Current Neurovascular Research - Volume 17, Issue 2, 2020

Background: Curcumin, the complex extracted from the traditional edible herb, has a wide range of pharmacological effects. A great deal of studies has demonstrated that curcumin could protect against cerebral ischemia-reperfusion (I/R) injury. In the present study, we aimed to test the hypothesis that curcumin reduces brain damage via regulating mitophagy and preserving mitochondrial function. To clarify the potential effect and mechanism of curcumin on cerebral I/R, we utilize MCAO followed by reperfusion rats and OGD/R neurons as cerebral I/R in vivo and in vitro, respectively. Methods: We determined the cellular ROS levels and mitochondrial function, including mitochondrial membrane potential (MMP), ATP levels, state 3 respiration and state 4 respiration. We also detected the levels of mitophagy by immunofluorescent staining and western blotting. Results: Results found that curcumin decreased neurological deficit scores, infarct volume and morphological changes of neurons in rats after brain I/R injury. Curcumin also reduced the levels of ROS while increased MMP, ATP levels and state 3 respiration to prevent the impairment of mitochondrial function from cerebral I/R. Furthermore, curcumin enhanced the co-localization of LC3B and mitochondrial marker VDAC1, the ratio of LC3-II to LC3-I, improving cerebral I/Rinduced mitophagy. Conclusion: In conclusion, our results suggest that curcumin protects against cerebral I/R injury by improving mitophagy and preserving mitochondrial function.

Background: Myocardial Infarction (MI), a kind of heart deficiency, is the main cause of death and disability. Autophagy, a metabolic process for the degradation of damaged proteins or organelles, is important for cardiac functions and regulated by several miRNAs including miRNA- 101. The aim of this research was to investigate the effects of miR-101 in myocardial infarctioninduced injury and the related mechanisms. Methods: MI model was induced by ligation of the left coronary artery. The in vitro model was established by hypoxia-induced H9c2 cells (rat myocardial cells). The overexpression of miR-101 was achieved by transfection. The expression of associated proteins was analyzed by Western blotting. The level of miR-101 was analyzed by reverse transcription-polymerase chain reaction (RTPCR). The target genes for miR-101 and the target sites were analyzed by TargetScan. Results: The results showed that miR-101 was decreased in MI mice (P<0.01). Autophagy and apoptosis were increased in MI-induced injury (in vivo) and in hypoxia treated myocardial cells (in vitro) (P<0.01). miR-101 overexpression inhibited the increase of autophagy and apoptosis in mice and myocardial cells (P<0.01). DDIT4 was a target gene of miR-101 and expressed increasingly in MI-induced injury mice and hypoxia treated myocardial cells. miR-101 could negatively regulate the expression of DDIT4. Conclusion: This research suggested that miR-101 attenuated- MI-induced injury by targeting DDIT4 to regulate autophagy, which indicated that miR-101 or DDIT4 may be potential therapeutic targets for heart injury.

Background: Whether preoperative midline shift and its growing rate are associated with outcomes of decompressive craniectomy in patients with malignant middle cerebral artery infarction is unknown. Methods: We retrospectively included patients: 1) who underwent decompressive craniectomy for malignant middle cerebral artery infarction in West China Hospital from August 2010 to December 2, 2018) who had at least two brain computed tomography scans before decompressive craniectomy. Midline shift was measured on the first and last preoperative computed tomography scans. Midline shift growing rate was calculated by dividing Δmidline shift value using Δ time. The primary outcome was inadequate decompression of the mass effect. Secondary outcomes were 3 month death and unfavorable outcomes. Results: Sixty-one patients (mean age 53.7 years, 57.4% (35/61) male) were included. Median time from onset to decompressive craniectomy was 51.8 h (interquartile range: 39.7-77.8). Rates of inadequate decompression, 3 month death, 3 month modified Rankin Scale 5-6 and 4-6 were 50.8% (31/61), 50.9% (29/57), 64.9% (37/57) and 84.2% (48/57), respectively. The inadequate decompression group had a higher midline shift growing rate than the adequate decompression group (median: 2.7 mm/8 h vs. 1.4 mm/8 h, P=0.041). No intergroup difference of 3 month outcomes was found in terms of preoperative midline shift growing rate. Conclusion: Higher preoperative midline shift growing rate was associated with inadequate decompression of decompressive craniectomy in patients with malignant middle cerebral artery infarction.

Background: Propofol is an intravenous drug commonly used in anesthesia procedures and intensive care in children. However, it also has neurotoxic effects on children. MicroRNA plays an important role in neurological diseases and neurotoxicity. Methods: In this study, primary rat hippocampal neurons were used to investigate the role of miR- 582-5p in propofol-induced neurotoxicity. Cell viability was monitored by 3-(4,5-dimethylthiazolyl)- 2,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while the expression of proteins was monitored by real-time quantitation polymerase chain reaction (RT-qPCR) and western blot. TargetScan and double luciferase report assay were used to predict the targeting relationship between miR-582-5p and Rho-associated serine-threonine protein kinase 1 (ROCK1). Results: In the present study, the viability of neurons and the expression of miR-582-5p were decreased in a time-dependent manner after propofol treatment. Besides, miR-582-5p overexpression significantly reduced the toxicity of propofol on neuron cells but had no significant effect on normal nerve cells. In addition, miR-582-5p overexpression significantly reversed the expression of apoptosis-related proteins (cleaved caspase 3 and cleaved caspase 9) induced by propofol but had no significant effect in normal nerve cells. TargetScan and Dual-luciferase report assay revealed that ROCK1 was a targeted regulatory gene for miR-582-5p, and propofol treatment up-regulated ROCK1 expression by inhibiting miR-582-5p expression. Notably, miR-582-5p overexpression significantly increased cell viability, while ROCK1 overexpression reversed the effect of miR-582- 5p. Conclusion: Taken together, these findings suggest that miR-582-5p alleviated propofol-induced apoptosis of newborn rat neurons by inhibiting ROCK1.

Background: Curcumin has anti-inflammatory, antioxidant and anticancer properties. Despite the considerable evidence showing that curcumin is an efficacious and safe compound for multiple medicinal benefits, there are some demerits with respect to the therapeutic effectiveness of curcumin, namely, poor stability and solubility, and its role in angiogenesis in vivo is still not yet clear. More recently, the biodegradable polymer nanoparticles have been developed. This offers promise for the therapeutic effectiveness of curcumin by increasing its bioavailability, solubility and retention time. Methods: Here, we compared the medicinal effectiveness of curcumin and nanocurcumin (NC), and found that nanocurcumin can inhibit angiogenesis more effectively than curcumin in zebrafish. Tests of proliferation and apoptosis showed no difference between nanocurcumin-treated and wildtype embryos. Results: qPCR and in situ hybridization experiments indicated that the VEGF signaling pathway genes, vegfa, VEGF-C and flt4 were all down-regulated after nanocurcumin treatment, and vegfa over-expression rescued the vascular defective phenotype. Moreover, hif1a expression also decreased and hif1a over-expression also rescued the vascular defective phenotype but the Notch signaling pathway had no difference after nanocurcumin treatment. Conclusion: These results indicate that nano curcumin inhibits angiogenesis in zebrafish by downregulating hif1a/vegfa signaling pathway. Hence, our work reveals the key role of nanocurcumin in angiogenesis in vivo.

Objective: The effects of mesenchymal stem cell (MSC)-derived exosomes on brain microvascular endothelial cells under oxygen-glucose deprivation (OGD), which mimic cells in deep hypothermic circulatory arrest (DHCA) in vitro, are yet to be studied. Methods: MSCs were co-cultured with primary rat brain endothelial cells, which were then exposed to OGD. Cell viability, apoptosis, the inflammatory factors (IL-1β, IL-6, and TNF-α), and the activation of inflammation-associated TLR4-mediated pyroptosis and the NF-ΚB signaling pathway were determined. Furthermore, exosomes derived from MSCs were isolated and incubated with endothelial cells to investigate whether the effect of MSCs is associated with MSCderived exosomes. Apoptosis, cell viability, and the inflammatory response were also analyzed in OGD-induced endothelial cells incubated with MSC-derived exosomes. Results: OGD treatment promoted endothelial cell apoptosis, induced the release of inflammatory factors IL-1β, IL-6, and TNF-α, and inhibited cell viability. Western blot analysis showed that OGD treatment-induced TLR4, and NF-ΚB p65 subunit phosphorylation and caspase-1 upregulation, while co-culture with MSCs could reduce the effect of OGD treatment on endothelial cells. As expected, the effect of MSC-derived exosomes on OGD-treated endothelial cells was similar to that of MSCs. MSC-derived exosomes alleviated the OGD-induced decrease in the viability of endothelial cells, and increased levels of apoptosis, inflammatory factors, and the activation of inflammatory and inflammatory focal pathways. Conclusion: Both MSCs and MSC-derived exosomes attenuated OGD-induced rat primary brain endothelial cell injury. These findings suggest that MSC-derived exosomes mediate at least some of the protective effects of MSCs on endothelial cells.

Background: Iron homeostasis disorder and neuroinflammation are the most commonly known factors that promote the occurrence and development of cognitive impairment in people. Dexmedetomidine has an anti-inflammatory effect, and it reduces the incidence of postoperative cognitive dysfunction. Therefore, the aim of this study is to verify whether dexmedetomidine could improve lipopolysaccharide-induced iron homeostasis disorder in aged mice, and show neuroprotective effect. Methods: First part, forty 12 month old male Kunming(KM) mice were divided into group N and group D: Normal saline group (group N), Dexmedetomidine group (group D). Second part, sixty 12-month-old male KM mice were divided into the following three groups: Normal saline group (group N), Lipopolysaccharide group (group LPS) and Dexmedetomidine + Lipopolysaccharide group (group D + LPS). The mice in group D + LPS were given dexmedetomidine, and given LPS intraperitoneally 2 h later. Mice underwent an oriented navigation test and a space exploration test in the Morris Water maze (MWM) test. The expression levels of Interleukin-6 ( IL-6), L-ferritin (FTL) and Transferrin receptor-1 (TfR1) in hippocampus were detected by the Western blot analysis; the hippocampal hepcidin mRNA was detected by Real-time PCR(RT-PCR); the reactive oxygen species (ROS) in the hippocampus was measured using ROS test kit. Results: Dexmedetomidine improved the cognitive decline induced by LPS. Dexmedetomidine reduced the level of hippocampal IL-6, and it attenuated the increase in their levels caused by LPS. It had no effect on hippocampal hepcidin mRNA, FTL, TfR1 and ROS but it could attenuate the increase caused by LPS. Conclusion: Dexmedetomidine has no effect on iron metabolism pathway, but it can improve the cognitive decline and the iron disorder by reducing neuroinflammation and oxidative stress. The research indicates that dexmedetomidine plays a neuroprotective role.

Background: Reduction in myocardial I/R injury has become the key to the therapy of ischemic cardiovascular disease. Isoflurane (ISO) preconditioning can mimic the major potent protective mechanisms and attenuate ischemia injury. Nevertheless, the mechanisms involved in the cardioprotective effects afforded by isoflurane preconditioning have never been evaluated systematically. Methods: Mice were randomly divided into an ISO preconditioning group and control group. The size of the infarcted region was measured, and comparisons between ISO preconditioning and control animals were made. The metabotropic glutamate receptor type 1(GRM1) expression levels in all groups were determined by quantitative PCR. GRM1 protein expression and DNA damage relative protein γ-H2AX were measured by western blot analysis. The oxidative stress was detected by immunofluorescence after staining with the Dihydroethidium (DHE). Results: ISO preconditioning significantly reduced the IR induced infarct volumes and reversed the GRM1 protein expression level in I/R induced myocardial injury. Moreover, ISO preconditioning has a protective effect in reducing the I/R induced DNA damage and oxidative stress. Conclusion: The results of the present study have demonstrated that the expression of GRM1 provides a protective role in ISO preconditioning against I/R-induced myocardial infarction by reducing the oxidative stress and DNA damage.

Background: People with migraine experience cognitive decline more often than healthy controls, resulting in a significant functional impact. Early identifying influencing factors that contribute to cognitive decline in migraineurs is crucial for timely intervention. Although migraine may onset early in childhood and early onset migraine is related to significant disability, there is no research investigating the association between the age of migraine onset and migraineurs’ cognitive decline. Therefore we aim to explore possible factors that correlate to the cognitive function of migraineurs, especially focus on age of migraine onset. Methods: 531 patients with migraine were included. Data on demographics and headache-related characteristics were collected and evaluated using face-to-face interviews and questionnaires. We used the Montreal Cognitive Assessment scale to assess cognitive function. In addition, we analyzed independent correlations between cognitive decline and the age of migraine onset in patients with migraine. And all patients completed the Headache Impact Test-6 to evaluate their quality of life. Results: Migraineurs with cognitive decline showed significant differences from those without in age (OR=1.26, P<0.0001), years of education (OR=0.89, P=0.0182), the intensity of headache (OR=1.03, P=0.0217), age of onset (OR=0.92, P<0.0001) and anxiety scores (OR=1.09, P=0.0235). Furthermore, there was no interaction in the age of onset between subgroups. Multivariate linear regression analyses of HIT-6 scores showed that the intensity of headache (β=0.18, P<0.0001) and depression scores (β=0.26, P=0.0009) had independent effects on decreased quality of life. Conclusion: Our findings suggest that younger age of migraine onset is independently related to migraineurs’ cognitive decline, and migraine accompanying anxiety symptoms significantly related to decreased quality of life in migraineurs.

Objective: Early exposure to general anesthesia in children might be a potentially highrisk factor for learning and behavioral disorders. The mechanism of neurotoxicity induced by general anesthesia was not defined. miR-496 could regulate cerebral injury, while the roles of miR- 496 in neurotoxicity were not elucidated. Therefore, we aimed to investigate the effects of miR- 496 in neurotoxicity induced by propofol. Methods: Primary Prefrontal Cortical (PFC) neurons were isolated from neonatal rats and treated with propofol to induce neurotoxicity. Cell viability was detected by (3-(4,5-Dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and cell apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The target relationship of miR-496 and Rho Associated Coiled-Coil Containing Protein Kinase 2 (ROCK2) was explored using luciferase assays. Results: Propofol decreased cell viability, promoted cell apoptosis, and decreased the expression of miR-496 in PFC neurons in a dose-dependent manner. Overexpression of miR-496 attenuated neurotoxicity induced by propofol in PFC neurons. ROCK2 was a target of miR-496, and miR-496 oppositely modulated the expression of ROCK2. Besides, propofol increased the expression of ROCK2 through inhibiting miR-496 in PFC neurons. Overexpression of miR-496 attenuated propofol- induced neurotoxicity by targeting ROCK2 in PFC neurons. Conclusion: miR-496 was decreased in PFC neurons treated with propofol, and overexpression of miR-496 attenuated propofol-induced neurotoxicity by targeting ROCK2. miR-496 and ROCK2 may be promising targets for protecting propofol-induced neurotoxicity.

Objective: As a brain-specific microRNA, the mechanism of miR-124 in depression has not been clarified so far. The present study aimed to explore the role of miR-124 in depression and its potential targets. Methods: The depression model was first replicated by the chronic unpredictable mild stress (CUMS) method. miR-124 antagomir was injected into the hippocampus of CUMS rats. Sucrose preference test (SPT), open-field test (OFT), elevated-plus maze (EPM), and forced swimming test (FST) were used to analyze the depression-like behavior. The content of norepinephrine (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT) in the hypothalamus was analyzed by ELISA. qRT-PCR and western blot assay were used for functional analysis. Results: miR-124 expression was up-regulated in the hippocampus of CUMS -induced depression model rats, while CREB1 and BDNF were down-regulated. Administration of miR-124 antagomir in the hippocampus inhibited miR-124 expression in the hippocampus of CUMS rats. Additionally, SPT, OFT, EPM, and FST also showed that miR-124 antagomir can reduce the depression-like behavior of CUMS rats. Furthermore, miR-124 antagomir injection increased the levels of NE, DA and 5-HT in the hypothalamus of CUMS rats. Moreover, miR-124 antagomir injection increased the expression of cyclic AMP-responsive element-binding protein1 (CREB1) and brain-derived neurotrophic factor (BDNF) in the hippocampus. Using the dual-luciferase reporter assay, it was confirmed that miR-124 directly targets 3'UTR of CREB1 and BDNF genes. Conclusion: Knockdown of miR-124 can improve depression-like behavior in CUMS-induced depressive rats, which may be related at least in part to the up-regulation of CREB1 and BDNF expression in the hippocampus.

Background: Myocardial Fibrosis (MF) is an important physiological change after myocardial infarction (MI). MicroRNA-26b (MiR-26b) has a certain inhibitory effect on pulmonary fibrosis. However, the role of miR-26b in MI-induced MF rats and underlying molecular mechanisms remain unknown. Methods: Forty male Sprague Dawley (SD) rats weighing 200-250 g were divided into four groups (n=10): Sham group, MF group, MF + negative control (NC) agomir group and MF + miR-26b agomir group. Cardiac fibroblasts were isolated from cardiac tissue. Fibrosis levels were detected by MASSON staining, while the expression of related genes was detected by RT-qPCR, Western blotting and Immunohistochemistry, respectively. TargetScan and dual-luciferase reporter assay were utilized to predict the relationship between miR-26b and high mobility group, AT-hook 2 (HMGA2). Results: The study found the expression of miR-26b to be down-regulated in the myocardium of MF rats (P<0.01). miR-26b overexpression in vitro significantly reduced the survival rate of cardiac fibroblasts and inhibited the expression of the fibrillar-associated protein (α-SMA alphasmooth muscle actin (α-SMA) and collagen I) (P<0.01). TargetScan indicated that HMGA2 was one of the target genes of miR-26b; dual-luciferase reporter assay further confirmed the targeted regulatory relationship (P<0.01). Moreover, miR-26b overexpression significantly reduced the expression of HMGA2 (P<0.01). Notably, HMGA2 overexpression reversed the inhibitory effect of miR-26b overexpression on cardiac fibroblast viability and the expression of α-SMA and collagen I (P<0.01). Animal experiments further indicated that miR-26b overexpression inhibited MIinduced rat MF by inhibiting the expression of HMGA2 (P<0.05, P<0.01). Conclusion: In short, these findings indicate that miR-26b targets HMGA2 to ameliorate MI-induced fibrosis by suppression of cardiac fibroblasts activation.