Nafamostat mesilate attenuates neuronal damage in a rat model of transient focal cerebral ischemia through thrombin inhibition.
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ABSTRACT:
SUBMITTER: Chen T
PROVIDER: S-EPMC4078306 | biostudies-other | 2014
REPOSITORIES: biostudies-other
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Project description:The serine proteases tissue plasminogen activator, plasmin, and thrombin and their receptors have previously been suggested to contribute to neuronal damage in certain pathological situations. Here we demonstrate that mice lacking protease-activated receptor 1 (PAR1) have a 3.1-fold reduction in infarct volume after transient focal cerebral ischemia. Intracerebroventricular injection of PAR1 antagonist BMS-200261 reduced infarct volume 2.7-fold. There are no detectable differences between PAR1-/- and WT mice in cerebrovascular anatomy, capillary density, or capillary diameter, demonstrating that the neuroprotective phenotype is not likely related to congenital abnormalities in vascular development. We also show that the exogenously applied serine proteases thrombin, plasmin, and tissue plasminogen activator can activate PAR1 signaling in brain tissue. These data together suggest that if blood-derived serine proteases that enter brain tissue in ischemic situations can activate PAR1, this sequence of events may contribute to the harmful effects observed. Furthermore, PAR1 immunoreactivity is present in human brain, suggesting that inhibition of PAR1 may provide a novel potential therapeutic strategy for decreasing neuronal damage associated with ischemia and blood-brain barrier breakdown.
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Project description:ObjectiveRho-associated kinase (ROCK) is a key regulator of numerous processes in multiple cell types relevant in stroke pathophysiology. ROCK inhibitors have improved outcome in experimental models of acute ischemic or hemorrhagic stroke. However, the relevant ROCK isoform (ROCK1 or ROCK2) in acute stroke is not known.MethodsWe characterized the pharmacodynamic and pharmacokinetic profile, and tested the efficacy and safety of a novel selective ROCK2 inhibitor KD025 (formerly SLx-2119) in focal cerebral ischemia models in mice.ResultsKD025 dose-dependently reduced infarct volume after transient middle cerebral artery occlusion. The therapeutic window was at least 3 hours from stroke onset, and the efficacy was sustained for at least 4 weeks. KD025 was at least as efficacious in aged, diabetic or female mice, as in normal adult males. Concurrent treatment with atorvastatin was safe, but not additive or synergistic. KD025 was also safe in a permanent ischemia model, albeit with diminished efficacy. As one mechanism of protection, KD025 improved cortical perfusion in a distal middle cerebral artery occlusion model, implicating enhanced collateral flow. Unlike isoform-nonselective ROCK inhibitors, KD025 did not cause significant hypotension, a dose-limiting side effect in acute ischemic stroke.InterpretationAltogether, these data show that KD025 is efficacious and safe in acute focal cerebral ischemia in mice, implicating ROCK2 as the relevant isoform in acute ischemic stroke. Data suggest that selective ROCK2 inhibition has a favorable safety profile to facilitate clinical translation.
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Project description:Whether the effect of miR-181a is sexually dimorphic in stroke is unknown. Prior work showed protection of male mice with miR-181a inhibition. Estrogen receptor-? (ER?) is an identified target of miR181 in endometrium. Therefore we investigated the separate and joint effects of miR-181a inhibition and 17?-estradiol (E2) replacement after ovariectomy. Adult female mice were ovariectomized and implanted with an E2- or vehicle-containing capsule for 14d prior to 1h middle cerebral artery occlusion (MCAO). Each group received either miR-181a antagomir or mismatch control by intracerebroventricular injection 24h before MCAO. After MCAO neurologic deficit and infarct volume were assessed. Primary male and female astrocyte cultures were subjected to glucose deprivation with miR-181a inhibitor or transfection control, and E2 or vehicle control, with/without ESR? knockdown with small interfering RNA. Cell death was assessed by propidium iodide staining, and lactate dehydrogenase assay. A miR-181a/ER? target site blocker (TSB), with/without miR-181a mimic, was used to confirm targeting of ER? by miR-181a in astrocytes. Individually, miR-181a inhibition or E2 decreased infarct volume and improved neurologic score in female mice, and protected male and female astrocyte cultures. Combined miR-181a inhibition plus E2 afforded greater protection of female mice and female astrocyte cultures, but not in male astrocyte cultures. MiR-181a inhibition only increased ER? levels in vivo and in female cultures, while ER? knockdown with siRNA increased cell death in both sexes. Treatment with ER? TSB was strongly protective in both sexes. In conclusion, the results of the present study suggest miR-181a inhibition enhances E2-mediated stroke protection in females in part by augmenting ER? production, a mechanism detected in female mice and female astrocytes. Sex differences were observed with combined miR-181a inhibition/E2 treatment, and miR-181a targeting of ER?.
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Project description:MicroRNAs (miRNAs) are approximately 22 nucleotides long, noncoding RNAs that control cellular function by either degrading mRNAs or arresting their translation. To understand their functional significance in ischemic pathophysiology, we profiled miRNAs in adult rat brain as a function of reperfusion time after transient middle cerebral artery occlusion. Of the 238 miRNAs evaluated, 8 showed increased and 12 showed decreased expression at least at 4 out of 5 reperfusion time points studied between 3 h and 3 days compared with sham. Of those, 17 showed >5 fold change. Bioinformatics analysis indicated a correlation between miRNAs altered to several mRNAs known to mediate inflammation, transcription, neuroprotection, receptors function, and ionic homeostasis. Antagomir-mediated prevention of mir-145 expression led to an increased protein expression of its downstream target superoxide dismutase-2 in the postischemic brain. In silico analysis showed sequence complementarity of eight miRNAs induced after focal ischemia to 877 promoters indicating the possibility of noncoding RNA-induced activation of gene expression. The mRNA expression of the RNases Drosha and Dicer, cofactor Pasha, and the pre-miRNA transporter exportin-5, which modulate miRNA biogenesis, were not altered after transient middle cerebral artery occlusion. Thus, the present studies indicate a critical role of miRNAs in controlling mRNA transcription and translation in the postischemic brain.
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Project description:Neuroinflammation is one of the important factors aggravating brain injury after ischemic stroke. We aimed to investigate the effects of cerebrolysin (CBL) on neuroinflammation in vivo and in vitro and the underlying mechanisms. The gene expressions of pro-inflammatory factors and anti-inflammatory factors were analyzed by real time PCR in rat transient middle cerebral artery occlusion (tMCAO) model, lipopolysaccharides-induced neuroinflammatory mice model and LPS-treated mouse primary microglia cells. The neuroprotective effects of CBL were evaluated by infarct size, Longa test and Rotarod test for long-term functional recovery in rats subjected to ischemia. The role of CREB/PGC-1? pathway in anti-neuroinflammatory effect of CBL was also determined by real time PCR and Western blotting. In the tMCAO model, administration of CBL at 3 h post-ischemia reduced infarct volume, promoted long-term functional recovery, decreased the gene expression of pro-inflammatory factors and increased the gene expression of anti-inflammatory factors. Correspondingly, in LPS-induced neuroinflammatory mice model, CBL treatment attenuated sickness behavior, decreased the gene expression of pro-inflammatory factors, and increased the gene expression of anti-inflammatory factors. In in vitro and in vivo experiments, CBL increased the protein expression levels of PGC-1? and phosphorylated CREB to play anti-inflammatory effect. Additionally, the application of the specific CREB inhibitor, 666-15 compound could effectively reverse the anti-inflammatory effect of CBL in primary mouse microglia cells and anti-ischemic brain injury of CBL in rats subjected to tMCAO. In conclusion, CBL ameliorated cerebral ischemia injury through reducing neuroinflammation partly via the activation of CREB/PGC-1? pathway and may play a therapeutic role as anti-neuroinflammatory agents in the brain disorders associated with neuroinflammation.
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Project description:Dimethyloxalylglycine (DMOG) is an inhibitor of prolyl-4-hydroxylase domain (PHD) enzymes that regulate the stability of hypoxia-inducible factor (HIF). We investigated the effect of DMOG on the outcome after permanent and transient middle cerebral artery occlusion (p/tMCAO) in the rat. Before and after pMCAO, rats were treated with 40 mg/kg, 200 mg/kg DMOG, or vehicle, and with 40 mg/kg or vehicle after tMCAO. Serial magnetic resonance imaging (MRI) was performed to assess infarct evolution and regional cerebral blood flow (rCBF). Both doses significantly reduced infarct volumes, but only 40 mg/kg improved the behavior after 24 hours of pMCAO. Animals receiving 40 mg/kg were more likely to maintain rCBF values above 30% from the contralateral hemisphere within 24 hours of pMCAO. DMOG after tMCAO significantly reduced the infarct volumes and improved behavior at 24 hours and 8 days and also improved the rCBF after 24 hours. A consistent and significant upregulation of both mRNA and protein levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) was associated with the observed neuroprotection, although this was not consistently related to HIF-1? levels at 24 hours and 8 days. Thus, DMOG afforded neuroprotection both at 24 hours after pMCAO and at 24 hours and 8 days after tMCAO. This effect was associated with an increase of VEGF and eNOS and was mediated by improved rCBF after DMOG treatment.
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Project description:Background Systemic innate immune priming is a recognized sequela of post-ischemic neuroinflammation and contributor to delayed neurodegeneration. Given mounting evidence linking acute stroke with reactive lung inflammation, we asked whether enhanced expression of the endogenous antioxidant extracellular superoxide dismutase 3 (SOD3) produced by alveolar type II pneumocytes would protect the lung from transient global cerebral ischemia and the brain from the delayed effects of ischemia-reperfusion. Methods and Results Following 15 minutes of global cerebral ischemia or sham conditions, transgenic SOD3 and wild-type mice were followed daily for changes in weight, core temperature, and neurological function. Three days after reperfusion, arterial and venous samples were collected for complete blood counts, flow cytometry, and SOD3 protein blotting, and immunohistochemistry was performed on lung and brain tissue to assess tissue injury, blood-brain barrier permeability, and neutrophil transmigration. Relative to ischemic controls, transgenic SOD3 mice performed better on functional testing and exhibited reduced peripheral neutrophil activation, lung inflammation, and blood-brain barrier leak. Once released from the lung, SOD3 was predominantly not cell associated and depleted in the venous phase of circulation. Conclusions In addition to reducing the local inflammatory response to cerebral ischemia, targeted enrichment of SOD3 within the lung confers distal neuroprotection against ischemia-reperfusion injury. These data suggest that therapies geared toward enhancing adaptive lung-neurovascular coupling may improve outcomes following acute stroke and cardiac arrest.
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