Association of admission neutrophil serine proteinases levels with the outcomes of acute ischemic stroke: a prospective cohort study.

BACKGROUND: Neutrophil serine proteinases (NSPs), released by activated neutrophils, are key proteins involved in the pathophysiologic processes of stroke. NSPs are also implicated in the process and response of thrombolysis. This study aimed to analyze three NSPs (neutrophil elastase, cathepsin G, and proteinase 3) in relation to acute ischemic stroke (AIS) outcomes and in relation to the outcomes of patients treated with intravenous recombinant tissue plasminogen activator (IV-rtPA). METHODS: Among 736 patients prospectively recruited at the stroke center from 2018 to 2019, 342 patients diagnosed with confirmed AIS were included. Plasma neutrophil elastase (NE), cathepsin G (CTSG), and proteinase 3 (PR3) concentrations were measured on admission. The primary endpoint was unfavorable outcome defined as modified Rankin Scale score 3-6 at 3 months, and the secondary endpoints were symptomatic intracerebral hemorrhage (sICH) within 48 h, and mortality within 3 months. In the subgroup of patients who received IV-rtPA, post-thrombolysis early neurological improvement (ENI) (defined as National Institutes of Health Stroke Scale score = 0 or decrease of ≥ 4 within 24 h after thrombolysis) was also included as the secondary endpoint. Univariate and multivariate logistic regression analyses were performed to evaluate the association between NSPs levels and AIS outcomes. RESULTS: Higher NE and PR3 plasma levels were associated with the 3-month mortality and 3-month unfavorable outcome. Higher NE plasma levels were also associated with the risk of sICH after AIS. After adjusting for potential confounders, plasma NE level > 229.56 ng/mL (odds ratio [OR] = 4.478 [2.344-8.554]) and PR3 > 388.77 ng/mL (OR = 2.805 [1.504-5.231]) independently predicted the 3-month unfavorable outcome. Regarding rtPA treatment, patients with NE plasma concentration > 177.22 ng/mL (OR = 8.931 [2.330-34.238]) or PR3 > 388.77 ng/mL (OR = 4.275 [1.045-17.491]) were over 4 times more likely to suffer unfavorable outcomes after rtPA treatment. The addition of NE and PR3 to clinical predictors of unfavorable functional outcome after AIS and the outcome after rtPA treatment improved discrimination as well as reclassification (integrated discrimination improvement = 8.2% and 18.1%, continuous net reclassification improvement = 100.0% and 91.8%, respectively). CONCLUSIONS: Plasma NE and PR3 are novel and independent predictors of 3-month functional outcomes after AIS. Plasma NE and PR3 also possess predictive value to identify patients with unfavorable outcomes after rtPA treatment. NE is probably an important mediator of the effects of neutrophils on stroke outcomes, which worth further investigation.

LC-MS/MS metabolomic profiling of the protective butylphthalide effect in cerebral ischemia/reperfusion mice.

OBJECTIVES: This study was designed to investigate metabolic biomarker changes and related metabolic pathways of Butylphthalide (NBP) on cerebral ischemia/reperfusion. METHODS: In this study, a mouse cerebral ischemia/reperfusion (I/R) model was prepared using the middle cerebral artery occlusion method, and neurobehavioral score and 2, 3, 5-triphenyltetrazolium chloride (TTC) staining experiments were used to confirm the obvious NBP anti-cerebral ischemia effect. The protective effect of NBP in the mouse cerebral I/R model and its metabolic pathway and mechanism were investigated using mouse blood samples. RESULTS: The metabolic profiles of mice in the I/R+NBP, I/R, and sham groups were significantly different. Under the condition that I/R vs. sham was downregulated and I/R + NBP vs. I/R was upregulated, 88 differential metabolites, including estradiol, ubiquinone-2, 2-oxoarginine, and L-histidine trimethylbetaine, were screened and identified. The related metabolic pathways involved arginine and proline metabolism, oxidative phosphorylation, ubiquitin and other terpenoid-quinone biosynthesis, and estrogen signaling. CONCLUSIONS: Metabolomics was used to elucidate the NBP mechanism in cerebral ischemia treatment in mice, revealing synergistic NBP pharmacological characteristics with multiple targets.

Intravenous antagomiR-494 lessens brain-infiltrating neutrophils by increasing HDAC2-mediated repression of multiple MMPs in experimental stroke.

Neutrophil infiltration and phenotypic transformation are believed to contribute to neuronal damage in ischemic stroke. Emerging evidence suggests that histone deacetylase 2 (HDAC2) is an epigenetic regulator of inflammatory cells. Here, we aimed to investigate whether microRNA-494 (miR-494) affects HDAC2-mediated neutrophil infiltration and phenotypic shift. MiR-494 levels in neutrophils from acute ischemic stroke (AIS) patients were detected by real-time PCR. Chromatin Immunoprecipitation (ChIP)-Seq was performed to clarify which genes are the binding targets of HDAC2. Endothelial cells and cortical neurons were subjected to oxygen-glucose deprivation (OGD), transwell assay was conducted to examine neutrophil migration through endothelial cells, and neuronal injury was examined after stimulating with supernatant from antagomiR-494-treated neutrophils. C57BL/6J mice were subjected to transient middle cerebral artery occlusion (MCAO) and antagomiR-494 was injected through tail vein immediately after reperfusion, and neutrophil infiltration and phenotypic shift was examined. We found that the expression of miR-494 in neutrophils was significantly increased in AIS patients. HDAC2 targeted multiple matrix metalloproteinases (MMPs) and Fc-gamma receptor III (CD16) genes in neutrophils of AIS patients. Furthermore, antagomiR-494 repressed expression of multiple MMPs genes, including MMP7, MMP10, MMP13, and MMP16, which reduced the number of brain-infiltrating neutrophils by regulating HDAC2. AntagomiR-494 could also exert its neuroprotective role through inhibiting the shift of neutrophils toward pro-inflammatory N1 phenotype in vivo and in vitro. Taken together, miR-494 may serve as an alternative predictive biomarker of the outcome of AIS patients, and antagomiR-494 treatment decreases the expression of multiple MMPs and the infiltration of neutrophils and inhibits the shift of neutrophils into N1 phenotype partly by targeting HDAC2.

sLOX-1: A Molecule for Evaluating the Prognosis of Recurrent Ischemic Stroke.

Several clinical parameters and biomarkers have been proposed as prognostic markers for stroke. However, it has not been clarified whether the risk factors affecting the prognosis of patients with recurrent and first-ever stroke are similar. In this study, we aimed to explore the relationship between soluble lectin-like oxidized low-density lipoprotein receptor 1 (sLOX-1) levels and the prediction of the functional outcome in patients with recurrent and first-ever stroke. A total of 266 patients with recurrent and first-ever stroke, who underwent follow-up for 3 months, were included in this study. Plasma samples were collected within 24 h after onset. The results showed that biomarkers for the prognosis of patients with recurrent stroke were different from that of those with first-ever stroke. sLOX-1 levels were correlated with modified Rankin Scale scores of patients with recurrent stroke alone (r = 0.3232, p = 0.001). sLOX-1 levels were also associated with an increased risk of unfavorable outcomes in patients with recurrent stroke with an adjusted odds ratio of 1.489 (95% confidence interval, 1.204-1.842, p < 0.0001). Combining the risk factors showed greater accuracy for prognosis, yielding a sensitivity of 93.2% and a specificity of 75%, with an area under the curve of 0.916, evaluated by the receiver operating characteristic curve. These findings suggest that the diagnosis and prognosis are different between patients with recurrent stroke and those with first-ever stroke, and sLOX-1 level is an independent prognostic marker in patients with recurrent stroke.

Prognostic significance of plasma IL-2 and sIL-2Rα in patients with first-ever ischaemic stroke.

BACKGROUND: An imbalance between circulating neuroprotective and neurotoxic T cell subsets leads to poor prognosis in acute ischaemic stroke (AIS). Preclinical studies have indicated that the soluble form of the interleukin-2 receptor α (sIL-2Rα)-IL-2 complex regulates T cell differentiation. However, the association between sIL-2Rα levels and AIS remains unclear. METHODS: A total of 201 first-ever AIS patients within 24 h after stroke onset and 76 control subjects were recruited. The National Institutes of Health Stroke Scale (NIHSS) score and 3-month functional outcome (modified Rankin Scale [mRS] score) at admission were assessed. Plasma sIL-2Rα and IL-2 levels at admission were measured. Prognostic significance was identified by using univariate and multivariate logistic regression analyses. RESULTS: Patients with poor functional outcomes at 3 months had significantly higher levels of sIL-2Rα and lower levels of IL-2 than patients with good outcomes. Moreover, sIL-2Rα levels showed a strong positive correlation with NIHSS and mRS scores (p < 0.0001), whereas IL-2 levels were negatively correlated with mRS scores (p < 0.01). Univariate analyses showed that higher sIL-2Rα and IL-2 levels were associated with an increased and reduced risk of unfavourable outcomes, respectively. After adjusting for confounding variables, the sIL-2Rα level remained independently associated with an increased risk of an unfavourable outcome, and adding sIL-2Rα levels to the conventional risk factor model significantly improved risk reclassification (net reclassification improvement 17.56%, p = 0.003; integrated discrimination improvement 5.78%, p = 0.0003). CONCLUSIONS: sIL-2Rα levels represent a novel, independent prognostic marker that can improve the currently used risk stratification of AIS patients. Our findings also highlight that elevated plasma sIL-2Rα and IL-2 levels manifested opposite correlations with functional outcome, underlining the importance of IL-2/IL-2R autocrine loops in AIS.

[Establish and use of an epilepsy model in larval zebrafish].

Epilepsy is a kind of neurogenic diseases with high prevalence and characterized by seizure, brain paradoxical discharge and convulsion in spontaneous, transient, recurrent and uncontrolled manner. Development of new anti-epilepsy drugs requires a new reliable and high-performance animal models in screening of leading compounds. In this study, an epilepsy model in larval zebrafish was established using pentylenetetrazole (PTZ) compound. The results show that PTZ induced epilepsy-like seizure behavior such as irregular circular swimming, exciting locomotion, high swim velocity and convulsion in zebrafish. Expression patterns of two epilepsy-related gene c-fos and lgi1 were analyzed using RT-PCR and in situ hybridization; c-fos was enhanced and extended and lgi1 expression was reduced in PTZ concentration-dependent in the larval brain. When the model larvae exposed to anticonvulsant valproate(VPA), the epilepsy-like symptom decreased or disappeared, the marker genes c-fos and lgi1, as well as NeuN protein recovered to the normal levels. These responses to PTZ and to antiepileptic drug VPA are consistent with the observations in clinical studies and mouse models. Using this model, we evaluated anti-epilepsy activity of compounds Y53 and BMT, two homolog of berberine. The results show that the model larvae seizure triggered by lighting was partly remedied by Y53; and the larval exciting locomotion under the condition of no stimulation was suppressed by BMT. The findings indicate that the zebrafish larval epilepsy model is able to distinguish compounds with different activities in eleptiform seizure. We conclude that the zebrafish epilepsy model may be as a reliable and useful platform in screening of new anti-epilepsy candidates, which is suitable for basic research in epilepsy pathogenesis.

Prognostic Significance of Plasma VEGFA and VEGFR2 in Acute Ischemic Stroke-a Prospective Cohort Study.

Enhancement of vascular remodeling in affected brain tissue is a novel therapy for acute ischemic stroke (AIS). However, conclusions regarding angiogenesis after AIS remain ambiguous. Vascular endothelial growth factor A (VEGFA) and VEGF receptor 2 (VEGFR2) are potent regulators of angiogenesis and vascular permeability. We aimed to investigate the association between VEGFA/VEGFR2 expression in the acute stage of stroke and prognosis of patients with AIS. We enrolled 120 patients with AIS within 24 h of stroke onset and 26 healthy controls. Plasma levels of VEGFA and VEGFR2 were measured by enzyme-linked immunosorbent assay (ELISA). The primary endpoint was an unfavorable outcome defined as a modified Rankin Scale (mRS) score > 2 at 3 months after AIS. Univariate and multivariate logistic regression analyses were used to identify risk factors affecting prognosis. Plasma VEGFA and VEGFR2 were significantly higher in patients with AIS than in health controls, and also significantly higher in patients with unfavorable than those with favorable outcomes. Moreover, both VEGFA and VEGFR2 showed a significantly positive correlation with mRS at 3 months. Univariate and multivariate analyses showed VEGFA and VEGFR2 remained associated with unfavorable outcomes, and adding VEGFA and VEGFR2 to the clinical model significantly improved risk reclassification (continuous net reclassification improvement, 105.71%; integrated discrimination improvement, 23.45%). The new risk model curve exhibited a good fit with an area under the receiver operating characteristic curve (ROC) curve of 0.9166 (0.8658-0.9674). Plasma VEGFA and VEGFR2 are potential markers for predicting prognosis; thus these two plasma biomarkers may improve risk stratification in patients with AIS.

Neutrophil migration participates in the side effect of recombinant human tissue plasminogen activator.

AIMS: Ischemic stroke remains a challenge in medical research because of the limited treatment options. Recombinant human tissue plasminogen activator (rtPA) is the primary treatment for recanalization. However, nearly 50% of the patients experience complications that result in ineffective reperfusion. The precise factors contributing to ineffective reperfusion remain unclear; however, recent studies have suggested that immune cells, notably neutrophils, may influence the outcome of rtPA thrombolysis via mechanisms such as the formation of neutrophil extracellular traps. This study aimed to explore the nonthrombolytic effects of rtPA on neutrophils and highlight their contribution to ineffective reperfusion. METHODS: We evaluated the effects of rtPA treatment on middle cerebral artery occlusion in rats. We also assessed neutrophil infiltration and activation after rtPA treatment in vitro and in vivo in a small cohort of patients with massive cerebral ischemia (MCI). RESULTS: rtPA increased neutrophil infiltration into the brain microvessels and worsened blood-brain barrier damage during ischemia. It also increased the neutrophil counts of the patients with MCI. CONCLUSION: Neutrophils play a crucial role in promoting ischemic injury and blood-brain barrier disruption, making them potential therapeutic targets.

Alteration of N6-methyladenosine modification profiles in the neutrophilic RNA following ischemic stroke.

BACKGROUND: N6-methyladenosine (m6A) is one of the most extensive RNA methylation modifications in eukaryotes and participates in the pathogenesis of numerous diseases including ischemic stroke. Peripheral blood neutrophils are forerunners after ischemic brain injury and exert crucial functions. This study aims to explore the transcriptional profiles of m6A modification in neutrophils of patients with ischemic stroke. RESULTS: We found that the expression levels of m6A regulators FTO and YTHDC1 were notably decreased in the neutrophils following ischemic stroke, and FTO expression was negatively correlated with neutrophil counts and neutrophil-to-lymphocyte ratio (NLR). The m6A mRNA&lncRNA epigenetic transcriptome microarray identified 416 significantly upregulated and 500 significantly downregulated mRNA peaks in neutrophils of ischemic stroke patients. Moreover, 48 mRNAs and 18 lncRNAs were hypermethylated, and 115 mRNAs and 29 lncRNAs were hypomethylated after cerebral ischemia. Gene ontology (GO) analyses identified that these m6A-modified mRNAs were primarily enriched in calcium ion transport, long-term synaptic potentiation, and base-excision repair. The signaling pathways involved were EGFR tyrosine kinase inhibitor resistance, ErbB, and base excision repair signaling pathway. MeRIP-qPCR validation results showed that NRG1 and GDPD1 were significantly hypermethylated, and LIG1, CHRND, lncRNA RP11-442J17.2, and lncRNA RP11-600P1.2 were significantly hypomethylated after cerebral ischemia. Moreover, the expression levels of major m6A regulators Mettl3, Fto, Ythdf1, and Ythdf3 were obviously declined in the brain and leukocytes of post-stroke mouse models. CONCLUSION: This study explored the RNA m6A methylation pattern in the neutrophils of ischemic stroke patients, indicating that it is an intervention target of epigenetic regulation in ischemic stroke.

Dihydroergotamine protects against ischemic stroke by modulating microglial/macrophage polarization and inhibiting inflammation in mice.

OBJECTIVES: The search for drugs that can protect the brain tissue and reduce nerve damage in acute ischemic stroke has emerged as a research hotspot. We investigated the potential protective effects and mechanisms of action of dihydroergotamine against ischemic stroke. METHODS: C57BL/6 mice were subjected to middle cerebral artery occlusion (MCAO), and dihydroergotamine at a dose of 10 mg/kg/day was intraperitoneally injected for 14 days. Adhesive removal and beam walking tests were conducted 1, 3, 5, 7, 10, and 14 days after MCAO surgery. Thereafter, the mechanism by which dihydroergotamine regulates microglia/macrophage polarization and inflammation and imparts ischemic stroke protection was studied using enzyme-linked immunosorbent assay, immunofluorescence staining, and western blotting. RESULTS: From the perspective of a drug repurposing strategy, dihydroergotamine was found to inhibit oxygen-glucose deprivation damage to neurons, significantly improve cell survival rate, and likely exert a protective effect on ischemic brain injury. Dihydroergotamine significantly improved neural function scores and survival rates and reduced brain injury severity in mice. Furthermore, dihydroergotamine manifests its protective effect on ischemic brain injury by reducing the expression of TNF-α and IL-1ß in mouse ischemic brain tissue, inhibiting the polarization of microglia/macrophage toward the M1 phenotype and promoting polarization toward the M2 phenotype. CONCLUSION: This study is the first to demonstrate the protective effect of dihydroergotamine, a first-line treatment for migraine, against ischemic nerve injury in vitro and in vivo.

Annexin A6 mitigates neurological deficit in ischemia/reperfusion injury by promoting synaptic plasticity.

AIMS: Alleviating neurological dysfunction caused by acute ischemic stroke (AIS) remains intractable. Given Annexin A6 (ANXA6)'s potential in promoting axon branching and repairing cell membranes, the study aimed to explore ANXA6's potential in alleviating AIS-induced neurological dysfunction. METHODS: A mouse middle cerebral artery occlusion model was established. Brain and plasma ANXA6 levels were detected at different timepoints post ischemia/reperfusion (I/R). We overexpressed and down-regulated brain ANXA6 and evaluated infarction volume, neurological function, and synaptic plasticity-related proteins post I/R. Plasma ANXA6 levels were measured in patients with AIS and healthy controls, investigating ANXA6 expression's clinical significance. RESULTS: Brain ANXA6 levels initially decreased, gradually returning to normal post I/R; plasma ANXA6 levels showed an opposite trend. ANXA6 overexpression significantly decreased the modified neurological severity score (p = 0.0109) 1 day post I/R and the infarction area at 1 day (p = 0.0008) and 7 day (p = 0.0013) post I/R, and vice versa. ANXA6 positively influenced synaptic plasticity, upregulating synaptophysin (p = 0.006), myelin basic protein (p = 0.010), neuroligin (p = 0.078), and tropomyosin-related kinase B (p = 0.150). Plasma ANXA6 levels were higher in patients with AIS (1.969 [1.228-3.086]) compared to healthy controls (1.249 [0.757-2.226]) (p < 0.001), that served as an independent risk factor for poor AIS outcomes (2.120 [1.563-3.023], p < 0.001). CONCLUSIONS: This study is the first to suggest that ANXA6 enhances synaptic plasticity and protects against transient cerebral ischemia.

Erythropoietin-derived peptide ARA290 mediates brain tissue protection through the ß-common receptor in mice with cerebral ischemic stroke.

AIM: To explore the neuroprotective effects of ARA290 and the role of ß-common receptor (ßCR) in a mouse model of middle cerebral artery occlusion (MCAO). METHODS: This study included male C57BL/6J mice that underwent MCAO and reperfusion. The neuroprotective effect of ARA290 on MCAO-induced brain injury was investigated using neurological function tests (Longa and modified neurological severity score). Cerebral infarction was examined by 2, 3, 5-triphenyl tetrazolium chloride staining, neuronal apoptosis was assessed by immunofluorescence staining, blood parameters were measured using a flow cytometry-based automated hematology analyzer, liquid chromatography with tandem mass spectrometry was used to identify the serum metabolomics signature, inflammatory cytokines and liver index were detected by commercially available kits, and the protein levels of the erythropoietin (EPO) receptor and ßCR were measured by western blot. RESULTS: ARA290 exerted a qualitatively similar neuroprotective effect after MCAO as EPO. ARA290 significantly reduced neuronal apoptosis and the level of inflammatory cytokines in the brain tissue. However, ARA290's neuroprotective effect was significantly suppressed following the injection of siRNA against ßCR. CONCLUSION: ARA290 provided a neuroprotective effect via ßCR in cerebral ischemic mice without causing erythropoiesis. This study provides novel insights into the role of ARA290 in ischemic stroke intervention.

Is the ß Common Receptor the Key Molecule for the Protective Effect of Erythropoietin?

Erythropoietin is generally assumed to have protective effects against multiple diseases, especially ischemic stroke, and myocardial infarctions. The theory behind Erythropoietin's (EPO) protective effects has been misconstrued in the scientific community to a degree, with assumptions made that the ß common receptor (ßcR) in the heteroreceptor EPO receptor (EPOR)/ßcR is responsible for these protective effects. Our purpose with this opinion article is to convey our concern for the general assumption of the importance of ßcR in EPO's protective effect and to emphasize the necessity of further research in this field.

Autophagic degradation of LOX-1 is involved in the maintenance of vascular integrity injured by oxLDL and protected by Berberine.

Damage to vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) caused by oxidized low-density lipoprotein (oxLDL) contributes to cardiovascular and cerebrovascular diseases. Protection effects of Berberine (BBR) on the cardiovascular system have been reported, however, the molecular mechanism of vascular protection is still unclear. In this study, we established two hyperlipidemia models in zebrafish and VEC-VSMC co-culture using high-cholesterol food (HCF) and oxLDL, respectively. We demonstrated that HCF doubled total cholesterol and total glyceride levels, and BBR decreased these indices in a concentration-dependent manner. Lipid staining and hematoxylin-eosin staining revealed that BBR inhibited oxLDL-induced VSMC bulge-like proliferation and migration toward VECs and prevented the HCF-induced trunk vascular obstruction in zebrafish. Immunoblot analysis, cell immunofluorescence, co-immunoprecipitation assays, and transmission electron microscopy showed that oxLDL/HCF increased lectin-like oxLDL receptor-1 (LOX-1) expression at least 5-fold and significantly inhibited autophagolysosome formation in the blood vessel cells and in zebrafish. These observations were associated with endothelial-to-mesenchymal transition (EMT) in VECs and triggered VE-cadherin ectopic expression in VSMCs, and they were responsible for aberrant VSMC migration and vascular occlusion. However, BBR, by promoting autolysosome formation and degradation of LOX-1, reversed the above events and maintained intracellular homeostasis of vessel cells and vascular integrity. In conclusion, regulation of autophagy may be an effective approach to treating oxLDL-induced cardiovascular diseases by reducing LOX-1 protein level. BBR can protect blood vessels by adjusting the oxLDL-LOX-1-EMT-autophagy axis. This study is a step toward the development of new applications of BBR.

Untargeted metabolomics uncovering neuroprotective effect of Dl-3-n-butylphthalide on improving cognitive impairment induced by chronic cerebral hypoperfusion in rats.

Chronic cerebral hypoperfusion (CCH) can cause cognitive impairments. Dl-3-n-butylphthalide (NBP) is widely used in neurological disorders; but, the role of NBP in CCH remains unclear. This study aimed to investigate the potential mechanism of NBP on CCH through untargeted metabolomics. Animals were divided into CCH, Sham, and NBP groups. A rat model of bilateral carotid artery ligation was used to simulate CCH. Cognitive function of the rats was assessed using the Morris water maze test. Additionally, we used LC-MS/MS to detect ionic intensities of metabolites between the three groups for off-target metabolism analysis and to screen for differential metabolites. The analysis showed an improvement in cognitive function in rats after NBP treatment. Moreover, metabolomic studies showed that the serum metabolic profiles of the Sham and CCH groups were significantly altered, and 33 metabolites were identified as potential biomarkers associated with the effects of NBP. These metabolites were enriched in 24 metabolic pathways.And the pathway of differential metabolite enrichment was further verified by immunofluorescence. Thus, the study provides a theoretical basis for the pathogenesis of CCH and the treatment of CCH by NBP, and supports a wider application of NBP drugs.

Explore the role of long noncoding RNAs and mRNAs in intracranial atherosclerotic stenosis: From the perspective of neutrophils.

CONTEXT: Circulating neutrophils and long noncoding RNAs (lncRNAs) play various roles in intracranial atherosclerotic stenosis (ICAS). OBJECTIVE: Our study aimed to detect differentially expressed (DE) lncRNAs and mRNAs in circulating neutrophils and explore the pathogenesis of atherosclerosis from the perspective of neutrophils. METHODS: Nineteen patients with ICAS and 15 healthy controls were enrolled. The peripheral blood of the participants was collected, and neutrophils were separated. The expression profiles of lncRNAs and mRNAs in neutrophils from five patients and five healthy controls were obtained, and DE lncRNAs and mRNAs were selected. Six lncRNAs were selected and validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and ceRNA and lncRNA-RNA binding protein (RBP)-mRNA networks were constructed. Correlation analysis between lncRNAs and mRNAs was performed. Functional enrichment annotations were also performed. RESULTS: Volcano plots and heat maps displayed the expression profiles and DE lncRNAs and mRNAs, respectively. The qRT-PCR results revealed that the four lncRNAs showed a tendency consistent with the expression profile, with statistical significance. The ceRNA network revealed three pairs of regulatory networks: lncRNA RP3-406A7.3-NAGLU, lncRNA HOTAIRM1-MVK/IL-25/GBF1/CNOT4/ANKK1/PLEKHG6, and lncRNA RP11-701H16.4-ZNF416. The lncRNA-RBP-mRNA network showed five pairs of regulatory networks: lncRNA RP11-701H16.4-TEK, lncRNA RP11-701H16.4-MED17, lncRNA SNHG19-NADH-ubiquinone oxidoreductase core subunit V1, lncRNA RP3-406A7.3-Angel1, and lncRNA HOTAIRM1-CARD16. CONCLUSIONS: Our study identified and verified four lncRNAs in neutrophils derived from peripheral blood, which may explain the transcriptional alteration of neutrophils during the pathophysiological process of ICAS. Our results provide insights for research related to the pathogenic mechanisms and drug design of ICAS.

Inhibition of the JAK2/STAT3 pathway and cell cycle re-entry contribute to the protective effect of remote ischemic pre-conditioning of rat hindlimbs on cerebral ischemia/reperfusion injury.

AIMS: Remote ischemic pre-conditioning (RIPC) protects against ischemia/reperfusion (I/R) injury. However, the mechanisms underlying this protection remain unclear. In the present study, we investigated the role of Janus-activated kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway and cell cycle arrest, and their relationship with neuronal apoptosis following RIPC. METHODS: A rat cerebral I/R injury model was induced by middle cerebral artery occlusion (MCAO), and AG490 was used to investigate the mechanisms of RIPC. p-JAK2-, p-STAT3-, cyclin D1-, and cyclin-dependent kinase 6 (CDK6) expression was assessed by Western blotting and immunofluorescence staining. RESULTS: RIPC reduced the infarct volume, improved neurological function, and increased neuronal survival. Furthermore, p-JAK2 and p-STAT3 were detected during the initial phase of reperfusion; the expression levels were significantly increased at 3 and 24 h after reperfusion and were suppressed by RIPC. Additionally, the MCAO-induced upregulation of the cell cycle regulators cyclin D1 and CDK6 was ameliorated by RIPC. Meanwhile, cyclin D1 and CDK6 were colocalized with p-STAT3 in the ischemic brain. CONCLUSION: RIPC ameliorates the induction of the JAK2/STAT3 pathway and cell cycle regulators cyclin D1 and CDK6 by MCAO, and this net inhibition of cell cycle re-entry by RIPC is associated with downregulation of STAT3 phosphorylation.

MicroRNA-193a-5p Rescues Ischemic Cerebral Injury by Restoring N2-Like Neutrophil Subsets.

Circulating neutrophils are activated shortly after stroke and in turn affect the fate of ischemic brain tissue, and microRNAs (miRNA) participate in regulating neuroinflammation. We probed the role of neutrophilic miRNA in ischemic stroke. miR-193a-5p was decreased in circulating neutrophils of acute ischemic stroke (AIS) patients and healthy controls. In another set of AIS patients treated with recombinant tissue plasminogen activator, higher neutrophilic miR-193a-5p levels were associated with favorable outcomes at 3 months and non-symptomatic intracerebral hemorrhage. An experimental stroke model and human neutrophil-like HL-60 cells were further transfected with agomiR-193a-5p/antagomiR-193a-5p or ubiquitin-conjugating enzyme V2 (UBE2V2)-siRNA prior to model induction for in vivo and in vitro studies. Results of 2,3,5-triphenyl tetrazolium chloride staining and neurological function evaluations at post-experimental stroke showed that intravenous agomiR-193a-5p transfusion protected against ischemic cerebral injury in the acute stage and promoted neurological recovery in the subacute stage. This protective role was suggested to correlate with neutrophil N2 transformation based on the N2-like neutrophil proportions in the bone marrow, peripheral blood, and spleen of the experimental stroke model and the measurement of neutrophil phenotype-associated molecule levels. Mechanistically, analyses indicated that UBE2V2 might be a target of miR-193a-5p. Cerebral injury and neuroinflammation aggravated by miR-193a-5p inhibition were reversed by UBE2V2 silencing. In conclusion, miR-193a-5p protects against cerebral ischemic injury by restoring neutrophil N2 phenotype-associated neuroinflammation suppression, likely, in part, via UBE2V2 induction.

CCA repair or ECA ligation-Which middle cerebral artery occlusion is better in the reperfusion mouse model?

A reliable animal model is essential for ischemic stroke research. The implications of the external carotid artery (ECA) transection or common carotid artery (CCA) ligation have been described. Thus, a modified animal model, the CCA-repair model, has been established, and studies have shown that the CCA-repair model has potential advantages over the CCA-ligation model. However, whether the CCA-repair model is superior to the ECA-ligation model remains unclear. Sixty male C57BL/6 mice were randomly assigned to establish the CCA-repair (n = 34) or ECA-ligation (n = 26) models. Cerebral blood flow before middle cerebral artery occlusion (MCAO), immediately after MCAO and reperfusion were monitored and the operation duration, postoperative body weight, and food intake within 7 days, and the number of intraoperative and postoperative deaths within 7 days were recorded in the two models. Modified neurological severity scores and Bederson (0-5) scores were used to evaluate postoperative neurological function deficits on Days 1/3/5/7. 2,3,5-Triphenyltetrazolium chloride staining was used to quantify lesion volume on Day 7 after the operation. We found the establishment of the CCA-repair model required a longer total operation duration (p = 0.0175), especially the operation duration of reperfusion (p < 0.0001). However, there was no significant difference in body weight and food intake development, lesion volume and intragroup variability, neurological function deficits, mortality, and survival probability between the two groups. The CCA-repair model has no significant advantage over the ECA-ligation model. The ECA-ligation model is still a better choice for focal cerebral ischemia.

Nrf2 Pathway and Autophagy Crosstalk: New Insights into Therapeutic Strategies for Ischemic Cerebral Vascular Diseases.

Cerebrovascular disease is highly prevalent and has a complex etiology and variable pathophysiological activities. It thus poses a serious threat to human life and health. Currently, pathophysiological research on cerebrovascular diseases is gradually improving, and oxidative stress and autophagy have been identified as important pathophysiological activities that are gradually attracting increasing attention. Many studies have found some effects of oxidative stress and autophagy on cerebrovascular diseases, and studies on the crosstalk between the two in cerebrovascular diseases have made modest progress. However, further, more detailed studies are needed to determine the specific mechanisms. This review discusses nuclear factor erythroid 2-related factor 2 (Nrf2) molecules, which are closely associated with oxidative stress and autophagy, and the crosstalk between them, with the aim of providing clues for studying the two important pathophysiological changes and their crosstalk in cerebrovascular diseases as well as exploring new target treatments.