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.

Methylene blue prevents osteoarthritis progression and relieves pain in rats via upregulation of Nrf2/PRDX1.

Oxidative stress-related cartilage degeneration, synovitis, and joint pain play vital roles in the progress of osteoarthritis (OA). Anti-oxidative stress agents not only prevent structural damage progression but also relieve OA-related pain. In this study, we investigated the therapeutic effect of methylene blue (MB), a classical and important anti-oxidant with strong neural affinity. Experimental OA was established in rats by radial transection of medial collateral ligament and medial meniscus (MCLT + MMT) of the right knee joint. The OA rats received intra-articular injection of MB (1 mg/kg) every week starting one week after surgery. We showed that MB administration exerted significant cartilage protection, synovitis inhibition as well as pain relief in OA rats. In human chondrocytes and fibroblast-like synoviocytes, MB significantly attenuated tert-butyl hydroperoxide (TBHP)-induced inflammatory response and oxidative stress. We demonstrated that these effects of MB resulted from dual targets of important antioxidant enzymes, Nrf2 and PRDX1, which also mutually reinforcing and participated in an interaction. Furthermore, we found that calcitonin gene-related peptide (CGRP), a neural inflammatory mediator, was accumulated around the vessel in synovium and subchondral bone in OA rats and in TBHP-treated primary cortical neurons; MB administration significantly inhibited CGRP expression through upregulation of Nrf2 and PRDX1. Taken together, these results suggest that MB ameliorates oxidative stress via Nrf2/PRDX1 regulation to prevent progression and relieve pain of OA.

Trehalose reduces bone loss in experimental biliary cirrhosis rats via ERK phosphorylation regulation by enhancing autophagosome formation.

Bone loss is a severe complication of primary biliary cirrhosis (PBC). Trehalose was intermittently administered in bile duct-ligated (BDL) male rats, a PBC-related osteoporosis model, for 4 weeks to reduce osteoporosis. Femoral bones were assessed ex vivo by micro computed tomography (CT) and histomorphometry. The potential mechanisms related to the reduction of osteoporosis were explored by evaluating the effect of trehalose on osteoblast autophagy, osteogenesis, osteoclastogenesis, and ERK phosphorylation. The results demonstrated that trehalose reduced osteoporosis of BDL rats and decreased osteoblast-mediated osteoclast differentiation by enhancing osteoblast autophagy to regulate osteoprotegerin (OPG) secretion. Hydroxychloroquine (HCQ) increased the expression of OPG and OPG/receptor activator genes for nuclear factor-κB ligand (RANKL) ratio, and reduced osteoblast-mediated osteoclastogenesis by inhibiting autophagy flux and inducing autophagosome formation. Furthermore, trehalose increased the phosphorylation of ERK1/2 in MC3T3-E1 cells, and the ERK inhibitor PD98059 reversed the upregulation of OPG gene and reduction of trehalose-induced osteoclastogeneis. The treatment with HCQ markedly increased the ERK phosphorylation. The correlation between autophagosome formation and ERK phosphorylation was confirmed in autophagy proteins (ATG) 4B or ATG5-deficient cells. Thus, trehalose could decrease osteoblast-mediated osteoclastogenesis and reduce PBC-related bone loss by regulating ERK phosphorylation via autophagosome formation.

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.

A C-Met chemical inhibitor promotes fracture healing through interacting with osteogenic differentiation via the mTORC1 pathway.

Currently, HGF/C-Met signaling inhibitors are being investigated to determine if they are useful for enhancing progenitor cell differentiation into osteoblasts, and one of them, BMS-777607, has been utilized to treat osteoporosis and bone loss in several types of diseases. However, whether BMS-777607 could be a potential treatment during fracture healing remains elusive. Here, we examined the therapeutic effects of BMS-777607 on bone fracture healing in a mouse model. In vivo radiological analysis showed that fractures treated with BMS-777607 exhibited accelerated osteotylus formation during the early stage of bone healing. Thereafter, the Safranin O staining evaluation indicated that the structure of the external callus in the Treatment group was larger than that in the Vehicle group at week 2. Furthermore, cellular proliferation of MC3T3-E1 was not significantly affected by low concentrations of BMS-777607. In addition, stimulation of osteoblast differentiation and mineralization was a result of BMS-777607 inducing the expression of Runx2 and Col1, and this osteogenic ability, at least in part, was mediated through the mammalian target of rapamycin complex 1 (mTORC1) signaling in vitro. Conclusively, BMS-777607 has been identified as a therapeutic agent to improve bone formation during fracture healing, and its osteogenic effects on osteoblast differentiation were mediated via the mTORC1 signaling pathway.

The effects of erythropoietin on neurogenesis after ischemic stroke.

Erythropoietin has been researched for its neuroprotective effects in ischemic stroke for over 30 years. Although erythropoietin can cause side effects that need to be controlled, it has been suggested to be effective in enhancing the prognosis of patients who are out of the therapeutic time window and have not received recombinant tissue plasminogen activator therapy. Studies on the mechanism of the function of erythropoietin have shown that it has various protective effects in ischemic brain injury after stroke, including promoting neurogenesis. In this review, we discuss the effects of erythropoietin on neurogenesis after ischemic brain injury and provide references for effective treatments for ischemic stroke, which is one of the leading causes of death worldwide.

The Chinese herb Fructus Broussonetiae aids learning and memory in chronic cerebral hypoperfusion by reducing proinflammatory microglia activation in rats.

The neuroprotective role of Fructus Broussonetiae in a model of chronic cerebral hypoperfusion with cognitive decline was focused on neural plasticity and microglia/macrophage polarization. Chronic cerebral hypoperfusion was induced by bilateral common carotid artery ligation. Fructus Broussonetiae shortened escape latency and added the number of platform crossings of rats, up-regulated the expression of synaptophysin in the gray matter and increased myelin basic protein expression in the white matter. Further mechanistic experiments were conducted to examine microglia activation and M1/M2 polarization. It was shown that Fructus Broussonetiae reduced the activation of microglia revealed by decreased expression of ionized calcium-binding adapter molecule-1, inhibited M1 polarization of microglia and improved microglial M2 polarization shown by down-regulated the expression of inducible nitric oxide synthase and Fc fragment of IgG receptor IIIa and up-regulated the expression of arginase-1. In conclusion, the Chinese herb Fructus Broussonetiae can improve cognitive function following chronic cerebral hypoperfusion by down-regulating the activation of microglia, inhibiting microglial M1 polarization, and improving neural plasticity.

Surface Engineering of Biodegradable Magnesium Alloys for Enhanced Orthopedic Implants.

Magnesium (Mg) alloys have been promised for biomedical implants in orthopedic field, however, the fast corrosion rate and mode challenge their clinical application. To push Mg alloys materials into practice, a composite coating with biodegradable and high compatible components to improve anticorrosion property of an Mg alloy (i.e., AZ31) is designed and fabricated. The inner layer is micro-nano structured Mg(OH)2 through hydrothermal treatment. Then stearic acid (SA) is introduced to modify Mg(OH)2 for better reducing the gap below a surface-degradation polymer layer of poly(1,3-trimethylene carbonate). Benefited by the SA modification effect, this sandwiched coating avoids corrosive medium penetration via enhancing the adhesion strength at the interface between outer and inner layers. Both in vitro and in vivo tests indicate that the composite coating modified AZ31 perform a better anticorrosion behavior and biocompatibility compared to bare AZ31. Strikingly, a 1.7-fold improvement in volume of newly formed bone is observed surrounding the composite coating modified implant after 12 week implantation. The sandwiched biocompatible coating strategy paves a hopeful way for future translational application of Mg alloys orthopedic materials in clinics.

L-3-n-Butylphthalide reduces ischemic stroke injury and increases M2 microglial polarization.

Overwhelming evidence suggests that microglia play an important role in ischemic injury and they polarize into two different phenotypes with distinct functions after ischemic stroke. We performed the present study to investigate whether L-3-n butylphthalide (NBP) has an effect on microglial polarization. Mice were subjected to transient middle cerebral artery occlusion (MCAO) for 45 min, and then immediately after reperfusion were treated with NBP or vehicle via the caudal vein for 7 consecutive days. 2,3,5-Triphenyltetrazolium chloride (TTC) staining showed that NBP treatment resulted in a tendency to decrease cerebral infarct volume at 1 day after MCAO, and significant decreased infarct volume at 3 days after MCAO. Sensorimotor function was evaluated by the adhesive removal test and balance beam test, which were superior in NBP-treated mice compared with vehicle-treated mice at 1 and 3 days after MCAO. Immunofluorescent staining further indicated that NBP treatment significantly increased the number of CD206+/Iba1+ M2 microglia/macrophages and reduced the number of CD16+/Iba1+ M1 cells at 3 and 7 days after MCAO reperfusion. Western blot also showed an elevation of M2 marker (arginase-1) in NBP-treated brains at 7 days after MCAO. In conclusion, our results clearly show that NBP treatment significantly mitigates ischemic brain damage and promotes recovery of neurological function in early phase after ischemic stroke, probably by skewing M1 microglia/macrophages polarization towards M2 phenotype. Thus, our study provides new evidence that NBP might be a promising candidate for ameliorating injury caused by ischemic stroke.

Long Noncoding RNA H19 Promotes Neuroinflammation in Ischemic Stroke by Driving Histone Deacetylase 1-Dependent M1 Microglial Polarization.

BACKGROUND AND PURPOSE: Long noncoding RNA H19 is repressed after birth, but can be induced by hypoxia. We aim to investigate the impact on and underlying mechanism of H19 induction after ischemic stroke. METHODS: Circulating H19 levels in stroke patients and mice subjected to middle cerebral artery occlusion were assessed using real-time polymerase chain reaction. H19 siRNA and histone deacetylase 1 (HDAC1) plasmid were used to knock down H19 and overexpress HDAC1, respectively. Microglial polarization and ischemic outcomes were assessed in middle cerebral artery occlusion mice and BV2 microglial cells subjected to oxygen-glucose deprivation. RESULTS: Circulating H19 levels were significantly higher in stroke patients compared with healthy controls, indicating high diagnostic sensitivity and specificity. Moreover, plasma H19 levels showed a positive correlation with National Institute of Health Stroke Scale score and tumor necrosis factor-α levels. After middle cerebral artery occlusion in mice, H19 levels increased in plasma, white blood cells, and brain. Intracerebroventricular injection of H19 siRNA reduced infarct volume and brain edema, decreased tumor necrosis factor-α and interleukin-1ß levels in brain tissue and plasma, and increased plasma interleukin-10 concentrations 24 hours poststroke. Additionally, H19 knockdown attenuated brain tissue loss and neurological deficits 14 days poststroke. BV2 cell-based experiments showed that H19 knockdown blocked oxygen-glucose deprivation-driven M1 microglial polarization, decreased production of tumor necrosis factor-α and CD11b, and increased the expression of Arg-1 and CD206. Furthermore, H19 knockdown reversed oxygen-glucose deprivation-induced upregulation of HDAC1 and downregulation of acetyl-histone H3 and acetyl-histone H4. In contrast, HDAC1 overexpression negated the effects of H19 knockdown. CONCLUSIONS: Our findings indicate that H19 promotes neuroinflammation by driving HDAC1-dependent M1 microglial polarization, suggesting a novel H19-based diagnosis and therapy for ischemic stroke.

Translocation of Rigid Rod-Shaped Virus through Various Solid-State Nanopores.

Nanopores have been used as a high throughput tool for characterizing individual biomolecules and nanoparticles. Here, we present the translocation of rigid rod-shaped tobacco mosaic virus (TMV) through solid-state nanopores. Interestingly, due to the high rigidity of TMV, three types of events with distinctive characteristics at the capture process and a strong current fluctuation during the translocation of TMV are observed. A kinetic model is then proposed to address the dynamics of the translocation, followed by corresponding dynamics simulations. The results reveal that TMV has to rotate to fit and pass the pore when it is captured by a nanopore with an angle larger than the maximum angle that allows it to pass through. Then, we investigate the dependence of the rotation of TMV on the conductance fluctuations at the blockade stage. The results show that the rotation of TMV during the passage through the pore affects the current signal significantly. This study gives a fundamental understanding of the dynamics of rod-shaped particles translocating through the nanopore and how the current responds to it. It opens a new possible way to characterize the rigidity of analytes by nanopores.

Error removal in microchip-synthesized DNA using immobilized MutS.

The development of economical de novo gene synthesis methods using microchip-synthesized oligonucleotides has been limited by their high error rates. In this study, a low-cost, effective and improved-throughput (up to 32 oligos per run) error-removal method using an immobilized cellulose column containing the mismatch binding protein MutS was produced to generate high-quality DNA from oligos, particularly microchip-synthesized oligonucleotides. Error-containing DNA in the initial material was specifically retained on the MutS-immobilized cellulose column (MICC), and error-depleted DNA in the eluate was collected for downstream gene assembly. Significantly, this method improved a population of synthetic enhanced green fluorescent protein (720 bp) clones from 0.93% to 83.22%, corresponding to a decrease in the error frequency of synthetic gene from 11.44/kb to 0.46/kb. In addition, a parallel multiplex MICC error-removal strategy was also evaluated in assembling 11 genes encoding ∼21 kb of DNA from 893 oligos. The error frequency was reduced by 21.59-fold (from 14.25/kb to 0.66/kb), resulting in a 24.48-fold increase in the percentage of error-free assembled fragments (from 3.23% to 79.07%). Furthermore, the standard MICC error-removal process could be completed within 1.5 h at a cost as low as $0.374 per MICC.

MicroRNA-424 protects against focal cerebral ischemia and reperfusion injury in mice by suppressing oxidative stress.

BACKGROUND AND PURPOSE: We previously showed that the microRNA miR-424 protects against permanent cerebral ischemic injury in mice by suppressing microglia activation. This study investigated the role of miR-424 in transient cerebral ischemia in mice with a focus on oxidative stress-induced neuronal injury. METHODS: Transient cerebral ischemia was induced in C57/BL6 mice by middle cerebral artery occlusion for 1 hour followed by reperfusion (ischemia/reperfusion). The miR-424 level in the peri-infarct cortex was quantified. Mice were also administered miR-424 angomir by intracerebroventricular injection. Cerebral infarct volume, neuronal apoptosis, and levels of oxidative stress markers and antioxidants were evaluated. In an in vitro experiment, primary cortical neurons were exposed to H2O2 and treated with miR-424 angomir, nuclear factor erythroid 2-related factor 2 siRNA, and superoxide dismutase (SOD) inhibitor; cell activity, lactate dehydrogenase release, malondialdehyde level, and manganese (Mn)SOD activity were then evaluated. RESULTS: MiR-424 levels in the peri-infarct cortex increased at 1 and 4 hours then decreased 24 hours after reperfusion. Treatment with miR-424 decreased infarct volume and inhibited neuronal apoptosis after ischemia/reperfusion, reduced reactive oxygen species and malondialdehyde levels in the cortex, and increased the expression and activation of MnSOD as well as the expression of extracellular SOD and the redox-sensitive transcription factor nuclear factor erythroid 2-related factor. In neuronal cultures, miR-424 treatment abrogated H2O2-induced injury, as evidenced by decreased lactate dehydrogenase leakage and malondialdehyde level and increased cell viability and MnSOD activity; the protective effects of miR-424 against oxidative stress were reversed by nuclear factor erythroid 2-related factor knockdown and SOD inhibitor treatment. CONCLUSIONS: MiR-424 protects against transient cerebral ischemia/reperfusion injury by inhibiting oxidative stress.

Intra-artery infusion of recombinant human erythropoietin reduces blood-brain barrier disruption in rats following cerebral ischemia and reperfusion.

OBJECTIVES: Intra-artery infusion of recombinant human erythropoietin (rhEPO) has recently been reported to confer neuroprotection against cerebral ischemia-reperfusion injury in animal models; however, the molecular mechanisms are still under investigation. The present study focused on the specific mechanism involved in blood-brain barrier (BBB) disruption. METHODS: Thirty-six male and nine female Sprague Dawley rats were subjected to middle cerebral artery (MCA) occlusion to induce focal cerebral ischemia, and administrated rhEPO at a dose of 800 U/kg through MCA infusion at the beginning of reperfusion. Neurobehavioral deficits, brain edema, and infarct volume were evaluated after 2 h of ischemia and 24 h of reperfusion. BBB permeability was assessed by quantifying the extravasation of Evans blue (EB) dye. The expression of tight junction proteins and matrix metalloproteinases (MMPs) (Claudin-5, Occludin, MMP-2, and MMP-9) in microvessels were detected by immunofluorescence and western blot. The activities of MMPs in the cerebral microvessels were determined by gelatin zymography. RESULTS: Treatment with rhEPO through the MCA strongly alleviated infarct volume, brain edema, and improved neurobehavioral outcomes in male and female rats. In addition, rhEPO remarkably suppressed the EB extravasation induced by brain ischemia. Furthermore, rhEPO prevented degradation of Claudin-5 and Occludin, and reduced the expression and activity of MMP-2 and MMP-9 in isolated brain microvessels. CONCLUSIONS: Treatment with rhEPO through MCA infusion prevented brain edema formation and infarction through inhibition of MMP-mediated BBB disruption in acute ischemic stroke.

Ischemic postconditioning relieves cerebral ischemia and reperfusion injury through activating T-LAK cell-originated protein kinase/protein kinase B pathway in rats.

BACKGROUND AND PURPOSE: Ischemic postconditioning (IPostC) protects against ischemic brain injury. To date, no study has examined the role of T-LAK-cell-originated protein kinase (TOPK) in IPostC-afforded neuroprotection. We explored the molecular mechanism related with TOPK in antioxidant effect of IPostC against ischemia/reperfusion. METHODS: Focal ischemia was induced in rats by transient middle cerebral artery occlusion. Reactive oxygen species production in the peri-infarct cortex was detected using dihydroethidium. Malondialdehyde, as a marker of lipid peroxidation, and 3-nitrotyrosine, as a marker of protein oxidation, were detected by ELISA. The expression or location of antioxidant proteins and signal molecules TOPK, phosphatase, and tensin homolog, and Akt was analyzed by Western blotting and immunofluorescence. RESULTS: Our results revealed that IPostC relieved transient middle cerebral artery occlusion-induced oxidative damage by reducing reactive oxygen species, malondialdehyde, and 3-nitrotyrosine accumulation in the peri-infarct cortex and raised levels of antioxidants perioxiredoxin-1, peroxiredoxin-2, and thioredoxin-1. In addition, IPostC increased p-AKT and p-TOPK levels, which colocalized in neural cells. In vitro TOPK knockdown by small interfering RNA decreased the levels of antioxidants peroxiredoxin-1, thioredoxin, and manganese superoxide dismutase activity in PC12 cells. In vivo intracerebroventricular injection of TOPK small interfering RNA reversed IPostC-induced neuroprotection by increasing infarct volume and nitric oxide content and reducing manganese superoxide dismutase activity. Moreover, IPostC-evoked Akt activation was blocked by TOPK small interfering RNA in vivo, but the decreased phosphorylated phosphatase and tensin homolog level in ischemia/reperfusion was not influenced by IPostC or by TOPK small interfering RNA treatment. CONCLUSIONS: Our results suggest that the antioxidative effects of TOPK/Akt might contribute to the neuroprotection of IPostC treatment against transient middle cerebral artery occlusion.

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.