GSDMD signaling pathway, reducing inflammatory cytokine release and protecting against cerebral microvascular injury.

Despite successful vascular recanalization in stroke, one-fourth of patients have an unfavorable outcome due to no-reflow. The pathogenesis of no-reflow is fully unclear, and therapeutic strategies are lacking. Upon traditional Chinese medicine, Tongxinluo capsule (TXL) is a potential therapeutic agent for no-reflow. Thus, this study is aimed to investigate the pathogenesis of no-reflow in stroke, and whether TXL could alleviate no-reflow as well as its potential mechanisms of action. Mice were orally administered with TXL (3.0 g/kg/d) after transient middle cerebral artery occlusion. We examined the following parameters: neurological function, no-reflow, leukocyte-endothelial cell interactions, HE staining, leukocyte subtypes, adhesion molecules, and chemokines. Our results showed stroke caused neurological deficits, neuron death, and no-reflow. Adherent and aggregated leukocytes obstructed microvessels as well as leukocyte infiltration in ischemic brain. Leukocyte subtypes changed after stroke mainly including neutrophils, lymphocytes, regulatory T cells, suppressor T cells, helper T type 1 (Th1) cells, Th2 cells, B cells, macrophages, natural killer cells, and dendritic cells. Stroke resulted in upregulated expression of adhesion molecules (P-selectin, E-selectin, and ICAM-1) and chemokines (CC-chemokine ligand (CCL)-2, CCL-3, CCL-4, CCL-5, and chemokine C-X-C ligand 1 (CXCL-1)). Notably, TXL improved neurological deficits, protected neurons, alleviated no-reflow and leukocyte-endothelial cell interactions, regulated multiple leukocyte subtypes, and inhibited the expression of various inflammatory mediators. Leukocyte-endothelial cell interactions mediated by multiple inflammatory factors are an important cause of no-reflow in stroke. Accordingly, TXL could alleviate no-reflow via suppressing the interactions through modulating various leukocyte subtypes and inhibiting the expression of multiple inflammatory mediators.

While polymorphonuclear leukocytes may contribute to the "no-reflow" phenomenon after focal cardiac and skeletal muscle ischemia/reperfusion, their contribution to acute focal cerebral ischemia is unresolved. We have examined the role of polymorphonuclear leukocytes in microvascular perfusion defects after focal cerebral ischemia/reperfusion in a baboon model of reversible middle cerebral artery occlusion with the anti-CD18 monoclonal antibody IB4, which inhibits neutrophil adherence to endothelium. Microvascular patency in the basal ganglia after 3-hour middle cerebral artery occlusion and 1-hour reperfusion (by india ink tracer perfusion) was quantified by computerized video imaging. Animals were randomized to receive intravenous IB4 infusion 15 minutes before reperfusion (n = 7) or to receive no treatment (n = 6). Binding of IB4 to baboon leukocytes was maximal within 5 minutes of infusion. In the untreated group, a significant reduction in patency was observed in microvessels less than 30 microns diameter: mean percent reflow was 51% in the capillary diameter class (4.0-7.5 microns) and 39% in the precapillary arteriole and postcapillary venule diameter class (7.5-30 microns). Infusion of IB4 before middle cerebral artery reperfusion increased reflow in microvessels of all size classes, most significantly in those 7.5-30 microns (p = 0.049) and 30-50 microns (p = 0.034) in diameter. These results suggest that CD18-mediated polymorphonuclear leukocyte-endothelium adherence contributes to no-reflow predominantly in noncapillary microvessels and at least partially to that in capillaries.

Acute neutrophil (PMN) recruitment to postischemic cardiac or pulmonary tissue has deleterious effects in the early reperfusion period, but the mechanisms and effects of neutrophil influx in the pathogenesis of evolving stroke remain controversial. To investigate whether PMNs contribute to adverse neurologic sequelae and mortality after stroke, and to study the potential role of the leukocyte adhesion molecule intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of stroke, we used a murine model of transient focal cerebral ischemia consisting of intraluminal middle cerebral artery occlusion for 45 min followed by 22 h of reperfusion. PMN accumulation, monitored by deposition of 111In-labeled PMNs in postischemic cerebral tissue, was increased 2.5-fold in the ipsilateral (infarcted) hemisphere compared with the contralateral (noninfarcted) hemisphere (P < 0.01). Mice immunodepleted of neutrophils before surgery demonstrated a 3.0-fold reduction in infarct volumes (P < 0.001), based on triphenyltetrazolium chloride staining of serial cerebral sections, improved ipsilateral cortical cerebral blood flow (measured by laser Doppler), and reduced neurological deficit compared with controls. In wild-type mice subjected to 45 min of ischemia followed by 22 h of reperfusion, ICAM-1 mRNA was increased in the ipsilateral hemisphere, with immunohistochemistry localizing increased ICAM-1 expression on cerebral microvascular endothelium. The role of ICAM-1 expression in stroke was investigated in homozygous null ICAM-1 mice (ICAM-1 -/-) in comparison with wild-type controls (ICAM-1 +/+). ICAM-1 -/- mice demonstrated a 3.7-fold reduction in infarct volume (P < 0.005), a 35% increase in survival (P < 0.05), and reduced neurologic deficit compared with ICAM-1 +/+ controls. Cerebral blood flow to the infarcted hemisphere was 3.1-fold greater in ICAM-1 -/- mice compared with ICAM-1 +/+ controls (P < 0.01), suggesting an important role for ICAM-1 in the genesis of postischemic cerebral no-reflow. Because PMN-depleted and ICAM-1-deficient mice are relatively resistant to cerebral ischemia-reperfusion injury, these studies suggest an important role for ICAM-1-mediated PMN adhesion in the pathophysiology of evolving stroke.

The infiltration of polymorphonuclear leukocytes (PMNs) in ischemia-reperfusion injury (I/RI) has been implicated as a critical component of inflammatory damage following ischemic stroke. However, successful blockade of PMN transendothelial migration (TEM) in preclinical studies has not translated to meaningful clinical outcomes. To investigate this further, leukocyte infiltration patterns were quantified, and these patterns were modulated by blocking platelet endothelial cell adhesion molecule-1 (PECAM), a key regulator of TEM. LysM-eGFP mice and microscopy were used to visualize all myeloid leukocyte recruitment following ischemia/reperfusion. Visual examination showed heterogeneous leukocyte distribution across the infarct at both 24 and 72 h post-I/RI. A semi-automated process was designed to precisely map PMN position across brain sections. Treatment with PECAM function-blocking antibodies did not significantly affect total leukocyte recruitment but did alter their distribution, with more observed at the cortex at both early and later time points (24 h: 89% PECAM blocked vs 72% control; 72 h: 69% PECAM blocked vs. 51% control). This correlated with a decrease in infarct volume. These findings suggest that TEM, in the setting of I/RI in the cerebrovascular, occurs primarily at the cortical surface. The reduction of stroke size with PECAM blockade suggests that infiltrating PMNs may exacerbate I/RI and indicate the potential therapeutic benefit of regulating the timing and pattern of leukocyte infiltration after stroke.

RATIONALE Adhesion molecules are key elements in stroke-induced brain injury by regulating the migration of effector immune cells from the circulation to the lesion site. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is an adhesion molecule highly expressed on endothelial cells and leukocytes, which controls the final steps of transendothelial migration. A functional role for PECAM-1 in post-ischemic brain injury has not yet been demonstrated. OBJECTIVE Using genetic Pecam-1 depletion and PECAM-1 blockade using a neutralizing anti-PECAM-1 antibody, we evaluated the role of PECAM-1 mediated transendothelial immune cell migration for ischemic injury, delayed brain atrophy, and brain immune cell infiltrates. Transendothelial immune cell migration was furthermore evaluated in cultured human cerebral microvascular endothelial cells. METHODS AND RESULTS Transient middle cerebral artery occlusion (tMCAO) was induced in 10-12-week-old male Pecam-1-/- and Pecam-1+/+ wildtype mice. PECAM-1 levels increased in the ischemic brain tissue due to the infiltration of PECAM-1+ leukocytes. Using magnetic resonance imaging, we observed smaller infarct volume, less edema formation, and less brain atrophy in Pecam-1-/- compared with Pecam-1+/+ wildtype mice. The transmigration of leukocytes, specifical neutrophils, was selectively reduced by Pecam-1-/-, as shown by immune fluorescence and flow cytometry in vivo and transmigration assays in vitro. Importantly, inhibition with an anti-PECAM-1 antibody in wildtype mice decreased neutrophil brain influx and infarct. CONCLUSION PECAM-1 controls the transendothelial migration of neutrophils in a mouse model of ischemic stroke. Antibody blockade of PECAM-1 after stroke onset ameliorates stroke severity in mice, making PECAM-1 an interesting target to dampen post-stroke neuroinflammation, reduce ischemic brain injury, and enhance post-ischemic brain remodeling.

Purpose of review In this review, we will describe how the combined ability of platelets and neutrophils to interact with each other drives ischemic stroke brain injury. Recent findings Neutrophils are one of the first cells to respond during ischemic stroke. Although animals stroke models have indicated targeting neutrophils improves outcomes, clinical trials have failed to yield successful strategies. Platelets play a critical role in recruiting neutrophils to sites of injury by acting as a bridge to the injured endothelium. After initial platelet adhesion, neutrophils can rapidly bind platelets through P-selectin and glycoprotein Ibα. In addition, recent data implicated platelet phosphatidylserine as a novel key regulator of platelet-neutrophil interactions in the setting of ischemic stroke. Inhibition of procoagulant platelets decreases circulating platelet-neutrophil aggregates and thereby reduces infarct size. Platelet binding alters neutrophil function, which contributes to the injury associated with ischemic stroke. This includes inducing the release of neutrophil extracellular traps, which are neurotoxic and pro-thrombotic, leading to impaired stroke outcomes. Summary Platelet-neutrophil interactions significantly contribute to the pathophysiology of ischemic stroke brain injury. Better understanding the mechanisms behind their formation and the downstream consequences of their interactions will lead to improved therapies for stroke patients.

Reperfusion injury of ischemic stroke, characterized by the uncontrolled production of reactive oxygen species (ROS) and inflammatory reactions, continues to present a major problem in clinical treatment. Neutrophils are forerunners to infiltrate cerebral ischemic regions and contribute to reperfusion injury. Herein, this study reports a tailored “burning the bridges” strategy by designing biomimetic nanozymes (D@HPB@SPM NPs) to diminish reperfusion injury of ischemic stroke. D@HPB@SPM NPs are composed of a sialic acid (SA)‐modified platelet membrane shell and a hollow Prussian blue nanoparticle core loaded with Deoxyribonuclease I (DNase I). Due to the unique binding affinity of SA to L‐selectin, which is abundantly expressed in circulating neutrophils, D@HPB@SPM NPs can effectively hitchhike on neutrophils across the blood‐brain barrier into the injured brain parenchyma after intravenous injection. Following this, neutrophils are activated and unleash D@HPB@SPM NPs through producing neutrophil extracellular traps (NETs). D@HPB@SPM NPs not only relieve oxidative stress injury by efficiently scavenging ROS, but also mitigate neutrophil‐induced reperfusion injury by degrading NETs in a manner similar to “burning the bridges”. The encouraging accumulation of D@HPB@SPM NPs to cerebral ischemic regions and their efficient therapeutic efficacy are systematically validated in ischemic stroke rats. This work offers a fresh insight for ischemic stroke treatment.

Sterile acute kidney injury (AKI) is common in the clinic and frequently associated with unexplained hypoxemia that does not improve with dialysis. AKI induces remote lung inflammation with neutrophil recruitment in mice and humans, but which cellular cues establish neutrophilic inflammation and how it contributes to hypoxemia is not known. Here we report that AKI induced rapid intravascular neutrophil retention in lung alveolar capillaries without extravasation into tissue or alveoli, causing hypoxemia by reducing lung capillary blood flow in the absence of substantial lung interstitial or alveolar edema. In contrast to direct ischemic lung injury, lung neutrophil recruitment during remote lung inflammation did not require cues from intravascular nonclassical monocytes or tissue-resident alveolar macrophages. Instead, lung neutrophil retention depended on the neutrophil chemoattractant CXCL2 released by activated classical monocytes. Comparative single-cell RNA-Seq analysis of direct and remote lung inflammation revealed that alveolar macrophages were highly activated and produced CXCL2 only in direct lung inflammation. Establishing a CXCL2 gradient into the alveolus by intratracheal CXCL2 administration during AKI-induced remote lung inflammation enabled neutrophils to extravasate. We thus discovered important differences in lung neutrophil recruitment in direct versus remote lung inflammation and identified lung capillary neutrophil retention that negatively affected oxygenation by causing a ventilation-perfusion mismatch as a driver of AKI-induced hypoxemia.

Highlights What are the main findings? NETs contribute to the activation of the coagulation cascade and have been successfully proved to be a potential driver of DIC in HS mice. What is the implication of the main finding? This work provides a novel alternative treatment strategy for the treatment of DIC in HS patients. Abstract Aims: Disseminated intravascular coagulation (DIC) is a common complication of heat stroke (HS) patients, and it is one of the important reasons leading to multiple organ failure and even death. The association between neutrophil extracellular traps (NETs) and DIC is unclear in HS mice. Methods and results: Here, HS was induced by the combination of hyperthermia (HT) and lipopolysaccharide (LPS). The DIC was evaluated by measuring prothrombin time (PT), D-dimer, thrombomodulin (TM), fibrinogen (FIB), and platelet (PLT). The expression of citrullinated-histone (CitH3) was analyzed by Western blotting. The formation of NETs was observed by immunofluorescence microscopy. The risk of HS-induced DIC was increased when HT was combined with LPS. The markers of NETs were significantly higher than those in the control group, and the NETs derived from HS promoted the development of DIC. DNase I improved coagulation dysfunction via the clearance of NETs caused by neutrophil aggregation. Conclusions: Degradation of NETs reduced the risk of developing DIC, and thus the survival rate of mice was improved. These results indicate that NETs may hold potential alternative therapeutic strategies for the treatment of DIC in HS patients.

Interleukin-17A (IL-17A) plays an important role in the progression of ischemic stroke. IL-17A mediates the endothelial inflammatory response, promotes water and sodium retention, and changes the electrophysiological structure of the atrium, accelerating the progression of ischemic stroke risk factors such as atherosclerotic plaques, hypertension, and atrial fibrillation. In the acute phase of ischemic stroke, IL-17A mediates neuronal injury through neutrophil chemotaxis to the site of injury, the induction of neuronal apoptosis, and activation of the calpain-TRPC-6 (transient receptor potential channel-6) pathway. During ischemic stroke recovery, IL-17A, which is mainly derived from reactive astrocytes, promotes and maintains the survival of neural precursor cells (NPCs) in the subventricular zone (SVZ), neuronal differentiation, and synapse formation and participates in the repair of neurological function. Therapies targeting IL-17A-associated inflammatory signaling pathways can reduce the risk of ischemic stroke and neuronal damage and are a new therapeutic strategy for ischemic stroke and its risk factors. In this paper, we will briefly discuss the pathophysiological role of IL-17A in ischemic stroke risk factors, acute and chronic inflammatory responses, and the potential therapeutic value of targeting IL-17A.

Clazosentan has been shown to prevent vasospasm and reduce mortality in patients after aneurysmal subarachnoid hemorrhage (SAH) and has been approved for clinical use in Japan; however, its systemic events in the elderly (aged ≥ 75 years) have not been well-documented. Here, we report serious/intolerable cardiopulmonary complications requiring discontinuation of drug therapy in elderly SAH patients. In this single-center case series study, medical records of consecutive SAH patients treated postoperatively with clazosentan (10 mg/h) between June 2022 and May 2023 were reviewed retrospectively. Thirty-three patients received clazosentan therapy, of whom six were elderly with a mean age of 80.3 ± 5.2 (range 75–89) years. Among them, despite no obvious medical history of systemic abnormalities, clazosentan was discontinued in three (50%) patients due to pleural effusion and hypoxemia with or without hypotension at 5 ± 3 days after therapy initiation, which was higher than the incidence for younger patients (15%). The elderly patients had significantly lower urine output (1935 ± 265 vs. 1123 ± 371 mL/day, p = 0.03) and greater weight gain (2.1 ± 1.1 vs. 4.2 ± 1.9 kg from baseline, p = 0.04) than patients who completed the therapy. One 89-year-old female developed congestive heart failure and hydrostatic pulmonary edema associated with increased intravascular and lung volumes even after therapy was discontinued, while the remaining two cases recovered within 2 days after drug cessation. These results suggest that elderly patients are more vulnerable to fluid retention and have a higher risk of cardiopulmonary complications during clazosentan therapy than younger patients. Careful monitoring of urine volume and weight gain and caution regarding age- and therapy-related hemodynamic insufficiencies are required.

Abstract Uromodulin (UMOD) is the most abundant renal protein secreted into urine by the thick ascending limb (TAL) epithelial cells of the loop of Henle. Genetic studies have demonstrated an association between UMOD risk variants and hypertension. We aimed to dissect the role of dietary salt in renal UMOD excretion in normotension and chronic hypertension. Normotensive Wistar–Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) (n=8/sex/strain) were maintained on 1% NaCl for 3 weeks. A subset of salt-loaded SHRSP was treated with nifedipine. Salt-loading in SHRSP increased blood pressure (ΔSBP 35 ± 5 mmHg, P<0.0001) and kidney injury markers such as kidney injury marker-1 (KIM-1; fold change, FC 3.4; P=0.003), neutrophil gelatinase-associated lipocalin (NGAL; FC, 2.0; P=0.012) and proteinuria. After salt-loading there was a reduction in urinary UMOD excretion in WKY and SHRSP by 26 and 55% respectively, compared with baseline. Nifedipine treatment reduced blood pressure (BP) in SHRSP, however, did not prevent salt-induced reduction in urinary UMOD excretion. In all experiments, changes in urinary UMOD excretion were dissociated from kidney UMOD protein and mRNA levels. Colocalization and ex-vivo studies showed that salt-loading increased intracellular UMOD retention in both WKY and SHRSP. Our study provides novel insights into the interplay among salt, UMOD, and BP. The role of UMOD as a cardiovascular risk marker deserves mechanistic reappraisal and further investigations based on our findings.

Abstract Objectives We investigated whether the totally video-assisted thoracoscopic mitral valve surgery provides superior clinical outcomes and less inflammatory injury reaction compared with conventional sternotomy. Methods A total of 504 consecutive patients admitted for mitral valve surgery from May 2014 through May 2019 in a single center were retrospectively analyzed according to two distinct procedure approach: the totally video-assisted thoracoscopic approach (group A, n = 127) and standard median sternotomy (group B, n = 377). The primary end point was the durations of cardiopulmonary bypass, aortic cross-clamping, the ventilation time and intensive care unit of stay; the secondary endpoints included inflammation indexes like high sensitivity C-reactive protein, neutrophil-lymphocyte ratio and metabolic injury parameters cardiac Troponin and lactate. Results There was only one in-hospital death due to diffuse intravascular coagulation in group A, but similar complications such as repair failure, re-thoracotomy and stroke in both groups. The durations of cardiopulmonary bypass and aortic cross-clamping were significantly longer in group A. In contrast, ventilation time and intensive care unit of stay were shortened compared with these in group B. In addition, postoperative equivalent lactate clearance but lower high sensitivity C-reactive protein, neutrophil-lymphocyte ratio and cardiac Troponin level was in group A than those in group B within postoperative 24 hours(P < 0.05). Conclusions The analysis of present study indicated despite relatively longer cardiopulmonary bypass time, the totally thoracoscopic mitral valve procedure seemed to be favorable with regard to the extent of inflammatory reaction, cardiac injury and postoperative recovery compared with conventional median sternotomy.

Endovascular reperfusion therapy is the primary strategy for acute ischemic stroke. No-reflow is a common phenomenon, which is defined as the failure of microcirculatory reperfusion despite clot removal by thrombolysis or mechanical embolization. It has been reported that up to 25% of ischemic strokes suffer from no-reflow, which strongly contributes to an increased risk of poor clinical outcomes. No-reflow is associated with functional and structural alterations of cerebrovascular microcirculation, and the injury to the microcirculation seriously hinders the neural functional recovery following macrovascular reperfusion. Accumulated evidence indicates that pathology of no-reflow is linked to adhesion, aggregation, and rolling of blood components along the endothelium, capillary stagnation with neutrophils, astrocytes end-feet, and endothelial cell edema, pericyte contraction, and vasoconstriction. Prevention or treatment strategies aim to alleviate or reverse these pathological changes, including targeted therapies such as cilostazol, adhesion molecule blocking antibodies, peroxisome proliferator-activated receptors (PPARs) activator, adenosine, pericyte regulators, as well as adjunctive therapies, such as extracorporeal counterpulsation, ischemic preconditioning, and alternative or complementary therapies. Herein, we provide an overview of pathomechanisms, predictive factors, diagnosis, and intervention strategies for no-reflow, and attempt to convey a new perspective on the clinical management of no-reflow post-ischemic stroke.

Studies have shown that a series of molecular events caused by oxidative stress is associated with ferroptosis and oxidation after ischemic stroke (IS). Differential analysis was performed to identify differentially expressed mRNA (DEmRNAs) between IS and control groups. Critical module genes were identified using weighted gene co-expression network analysis (WGCNA). DEmRNAs, critical module genes, oxidative stress-related genes (ORGs), and ferroptosis-related genes (FRGs) were crossed to screen for intersection mRNAs. Candidate mRNAs were screened based on the protein–protein interaction (PPI) network and the MCODE plug-in. Biomarkers were identified based on two types of machine learning algorithms, and the intersection was obtained. Functional items and related pathways of the biomarkers were identified using gene set enrichment analysis (GSEA). Finally, single-sample GSEA (ssGSEA) and Wilcoxon tests were used to identify differential immune cells. An miRNA-mRNA-TF network was created. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to verify the expression levels of biomarkers in the IS and control groups. There were 8287 DE mRNAs between the IS and control groups. The genes in the turquoise module were selected as critical module genes for IS. Thirty intersecting mRNAs were screened for overlaps. Seventeen candidate mRNAs were also identified. Four biomarkers (CDKN1A, GPX4, PRDX1, and PRDX6) were identified using two types of machine-learning algorithms. GSEA results indicated that the biomarkers were associated with steroid biosynthesis. Nine types of immune cells (activated B cells and neutrophils) were markedly different between the IS and control groups. We identified 3747 miRNA-mRNA-TF regulatory pairs in the miRNA-mRNA-TF regulatory network, including hsa-miR-4469-CDKN1A-BACH2 and hsa-miR-188-3p-GPX4-ATF2. CDKN1A, PRDX1, and PRDX6 were upregulated in IS samples compared with control samples. This study suggests that four biomarkers (CDKN1A, GPX4, PRDX1, and PRDX6) are significantly associated with IS. This study provides a new reference for the diagnosis and treatment of IS.

Strokes are conditions with a high degree of morbidity and mortality worldwide. These conditions profoundly affect the quality of life of patients; in addition to physical disabilities, patients present various mental disorders, such as mood disorders, anxiety, depression, behavioral disorders, fatigue, etc. Microscopic lesions of the brain parenchyma explain the clinical symptoms and correlate with the severity of the stroke. Our study consisted of the histopathological (HP) and immunohistochemical analysis of brain fragments, collected from 23 patients, with a clinical and imagistic diagnosis of stroke, who died during hospital admission. The microscopic analysis showed that both neurons and glial cells are affected in the ischemic focus. Neuronal death in the ischemic focus was mostly caused by cell necrosis and only about 10% by apoptosis. Regarding vascular lesions, it was observed that the most frequent HP lesion of intracerebral arterioles was arteriosclerosis. The lumen of the arterioles was reduced, and the vascular endothelium had a discontinuous aspect, which indicates a change in the blood–brain barrier. Sometimes the arteriole lumen was completely obstructed, with fibrinoid necrosis in the internal and middle tunic, or with the proliferation of fibroblasts and the formation of young intraluminal connective tissue. Intraparenchymal blood capillaries in the ischemic area showed endothelium discontinuities, lumen collapse, and sometimes massive perivascular edema. As for neuroinflammation, the presence of numerous neutrophils, lymphocytes, plasma cells and macrophages was found in the ischemic focus, forming a complex and inhomogeneous cellular mixture. Of the inflammatory cells present in the ischemic focus and in the ischemic penumbra area, the most numerous were the macrophages. The HP analysis showed that neuroinflammation is very complex and different in intensity from one patient to another, most likely due to associated comorbidities, age, treatment administered until death, etc.

Acute ischemic stroke (AIS) is a common neurological disease that seriously endangers both the physical and mental health of human. After AIS, activated immune cells are recruited to the stroke site, where inflammatory mediators are released locally, and severe immune inflammatory reactions occur within a short time, which affects the progress and prognosis of IS. Circular RNA (circRNA) is a type of non-coding RNA (ncRNA) with a closed-loop structure and high stability. Studies have found that circRNA can affect the course of IS. However, there is no report on ceRNA’s pathogenesis in AIS that is mediated by circRNA. In this study, the CIBERSORT algorithm was used to analyze the distribution of immune cells in patients with AIS. mRNA dataset was downloaded from the GEO database, and the weighted gene co-expression network analysis (WGCNA) method was used to construct weighted gene co-expression to determine 668 target genes, using GO, KEGG enrichment analysis, construction of protein-protein interaction (PPI) network analysis, and molecular complex detection (MCODE) plug-in analysis. The results showed that the biological function of the target gene was in line with the activation and immune regulation of neutrophils; signal pathways were mostly enriched in immune inflammation-related pathways. A Venn diagram was used to obtain 52 intersection genes between target genes and disease genes. By analyzing the correlation between the intersection genes and immune cells, we found that the top 5 hub genes were TOM1, STAT3, RAB3D, MDM2, and FOS, which were all significantly positively correlated with neutrophils and significantly negatively correlated with eosinophils. A total of 52 intersection genes and the related circRNA and miRNA were used as input for Cytoscape software to construct a circRNA-mediated ceRNA competition endogenous network, where a total of 18 circRNAs were found. Further analysis of the correlation between circRNA and immune cells found that 4 circRNAs are positively correlated with neutrophils. Therefore, we speculate that there may be a regulatory relationship between circRNA-mediated ceRNA and the immune mechanism in AIS. This study has important guiding significance for the progress, outcome of AIS, and the development of new medicine.

Thrombotic diseases remain the major cause of death and disability worldwide, and the contribution of inflammation is increasingly recognized. Thromboinflammation has been identified as a key pathomechanism, but an unsupervised map of immune-cell states, trajectories, and intercommunication at a single-cell level has been lacking. Here, we reveal innate leukocyte substates with prominent thrombolytic properties by employing single-cell omics measures on human stroke thrombi. Using in vivo and in vitro thrombosis models, we propose a pro-resolving monocyte-neutrophil axis, combining two properties: (1) NR4A1hi non-classical monocytes acquire a thrombolytic and neutrophil-chemoattractive phenotype, and (2) blood neutrophils are thereby continuously recruited to established thrombi through CXCL8-CXCR1 and CXCR2 and adopt a hypoxia-induced thrombus-resolving urokinase receptor (PLAUR)+ phenotype. This immunothrombolytic axis results in thrombus resolution. Together, with this immune landscape of thrombosis, we provide a valuable resource and introduce the concept of "immunothrombolysis" with broad mechanistic and translational implications at the crossroad of inflammation and thrombosis.

Abstract Platelets are increasingly being recognized for playing roles beyond thrombosis and hemostasis. Today we know that they mediate inflammation by direct interactions with innate immune cells or secretion of cytokines/chemokines. Here we review their interactions with neutrophils and monocytes/macrophages in infection and sepsis, stroke, myocardial infarction and venous thromboembolism. We discuss new roles for platelet surface receptors like GPVI or GPIb and also look at platelet contributions to the formation of neutrophil extracellular traps (NETs) as well as to deep vein thrombosis during infection, e.g. in COVID-19 patients.

Background Systemic immune-inflammation index (SII) and neutrophil-to-lymphocyte ratio (NLR) are novel inflammatory markers based on neutrophil, platelet and lymphocyte counts. Atherosclerosis is a chronic inflammatory vascular disease. This study aimed to verify the predictive value of the clinical parameters such as systemic immune-inflammation index (SII) and neutrophil-to-lymphocyte ratio (NLR) for the severity in Large Artery Atherosclerosis (LAA) stroke patients. Methods The SII is defined as platelet × (neutrophil count/lymphocyte count), the NLR is defined as neutrophil count/lymphocyte count. Univariate logistic regression was used to analyze the association between SII and NLR and NIHSS score in patients with LAA stroke. Multiple logistic regression was used to analyze the risk factors for the severity of LAA stroke. We plotted receiver operating characteristic curves to determine the diagnostic role of SII and NLR in differentiating stroke disease severity. Results We included 283 LAA stroke patients, the SII and NLR in the moderate-to-severe stroke group were significantly higher than the mild stroke group. Multiple logistic regression analysis showed that SII (OR 1.051 95% CI (1.035–1.066), P < 0.001), NLR (OR 1.077,95% CI (1.032–1.123), P < 0.001) were significantly associated with stroke severity. The SII values under the receiver operating characteristic curve (0.701, 95% CI (0.649–0.791, P < 0.001, cut-off value 912.97) and NLR values under the receiver operating characteristic curve (0.604,5% CI (0.519–0.689), P < 0.01, cut-off value 1.461), and SII values had high discrimination ability. Both SII and NLR had high diagnostic and predictive value for stroke severity, and SII was better than NLR. Conclusion The higher SII and NLR, the more severity in LAA stroke patients. SII and NLR are independent risk factors for LAA stroke, and they can also effectively predict stroke severity; moreover, SII has a higher diagnostic efficacy than NLR. However, multicenter studies with large sample size are still needed to confirm this conclusion.

The neutrophil-to-albumin ratio (NPAR) is a relatively novel composite biomarker of inflammation, which has been used for prognostication in cardiovascular diseases and may also be associated with stroke. A cross-sectional analysis was conducted using data from the National Health and Nutrition Examination Survey (NHANES) 1999–2018, including 48,734 individuals with complete NPAR and stroke data. The association between stroke prevalence and NPAR values was assessed through multivariate regression analysis. The relationship between these variables was further visualized using restricted cubic splines (RCS). Additionally, potential factors influencing this relationship were explored through subgroup analysis. The regression model revealed a significant association between NPAR and stroke prevalence, even after adjusting for other covariates [1.06 (1.04, 1.08)]. Stroke prevalence was 62% higher in the highest NPAR group compared to the lowest [1.62 (1.40, 1.89)]. The RCS analysis further confirmed this positive correlation. Subgroup analysis showed that this association was not significantly influenced by other factors. This study establishes a strong association between NPAR and stroke prevalence. However, further studies are needed to clarify the underlying mechanisms and establish a direct causal link.

Abstract Two-photon microscopy (TPM) plays an important role in the study of the changes of the two important components of neurovascular units (NVU) – neurons and blood vessels after ischemic stroke (IS). IS refers to sudden neurological dysfunction caused by focal cerebral ischemia, which is one of the leading causes of death and disability worldwide. TPM is a new and rapidly developing high-resolution real-time imaging technique used in vivo that has attracted increasing attention from scientists in the neuroscience field. Neurons and blood vessels are important components of neurovascular units, and they undergo great changes after IS to respond to and compensate for ischemic injury. Here, we introduce the characteristics and pre-imaging preparations of TPM, and review the common methods and latest progress of TPM in the neuronal and vascular research for injury and recovery of IS in recent years. With the review, we clearly recognized that the most important advantage of TPM in the study of ischemic stroke is the ability to perform chronic longitudinal imaging of different tissues at a high resolution in vivo. Finally, we discuss the limitations of TPM and the technological advances in recent years.

Recanalization is the mainstay of ischemic stroke treatment. However, even with timely clot removal, many stroke patients recover poorly. Leptomeningeal collaterals (LMCs) are pial anastomotic vessels with yet-unknown functions. We applied laser speckle imaging, ultrafast ultrasound, and two-photon microscopy in a thrombin-based mouse model of stroke and fibrinolytic treatment to show that LMCs maintain cerebral autoregulation and allow for gradual reperfusion, resulting in small infarcts. In mice with poor LMCs, distal arterial segments collapse, and deleterious hyperemia causes hemorrhage and mortality after recanalization. In silico analyses confirm the relevance of LMCs for preserving perfusion in the ischemic region. Accordingly, in stroke patients with poor collaterals undergoing thrombectomy, rapid reperfusion resulted in hemorrhagic transformation and unfavorable recovery. Thus, we identify LMCs as key components regulating reperfusion and preventing futile recanalization after stroke. Future therapeutic interventions should aim to enhance collateral function, allowing for beneficial reperfusion after stroke.

Abstract Background Predicting stroke-associated pneumonia (SAP) is crucial for intensifying preventive measures and decreasing morbidity and mortality. This meta-analysis aims to evaluate the association between baseline neutrophil-lymphocyte ratio (NLR), monocyte-lymphocyte ratio (MLR), and platelet-lymphocyte ratio (PLR) with SAP and to determine the strength of the association. Methods The Web of Science, SCOPUS, and PUBMED databases were searched to find eligible studies. The standardized mean difference (SMD) and 95% confidence interval (CI) were used to evaluate the differences in NLR, MLR, and PLR levels between SAP and non-SAP patients. The meta-analysis was conducted using the software “Review Manager” (RevMan, version 5.4.1, September 2020). The random-effect model was used for the pooling analysis if there was substantial heterogeneity. Otherwise, the fixed-effect model was adopted. Results Twelve studies comprising 6302 stroke patients were included. The pooled analyses revealed that patients with SAP had significantly higher levels of NLR, MLR, and PLR than the non-SAP group. The SMD, 95% CI, p-value, and I2 for them were respectively reported as (0.88, 0.70–1.07, .00001, 77%); (0.94, 0.43–1.46, .0003, 93%); and (0.61, 0.47–0.75, .001, 0%). Subgroup analysis of NLR studies showed no significant differences in the effect size index between the severity of the stroke, the sample size, and the period between the stroke onset and the blood sampling. Conclusion This systematic review and meta-analysis suggest that an elevated NLR, MLR, and PLR were associated with SAP, indicating that they could be promising blood-based biomarkers for predicting SAP. Large-scale prospective studies from various ethnicities are recommended to validate this association before they can be applied in clinical practice.

Background and Purpose The aim of this study was to explore the relationship between functional prognosis and the neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), lymphocyte-to-monocyte ratio (LMR) and systemic inflammatory response index (SIRI) in patients with acute ischemic stroke (AIS) at discharge. Methods A total of 861 patients with AIS were enrolled between January 2019 and December 2021. Blood cell counts were collected on admission. Logistic regression analysis was performed to assess the relationship between NLR, PLR, LMR, SIRI and adverse functional outcomes (modified Rankin scale score of 3–6) at discharge. We also used receiver operating characteristic (ROC) curves to estimate the overall ability of NLR, PLR, LMR and SIRI to judge short-term functional outcomes. Associations between NLR, PLR, LMR, and SIRI with length of hospital stay were analyzed by Spearman correlation test. Results A total of 194 patients (22.5%) had poor functional outcomes at discharge. Multivariate logistic regression analysis showed that NLR (odds ratio [OR], 1.060; 95% confidence interval [CI] 1.004–1.120, P=0.037), PLR (OR, 1.003; 95% CI 1.000–1.005, P=0.018), LMR (OR, 0.872; 95% CI 0.774–0.981, P=0.023) and SIRI (OR, 1.099; 95% CI 1.020–1.184, P=0.013) were independent factors for poor functional outcome. The odds ratios of the highest versus lowest quartiles of NLR, PLR and SIRI were 2.495 (95% CI 1.394–4.466), 1.959 (95% CI 1.138–3.373) and 1.866 (95% CI 1.106–3.146), respectively. The odds ratio of the lowest versus highest quartile of LMR was 2.300 (95% CI 1.331–3.975). The areas under the curve (AUCs) of the NLR, PLR, LMR, and SIRI to discriminate poor functional prognosis were 0.644, 0.587, 0.628, and 0.651, respectively. NLR, LMR, and SIRI were related with the length of hospital stay (P<0.05). Conclusion NLR, PLR, LMR, and SIRI were associated with functional outcome at discharge in AIS patients. NLR, LMR and SIRI were related to hospitalization days in patients with AIS.

BACKGROUND The neutrophil-to-lymphocyte ratio (NLR) and mean platelet volume (MPV) reflect systemic inflammation, which plays an important role in the process of treating ischemic strokes. Few studies have evaluated the association between blood biomarkers and clinical outcomes in ischemic strokes in intensive care units (ICUs). OBJECTIVES This retrospective study aims to explore the relationship between blood biomarkers and the clinical outcomes of acute ischemic stroke (AIS) patients. MATERIAL AND METHODS Basic descriptive statistics of the patients admitted to the ICU with the diagnosis of AIS according to sociodemographic, clinical and laboratory findings were collected. Receiver operating characteristic (ROC) curve analysis was used to determine the cutoff point for NLR and MPV variables based on the diagnosis in statistical analyses and crosstab analyses of variables. The χ2 and Fisher's exact tests were used to assess the statistical relationship between categorical variables. In addition, the odds ratio (OR) was utilized to show the strength of the relationship between the categorical NLR, MPV and modified Rankin Scale (mRS) variables. Finally, the Mann-Whitney U test was used to compare the medians of 2 independent groups. RESULTS A total of 1,379 records were identified in the database search. Eighty-seven patients who met the inclusion criteria and were hospitalized in the ICU were included in the study. The optimal cutoff point was determined to be 4.0 for NLR and 9.0 for the MPV. A statistically significant relationship was found between high medians of the NLR and the MPV and unfavorable functional outcomes using a 5% significance level (p < 0.001 and p < 0.001, respectively). CONCLUSIONS We showed that the NLR and MPV are associated with stroke severity, unfavorable functional outcomes and mortality in AIS. These findings provide new insights into the mechanisms and treatment strategies of AIS. The results show that these accessible values can be used as independent predictive biomarkers.

To investigate the association of neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), and lymphocyte to monocyte ratio (LMR) with post-thrombolysis early neurological outcomes including early neurological improvement (ENI) and early neurological deterioration (END) in patients with acute ischemic stroke (AIS). AIS patients undergoing intravenous thrombolysis were enrolled from April 2016 to September 2019. Blood cell counts were sampled before thrombolysis. Post-thrombolysis END was defined as the National Institutes of Health Stroke Scale (NIHSS) score increase of ≥ 4 within 24 h after thrombolysis. Post-thrombolysis ENI was defined as NIHSS score decrease of ≥ 4 or complete recovery within 24 h. Multinomial logistic regression analysis was performed to explore the relationship of NLR, PLR, and LMR to post-thrombolysis END and ENI. We also used receiver operating characteristic curve analysis to assess the discriminative ability of three ratios in predicting END and ENI. Among 1060 recruited patients, a total of 193 (18.2%) were diagnosed with END and 398 (37.5%) were diagnosed with ENI. Multinomial logistic model indicated that NLR (odds ratio [OR], 1.385; 95% confidence interval [CI] 1.238–1.551, P = 0.001), PLR (OR, 1.013; 95% CI 1.009–1.016, P = 0.001), and LMR (OR, 0.680; 95% CI 0.560–0.825, P = 0.001) were independent factors for post-thrombolysis END. Moreover, NLR (OR, 0.713; 95% CI 0.643–0.791, P = 0.001) served as an independent factor for post-thrombolysis ENI. Area under curve (AUC) of NLR, PLR, and LMR to discriminate END were 0.763, 0.703, and 0.551, respectively. AUC of NLR, PLR, and LMR to discriminate ENI were 0.695, 0.530, and 0.547, respectively. NLR, PLR, and LMR were associated with post-thrombolysis END. NLR and PLR may predict post-thrombolysis END. NLR was related to post-thrombolysis ENI.

Objective: Neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) have been emerging as the novel inflammatory biomarkers for determining the prognosis of various diseases. This study aimed to investigate the individual and joint effects of NLR and PLR on functional outcomes of acute ischemic stroke (AIS). Methods: Our study involved 448 eligible patients with first-ever AIS. Clinical and laboratory data were collected on admission within 72 h from stroke onset. Unfavorable functional outcome was defined as a modified Rankin Scale score of 3–6 at 3 months after AIS. Cox proportional hazard model and spline regression models was used to estimate the effect of NLR and PLR on risk of adverse outcomes after the last patient who completed a 3-months follow-up was enrolled. Results: After adjusting confounders, NLR were significantly associated with the unfavorable functional outcomes (P-trend < 0.001). So were PLR (P-trend < 0.001). NLR was discovered to have higher predictive value than PLR (AUC = 0.776, 95%CI = 0.727–0.825, P < 0.001; AUC = 0.697, 95%CI = 0.641–0.753, P < 0.001). The optimal cutoff values for NLR and PLR was 3.51 and 141.52, respectively. Stratified analysis performed by cox proportional hazard model showed that high level of NLR and PLR (NLR ≥ 3.51, PLR ≥ 141.52) presented the highest risk of unfavorable functional outcomes (adjusted HR, 3.77; 95% CI: 2.38–5.95; P < 0.001). Followed by single high level of NLR (adjusted HR, 2.32; 95% CI: 1.10–4.87; P = 0.027). Single high level of PLR (NLR < 3.51, PLR ≥ 141.52) also showed higher risk than low level of the combination, but it did not reach statistical significance (adjusted HR, 1.42; 95% CI: 0.75–2.70; P = 0.285). No obvious additive [relative excess risk due to interaction (RERI) not significant] or multiplicative (adjusted HR, 0.71; 95%CI: 0.46–1.09; P = 0.114) interaction was found between the effects of NLR and PLR on the risk of unfavorable functional outcomes. Conclusion: This study demonstrated that both NLR and PLR were independent predictors of 3-months functional outcomes of AIS. They may help to identify high-risk patients more forcefully when combined together.

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