Current Neurovascular Research - Volume 17, Issue 1, 2020

Background: Hemorrhagic transformation (HT) is a frequent complication of acute ischemic stroke (AIS). Mean platelet volume (MPV) is a marker of platelet function. The relationship between MPV and HT remains unclear. Methods: From January 1st, 2012 to December 31st 2016, we consecutively enrolled AIS patients admitted to the Department of Neurology of West China Hospital. MPV was measured on admission. HT was diagnosed by brain imaging and classified into hemorrhagic infarct (HI) and parenchymal hematoma (PH). Moreover, subjects were divided into tertiles according to MPV levels. Confounders were identified by univariate analysis and multivariate logistic regression was performed to explore the association between MPV and HT as well as HT subtypes. Also, a generalized additive model was used to investigate whether a non-linear association existed between MPV and HT. Results: A total of 783 AIS patients were included. 63 patients (8.0%) developed HT: 34 (4.3%) HI and 29 (3.7%) PH. It was observed that MPV positively correlated with HT. After adjustment for confounders, patients in the highest MPV tertile had a significantly increased risk of HT compared to patients in the lowest tertile (odds ratio 2.3, 95% confidence interval 1.0-5.4, P=0.04). The risk of HT increased step-wise across MPV tertiles (P for trend=0.04). MPV tertiles significantly correlated with HI rather than PH. The generalized additive model demonstrated a nonlinear association between MPV and HT (P=0.02). Conclusion: The risk of HT increased with increasing MPV level in a dose-dependent manner. Patients with elevated MPV levels were more likely to develop HI rather than PH.

Purpose: Myocardial infarction is a common cardiovascular disease. MicroRNA-16-5p (miR-16-5p) was upregulated in heart and kidney hypoxia/reoxygenation (H/R) injury. However, the role of miR-16-5p in myocardial infarction injury is still unclear. Methods: Human adult ventricular cardiomyocytes (AC16) were treated with ischemia/reperfusion (H/R). The miR-16-5p level was evaluated through real-time PCR. The activity of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) was detected via LDH and CK-MB monitoring kits. Cell viability was examined with 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetra-zolium bromide (MTT) assay. Western blotting was used to analyze the protein levels. The luci-ferase report assay confirmed the relative luciferase activity. Results: miR-16-5p was elevated in H/R-treated AC16 cells. miR-16-5p overexpression and knockdown were carried out. miR-16-5p knockdown repressed cell apoptosis, attenuated LDH and CK-MB activities, and enhanced cell viability in H/R-treated AC16 cells. Moreover, miR-16-5p knockdown promoted angiogenesis in human microvascular endothelial cells (HMVEC), causing elevation of vascular endothelial growth factor (VEGF), insulin receptor substrates 1 (IRS1), minichromosome maintenance complex component 2 (MCM2) and proliferating cell nuclear antigen (PCNA) protein levels. Moreover, miR-16-5p was testified to target IRS1. IRS1 silencing alleviated miR-16-5p knockdown-mediated inhibition of apoptosis in AC16 cells. Conclusion: miR-16-5p knockdown increased cell viability and angiogenesis, as well as inhibited cell apoptosis by increasing IRS1. These findings indicated that miR-16-5p knockdown may be a new therapeutic target for myocardial infarction.

Objective: We investigated whether intravenous thrombolysis (IVT) affected the outcomes and complications of mechanical thrombectomy (MT), specifically focusing on thrombus fragmentation. Methods: The patients who underwent MT for large artery occlusion (LAO) were classified into two groups: MT with prior IVT (MT+IVT) group and MT without prior IVT (MT-IVT) group. The clinical outcome, successful recanalization with other radiological outcomes, and complications were compared, between two groups. Subgroup analysis was also performed for patients with simultaneous application of stent retriever and aspiration. Results: There were no significant differences in clinical outcome and successful recanalization rate, between both groups. However, the ratio of pre- to peri-procedural thrombus fragmentation was significantly higher in the MT+IVT group (14.6% and 16.2%, respectively; P=0.004) compared to the MT-IVT group (5.1% and 6.8%, respectively; P=0.008). The MT+IVT group required more second stent retriever (16.2%), more stent passages (median value = 2), and more occurrence of distal emboli (3.9%) than the MT-IVT group (7.9%, median value = 1, and 8.1%, respectively) (P=0.004, 0.008 and 0.018, respectively). In subgroup analysis, the results were similar to those of the entire patients. Conclusion: Thrombus fragmentation of IVT with t-PA before MT resulted in an increased need for additional rescue therapies, and it could induce more distal emboli. The use of IVT prior to MT does not affect the clinical outcome and successful recanalization, compared with MT without prior IVT. Therefore, we need to reconsider the need for IVT before MT.

Background: Inflammation is one of the causes of neuroblastoma progression. Propofol attenuates inflammation by repressing nuclear transcription factor ΚB (NF-ΚB) in different diseases. But its effect on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced inflammation is not known. Objective: This study investigated the role and mechanism of action of propofol on OGD/Rinduced inflammation in mouse N2A neuroblastoma cells. Methods: MTT was performed on mouse neuroblastoma cells N2A to assess and select the maximum safe dose of propofol. Next, N2A cells were pretreated with propofol and then, exposed to the OGD condition for 3 h and reoxygenated for 6 h. The content of the inflammatory factors, interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α), in the medium was measured by ELISA, while their protein expression was detected by western blot and immunofluorescence. The protein expression of P65, p-P65, IKBα and p-IKBα belonging to the NF-ΚB pathway was also determined by western blot in N2A cells. To further confirm the mechanism of propofol on OGD/R-induced inflammation in mouse N2A cells, P65 was over-expressed and the above experiments were repeated. Results: Propofol did not affect cell viability of N2A cells even at the maximum concentration used (30 μM), thus, 30 μM of propofol was selected to perform our experiments. Besides, OGD/R induced inflammation and activation of NF-ΚB pathway with increased p-P65 and p-IKBα expression, and propofol pretreatment inhibited OGD/R induced inflammation and activation of NF-ΚB pathway in N2A cells. Over-expression of P56 abolished the effects of propofol on OGD/Rinduced inflammation and activation of NF-ΚB pathway in N2A cells. Conclusion: Our work demonstrated for the first time that propofol pretreatment ameliorated OGD/R induced inflammation via NF-ΚB pathway modulation in mouse neuroblastoma N2A cells, indicating that propofol might be considered as a potential therapeutic approach to reduce inflammation in neuroblastoma.

Introduction: Takotsubo syndrome (TTS) is a stress-induced cardiomyopathy, but the accurate cause of this syndrome is still unknown. Methods: β-adrenergic agonist isoproterenol (ISO) is used to establish the TTS rats model. TTS rats were treated with or without LY294002 or Rapamycin. The rat cardiomyoblast cell line H9C2 was subjected to infect with constitutively active Akt (myr-Akt) or dominant-negative mutant Akt (dn-Akt) and then, treated with ISO. Cell apoptosis was assessed using the Bax/ Bcl-2 ratio. In addition, reactive oxygen species (ROS) levels were measured using dihydroethidium (DHE). Mitochondrial superoxide generation and membrane potential were assayed by MitoSOX and JC-1 fluorescence intensity. Results: ISO might induce the erratic acute cardiac dysfunction and overexpression of PI3K/AKT/mTOR. Moreover, it also increased the oxidative stress and apoptosis in TTS rats. The Akt inhibitor significantly reversed the cardiac injury effect, which triggered by ISO treatment. In H9C2 cells, the inhibition of Akt provides a protective role against ISO-induced injury by reducing oxidative stress, apoptosis and mitochondrial dysfunction. Conclusion: This study provided new insight into the protective effects of myocardial dysfunction in TTS rats via chronic inhibition of the PI3K/AKT/mTOR expression, which could reduce mitochondrial ROS and oxidative stress-induced apoptosis. PI3K/AKT/mTOR inhibitor could be a therapeutic target to treat cardiovascular dysfunction induced by stress cardiomyopathy.

Background: Perihematomal edema (PHE) is a major threat leading to poor functional outcomes after intracerebral hemorrhage (ICH). TIMP-2 is considered to participate in the formation of PHE after ICH by antagonizing the damaging effects of MMP-2. In the early study, the polymorphisms of TIMP-2 rs8179090 have shown to influence the expression of TIMP-2. Objective: To prove that the severity of PHE was different in ICH patients with different TIMP-2 rs8179090 genotypes. Methods: In this prospective study, 130 hypertensive ICH patients were enrolled. The poly phisms of rs8179090 in TIMP-2 were determined. The hematoma volume and PHE volume were measured by computed tomography (CT) scan immediately after the onset of ICH, and were measured again one week and two weeks after the onset. Then, the comparison of TIMP-2 rs8179090 genotypes was made. Results: TIMP-2-418 position (rs8179090) had two genotypes in the studied population, GC and GG. Patients with the GC genotype developed more severe PHE, with a higher incidence of delayed cerebral edema in cerebral hemorrhage than those with the GG genotype. Conclusion: We have found that the GC genotype group may develop more severe PHE, with an increased incidence of delayed cerebral edema in cerebral hemorrhage.

Aim: Epilepsy is a common and serious neurological disease that causes recurrent episodes, but its molecular mechanism remains unclear. Abnormal miRNA expression is associated with epilepsy, including miR-451. This research investigated the role of miR-451 in seizure and its detailed mechanism. Methods: The seizure mice model was induced by kainic acid (KA) injection to the right lateral cerebral ventricle. Behavioral changes in mice were observed and evaluated by the Racine Scale. The miR-451 knockout mice were established by adenovirus infection. The in vitro model was performed by miR-451 mimics transfected HEK-293 cells. The amount of neuronal death and morphological changes were evaluated by Nissl staining and H&E staining. Results: The results showed that miR-451 is up regulated in KA-induced seizure models and miR- 451 knockout decreased the behavior score and improved the pathological changes of the hippocampus. Besides, MiR-451 knockout inhibited the apoptosis of hippocampal neurons. Bioinformatics studies have shown that glial cell line-derived neurotrophic factor (GDNF) is a target gene of miR-451. MiR-451 could negatively regulate the expression of GDNF. GDNF overexpression could reverse the effect of miR-451 on KA induced brain injury and neuronal apoptosis. Conclusion: This research demonstrates that miR-451 can affect the behavior of KA-induced epilepsy mice and hippocampal neuronal damage by regulating GDNF expression. The results would provide an experimental foundation for further research about the potential contribution of mi- RNAs to epilepsy pathophysiology.

Background: Endovascular treatment of intracranial aneurysms (IAs) by flow diverter (FD) stents depends on flow modification. Patient-specific modeling of FD deployment and computational fluid dynamics (CFD) could enable a priori endovascular strategy optimization. We developed a fast, simplistic, expansion-free balls-weeping algorithm to model FDs in patientspecific aneurysm geometry. However, since such strong simplification could result in less accurate simulations, we also developed a fast virtual stenting workflow (VSW) that explicitly models stent expansion using pseudo-physical forces. Methods: To test which of these two fast algorithms more accurately simulates real FDs, we applied them to virtually treat three representative patient-specific IAs. We deployed Pipeline Embolization Device into 3 patient-specific silicone aneurysm phantoms and simulated the treatments using both balls-weeping and VSW algorithms in computational aneurysm models. We then compared the virtually deployed FD stents against experimental results in terms of geometry and post-treatment flow fields. For stent geometry, we evaluated gross configurations and porosity. For post-treatment aneurysmal flow, we compared CFD results against experimental measurements by particle image velocimetry. Results: We found that VSW created more realistic FD deployments than balls-weeping in terms of stent geometry, porosity and pore density. In particular, balls-weeping produced unrealistic FD bulging at the aneurysm neck, and this artifact drastically increased with neck size. Both FD deployment methods resulted in similar flow patterns, but the VSW had less error in flow velocity and inflow rate. Conclusion: In conclusion, modeling stent expansion is critical for preventing unrealistic bulging effects and thus should be considered in virtual FD deployment algorithms. Also endowed with its high computational efficiency and superior accuracy, the VSW algorithm is a better candidate for implementation into a bedside clinical tool for FD deployment simulation.

Background: Electroacupuncture (EA) can promote nerve and vascular regeneration, confer neuroprotection, inhibit apoptosis and inflammatory reactions, reduce oxidative stress injury, regulate neurochemicals and inhibit the formation of brain oedema in cerebral ischemic. However, the precise site of EA stimulation in the treatment of cerebral ischemic is unclear. Objective: In the present study, we investigated the effect of EA at the acupoints of different meridians in motor function recovery and the involvement of Vascular Endothelial Growth Factor (VEGF), phosphorylated Protein Kinase B (P-Akt), phosphorylated endothelial nitric oxide synthase (p-eNOS) and Platelet Endothelial Cell Adhesion Molecule-1(CD31) were examined in the peri-infarction cortex of rats. Methods: The Middle cerebral artery occlusion (MCAO) model or sham surgery was performed in a total of Ninety male Sprague-Dawley rats. Rats were randomly divided into five groups: a sham group, a middle cerebral artery occlusion (MCAO) group, a Yang meridian group, a Yin meridian group and a combined Yang and Yin meridian group. EA stimulus was given during the middle cerebral artery occlusion. The neurobehavioural function was measured using Modified Neurological Severity Scores (mNSS), the rotarod test and the ladder rung walking test, and the protein expression of VEGF, P-Akt, p-eNOS in the peri-infarction cortex was detected by Western blot. Immunofluorescence was used to measure the vascular density of the peri-infarction cortex. Results: EA at different meridian acupoints has no effect on the infarction volume, while EA at Yin meridian acupoints significantly promoted neurobehavioural functional recovery, increased the vascular density and enhanced protein kinase B/Endothelial nitric oxide synthase (Akt/eNOS) phosphorylation and VEGF expression. Conclusion: In the early stage of stroke, EA at Yin meridian acupoints can improve neurobehavioural functional recovery and the mechanism of this effect may be related to the enhanced expression of VEGF, P-Akt and p-eNOS in the peri-infarction cortex of rats.

Purpose: The current study was conducted in order to investigate the role of Forkhead box O1 and p21-mediated macrophage polarization in postoperative cognitive dysfunction induced by sevoflurane. Methods: There involved a total of 30 healthy mice that were randomly divided into two groups: control group (without any treatment) and anaesthesia group (treated with sevoflurane inhalation). The effects of sevoflurane on cognitive function (memory) in mice were studied by trace fear conditioned reflex, and the effects of systemic inflammation and behavior after operation were measured by enzyme-linked immunosorbent assay (ELISA), the concentrations of CD163 and tumor necrosis factor-α (TNF-α) were measured. The expression of macrophage phenotype was observed by immunofluorescence staining, the expression levels of M1 and M2 markers mRNA were detected by real-time fluorescence quantitative PCR (RT-PCR), and the expression levels of FoxO1 and p21 were analyzed by immunoblotting (Western blot). Results: Compared with the control group, the freezing time in the anesthesia group was lower than that in the control group (P<0.01), indicating that sevoflurane anesthesia led to the decrease of cognitive ability. The blood concentrations of CD163 and TNF-α increased significantly at 24 h after the operation with sevoflurane anesthesia (P<0.05). Fluorescence microscopic observation showed that M2 was the main type of macrophages in normal tissues, while M1 and M2 phenotypes were highly expressed in sevoflurane anesthetized tissues at the same time, especially in M1 phenotypes (P<0.01). The polarization of macrophages in the anesthetic group showed the high level of M1 mRNA, and the expression levels of TNF-α, monocyte chemotactic protein 1(MCP-1) and Interleukin-6 (IL-6)mRNA in the anesthetic group were significantly higher than those in the control group (P<0.05). The expression levels of M2 mRNA such as transforming growth factor-β (TGF-β) and IL-10 were significantly lower than those in the control group (P<0.05). Compared with the control group, the expression of FoxO1 and p21 protein in the anesthesia group was significantly lower than that in the control group with a significant statistical difference (P<0.01). Conclusion: This study offers a theoretical basis and insight for further understanding of the prevention and treatment of cognitive dysfunction induced by anesthetic drugs.

Background: Alzheimer’s disease (AD) is the most common neurodegenerative disease, and the accumulation of amyloid-β is the initial process in AD. MicroRNAs (miRNAs) are widely known as key regulators of the accumulation of amyloid-β in AD. This study analyzed the potential effects and possible internal mechanisms of miR-340 on AD. Methods: The expression of miR-340 in senescence-accelerated mouse prone-8 (SAMP8) mouse and senescence-accelerated mice/resistant-1 (SAMR1) mouse was evaluated by qRT-PCR (quantitative real-time polymerase chain reaction). The expression of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) was determined by qRT-PCR and western blot. The binding ability between miR-340 and BACE1 was verified by dual-luciferase reporter assay. In vitro cell model of AD was established in human neuroblastoma SH-SY5Y cells transfected with Swedish mutant form of amyloid precursor protein (APPswe). The effect of miR-340 on the accumulation of amyloid- β was investigated by western blot analysis. Flow cytometry was conducted to detect cell apoptosis. Results: MiR-340 was down-regulated in the hippocampus of AD model SAMP8 mouse compared to SAMR1 mouse, while BACE1 was up-regulated in SAMP8, suggesting a negative correlation between miR-340 and BACE1 in SAMP8 mouse. MiR-340 could directly bind with BACE1, and over-expression of miR-340 decreased expression of BACE1 in SH-SY5Y/APPswe cells. MiR- 340 reduced the accumulation of amyloid-β and suppressed cell apoptosis through targeting BACE1 in SH-SY5Y/APPswe cells. Conclusion: MiR-340 was downregulated in AD and reduced the accumulation of amyloid-β through targeting BACE1, suggesting a potential therapeutic target for AD.

Objective: Alzheimer's disease (AD), also known as senile dementia, is a common neurodegenerative disease characterized by progressive cognitive impairment and personality changes. Numerous evidences have suggested that microRNAs (miRNAs) are involved in the pathogenesis and development of AD. However, the exact role of miR-335-5p in the progression of AD is still not clearly clarified. Methods: The protein and mRNA levels were measured by western blot and RNA extraction and quantitative real-time PCR (qRT-PCR), respectively. The relationship between miR-335-5p and c-jun-N-terminal kinase 3 (JNK3) was confirmed by dual-luciferase reporter assay. SH-SY5Y cells were transfected with APP mutant gene to establish the in vitro AD cell model. Flow cytometry and western blot were performed to evaluate cell apoptosis. The APP/PS1 transgenic mice were used as an in vivo AD model. Morris water maze test was performed to assess the effect of miR- 335-5p on the cognitive deficits in APP/PS1 transgenic mice. Results: The JNK3 mRNA expression and protein levels of JNK3 and β-Amyloid (Aβ) were significantly up-regulated, and the mRNA expression of miR-335-5p was down-regulated in the brain tissues of AD patients. The expression levels of miR-335-5p and JNK3 were significantly inversely correlated. Further, the dual Luciferase assay verified the relationship between miR-335- 5p and JNK3. Overexpression of miR-335-5p significantly decreased the protein levels of JNK3 and Aβ and inhibited apoptosis in SH-SY5Y/APPswe cells, whereas the inhibition of miR-335-5p obtained the opposite results. Moreover, the overexpression of miR-335-5p remarkably improved the cognitive abilities of APP/PS1 mice. Conclusion: The results revealed that the increased JNK3 expression, negatively regulated by miR-335-5p, may be a potential mechanism that contributes to Aβ accumulation and AD progression, indicating a novel approach for AD treatment.

Background: Neonatal pneumonia is a common disease in the neonatal period with a high incidence and death. This study aimed to investigate the molecular mechanism and effect of microRNA (miR)-429 in neonatal pneumonia. Methods: The peripheral blood was collected from neonatal pneumonia and healthy patients, respectively. Human lung fibroblast WI-38 cells were treated with lipopolysaccharide (LPS) to establish neonatal pneumonia cell model. Then, the miR-429 expression was detected by quantitative real-time polymerase chain reaction (qRT-PCR). In addition, the relationship between miR- 429 and kruppel-like factor 4 (KLF4) was confirmed by dual luciferase reporter assay. Cell viability, the level of interleukin 6 (IL-6), IL-1β and tumor necrosis factor α (TNF-α) and apoptosis were measured by Cell Counting Kit-8 (CCK-8), enzyme linked immunosorbent assay (ELISA) and flow cytometry. Meanwhile, apoptosis and nuclear factor kappa-B (NF-ΚB) pathway related proteins expression were analyzed by western blot. Results: MiR-429 expression level was increased in neonatal peripheral blood and LPS-stimulated WI-38 cells. Then, miR-429 overexpression increased apoptosis, the level of IL-6, IL-1β, TNF-α, Bax and cleaved caspase-3, while reduced cell viability in LPS-stimulated WI-38 cells. Besides, KLF4 was identified as the target gene of miR-429, and reversed the changes caused by miR-429 overexpression. Finally, miR-429 suppressor down-regulated p-NF-ΚB level in LPS-stimulated cells and KLF4 knockdown reversed these reductions. Conclusion: MiR-429 promotes inflammatory injury, apoptosis and activates the NF-ΚB signaling pathway by targeting KLF4 in neonatal pneumonia, and then these results provide evidence for clinical diagnosis and treatment for neonatal pneumonia.