Irisin Attenuates Apoptosis Following Ischemia-Reperfusion Injury Through Improved Mitochondria Dynamics and ROS Suppression Mediated Through the PI3K/Akt/mTOR Axis.

Irisin is a muscle-derived hormone that promotes the survival of motor neurons and enhances muscle size following injury. In this study, we investigated the beneficial effects and mechanism(s) of action of irisin in response to cerebral ischemia-reperfusion injury (CIRI). Right-middle cerebral artery occlusion (MCAO) and hypoxia/reoxygenation (H/R) models were generated in C57BL/6 J mice. Mouse neuronal cell lines (NSC-34) were used to confirm the molecular mechanisms of the protection afforded by irisin in response to CIRI. We found that irisin (250 µg/kg) improved cerebral function and reduced the cerebral infarct volume following CIRI. Irisin also protected neuronal cells against ischemia-reperfusion (I/R) induced apoptosis, assessed via TUNEL, and cleaved Caspase-3 staining. Western blotting of neuronal tissue from irisin treated I/R mice showed lower expression of pro-apoptotic Bax and caspase-9 (P < 0.001 and P < 0.01) and increased levels of the pro-survival protein Bcl-2 (P < 0.01 & P < 0.001 vs. I/R). Irisin also reduced the levels of reactive oxygen species (ROS) characterized through malondialdehyde (MDA) assays. Irisin was found to maintain mitochondrial homeostasis through the suppression of mitochondrial fission-linked dynamin-related protein 1 in CIRI mice (P < 0.01 and P < 0.05 v. I/R cohort). Moreover, mitochondrial fusion-related protein (Mfn2) and Opa1 expression were rescued following irisin treatment (P < 0.001 and P < 0.01 v. I/R cohort). Cell-based assays showed that irisin activates PI3K/AKT/mTOR signaling in the neurons of CIRI mice. Furthermore, the beneficial effects of irisin on NSC-34 cell-survival, mitochondrial function, and ROS generation were reversed by VS-5584, a highly specific PI3K/AKT/mTOR inhibitor. Collectively, these data highlight the ability of irisin to alleviate CIRI in vivo and in vitro. The mechanisms of action of irisin include the attenuation of apoptosis through the prevention of mitochondrial fission and increased mitochondrial fusion and the alleviation of oxidative stress through activation of the PI3K/AKT/mTOR axis. We therefore identify irisin as a much-needed therapeutic for CIRI.

Rodent models for intravascular ischemic cerebral infarction: a review of influencing factors and method optimization.

Rodent models for cerebral infarction are useful for studying human focal ischemic cerebral infarction, by simulating etiological and pathophysiological mechanisms. However, differences in the selection of anesthetic drugs, surgical methods and other factors may affect the extent to which preclinical models reflect the human condition. This review summarizes these factors. We searched pertinent literature from the MEDLINE and Web of Science databases, and reviewed differences in rodent strain, anesthesia method, sex, surgical method, timing of surgery, and factors influencing postoperative evaluation. In particular, circadian rhythm was found to have a significant impact on the outcome of cerebral infarction in rodent models. This information will enable researchers to quickly and clearly select appropriate modeling methods, acquire reliable quantitative experimental results, and obtain basic data for fundamental mechanism research.

Inhibition of microRNA-9-5p and microRNA-128-3p can inhibit ischemic stroke-related cell death in vitro and in vivo.

Ischemic stroke is the major form of stroke and is accentuated by multiple comorbidities. It has been previously shown that different microRNAs (miRNAs) regulate separate aspects of ischemic stroke. Differential miRNA expression analysis in cerebrospinal fluid of stroke patients had revealed upregulation of miR-124-3p, miR-9-3p, miR-9-5p, and miR-128-3p. However, whether the overexpression is correlative or causative was not known. Here, using an in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) neuronal cell model, we saw OGD/R-induced injury was associated with significant upregulation of the aforementioned four miRNAs. Target gene prediction using in situ algorithms and gene set enrichment analysis revealed significant enrichment of FOXO and Relaxin signaling pathways and regulatory processes associated with endothelial cell migration, which are all known to associate with apoptotic pathways. In situ protein-protein interaction network analysis confirmed the findings of gene set enrichment analysis. TUNEL analysis showed that OGD/R-induced injury resulted in significant apoptosis, which was significantly inhibited in neuronal cells pretransfected with inhibitors of either miR-9-5p or miR-128-3p. Further testing in an in vivo middle cerebral artery occlusion (MCAO) mouse model of ischemic stroke showed that inhibiting miR-9-5p or miR-128-3p significantly decreases MCAO-induced infraction volume and inhibited apoptotic response as revealed by decreased cleaved Caspase-3 protein expression in immunohistochemical analysis. Combined inhibition of miR-9-5p and miR-128-3p resulted in a synergistic decrease in cell death and infraction volume in vitro and in vivo, respectively. Cumulatively, our results provide critical knowledge about the mechanism by which elevated miR-9-5p and miR-128-3p causes brain damage in ischemic stroke and provides evidence of them being attractive therapeutic targets.

[Effect of scalp acupuncture stimulation on cerebral infarct volume and expression of IL-10, IL-6 and IL-1ß in para-hippocampal gyrus in acute ischemic cerebrovascular disease rats].

OBJECTIVE: To observe the influence of scalp acupuncture on cerebral infarct size and expression of IL-10, IL-6, and IL-1ß in the para-hippocampal gyrus in acute ischemic cerebrovascular disease(AICD) rats, so as to investigate its mechanisms underlying improvement of AICD. METHODS: Forty-eight male SD rats were randomly allocated to normal control (control), AICD model, medication, and scalp acupuncture groups (n＝12 per group). The AICD model was established by occlusion of the middle cerebral artery (MCAO). Rats of the medication group received intraperitoneal injection of Ammonium 1-Pyrrolidinedithiocarbamate (APDC, 100 mg•kg－1•d－1), once daily for 7 days. Scalp acupuncture stimulation was applied to bilateral "Dingnieqianxiexian" (MS6) once daily for 7 days. Before and after intervention, the neurologic deficit score (NDS) and the neurological score (NS) were evaluated according to Longa's and Schäbitz's methods, respectively. At the end of the intervention, the para-hippocampal gyrus and whole brain were collected respectively. The expression levels of IL-10, IL-6 and IL-1ß in the para-hippocampal gyrus tissue were detected by immunohistochemistry, and the cerebral infarct volume of the brain was detected by triphenyltetrazollium chloride (TTC) staining after sectioning. RESULTS: Following modeling, the NDS, NS and the expression of IL-10, IL-6 and IL-1ß in para-hippocampal gyrus were significantly increased in the model group compared with the control group (P<0.01). After the intervention, the NDS, NS and infarct volume, and the expression levels of IL-6 and IL-1ß in the para-hippocampal gyrus were significantly decreased in both medication and scalp acupuncture groups compared with the model group (P<0.05), and the expression of IL-10 was further obviously up-regulated in the scalp acupuncture group (P<0.05) rather than in the medication group (P>0.05). The effect of scalp acupuncture was obviously superior to that of medication in up-regulating IL-10 expression level (P<0.05). No significant differences were found between the medication and scalp acupuncture groups in the levels of NDS, NS, infarct volume, IL-6 and IL-1ß proteins (P>0.05). CONCLUSION: Scalp acupuncture can improve neurological function and reduce infarct volume in AICD rats, which may be associated with its function in up-regulating the expression of IL-10 and in inhibiting the expression of IL-6 and IL-1ß to reduce inflammation reaction.

[Scalp-acupuncture improves neurological function by regulating expression of IL-10 mRNA, IL-6 mRNA and TNF-α of parahippocampal gyrus in cerebral ischemic rats].

OBJECTIVE: To observe the effect of scalp-acupuncture intervention on the expression of Interleukin (IL)-10 mRNA, IL-6 mRNA and tumor necrosis factor (TNF) - α in the parahippocampal gyrus of cerebral ischemia (CI) rats, so as to explore its molecular mechanisms underlying improvement of CI. METHODS: A total of 64 male SD rats were randomized into normal control, model, medication and scalp-acupuncture groups (n＝16 rats in each group). The focal CI model was established by middle cerebral artery occlusion. Intraperitoneal injection of Ammonium Pyrrolidine Dithiocarbamate (100 mg•kg－1•d－1) was administrated for rats in the medication group, once a day for 7 days. For rats of the scalp-acupuncture group, the acupuncture needles were rapidly inserted into bilateral Dingnieqianxiexian (MS6), followed by twirling the needles at 100 cycles/min for 1 min, once again every 10 min during 20 min' needle retention. The treatment was conducted once a day for 7 days. The neurologic deficit score (0－4 points, impaired consciousness, death, etc.) and neurological function score (motor, sensory and sensory tests, 0－10 points) were assessed according to Longa's (1989) and Schabitz's (2004) methods, respectively. The expression levels of IL-10 mRNA and IL-6 mRNA were detected by fluorescence quantitative PCR, and the expression of TNF-α was detected by immunohistochemistry. RESULTS: After modeling, the neurologic deficit and neurological function scores and the expression levels of IL-10 mRNA, IL-6 mRNA and TNF-α protein in the parahippocampus were significantly increased in the model group than in the normal control group (P<0.01). Following the intervention, the neurologic deficit and neurological function scores as well as IL-6 mRNA and TNF-α protein expression were significantly down-regulated in both scalp-acupuncture and medication groups (P<0.05), and the expression of IL-10 mRNA was obviously increased (P<0.05) relevant to the model group. CONCLUSION: Scalp-acupuncture can improve neurologic function in CI rats, which is related to its effects in up-regulating the expression of IL-10, then down-regulating the expression of IL-6 and TNF-α (reducing inflammatory response) in the parahippocampal gyrus.

[Scalp-acupuncture Improves Neurological Function by Regulating Expression of NF-κB p 65 mRNA, IκB mRNA and IL-1 ß and TNF-α in Parahippocampal Gyrus of Cerebral Ischemia Rats].

OBJECTIVE: To observe the effect of scalp-acupuncture intervention on the expression of parahippocampal factor-κB p 65 mRNA (NF-κB p 65 mRNA), IκB mRNA, interleukin-1 ß (IL-1 ß) and tumor necrosis factor-α (TNF-α) in rats with cerebral ischemia (CI), so as to investigate its molecular mechanisms underlying improving CI by reducing inflammatory response. METHODS: A total of 64 SD rats were randomized into normal control, model, medication and scalp-acupuncture groups, with 16 rats in each group. The focal CI model was established by middle cerebral artery occlusion (MCAO). Intraperitoneal injection of Pyrrolidine Dithiocarbamate (100 mg•kg－1•d－1) was administrated for rats in the medication group, once a day for 7 days. For rats of the scalp-acupuncture group, the acupuncture needles were rapidly inserted into bilateral Dingnieqianxiexian (MS 6) and Dingniehouxiexian (MS 7), followed by twirling the needles at 200 cycles/min for 1 min, once again every 10 min during 30 min's needle retention. The treatment was conducted once a day for 7 days. The neurologic deficit score (0－5 points, impaired consciousness, death, etc.) and neurological function score (motor, sensory and sensory tests, 0－10 points) were assessed according to Longa's (1989) and Schäbitz's (2004) methods, respectively. The expression levels of NF-κB p 65 mRNA and IκB mRNA in the parahippocampus gyrus tissue were detected by fluorescence quantitative reverse transcription-PCR, and IL-1 ß and TNF-α proteins in the parahippocampus gyrus tissue were detected by immunohistochemistry. RESULTS: After modeling, the neurologic deficit and neurological function scores and the expression levels of NF-κB p 65 mRNA, IL-1 ß and TNF-α in the parahip-pocampus were significantly increased in the model group than in the normal group (P<0.01), while the expression of IκB mRNA was considerably down-regulated (P<0.01). Following treatment intervention, the neurologic deficit and neurological function scores as well as NF-κB p 65 mRNA, and IL-1 ß and TNF-α protein expression were significantly decreased in both scalp-acupuncture and medication groups compared with the model group (P<0.05, P<0.01), and the decreased expression of IκB mRNA was obviously increased (P<0.05)．. CONCLUSION: Scalp-acupuncture can improve neurologic function in cerebral ischemic rats, which is related with its effects in up-regulating the expression of IκB to inhibit the dissociation of NF-κB, then decreasing the expression of IL-1 ß and TNF-α (reducing inflammatory response) in the parahippocampal gyrus tissue.

Apolipoprotein E polymorphisms are associated with ischemic stroke susceptibility in a Northwest China Han population.

Ischemic stroke (IS), the leading neurology cause of death and disability worldwide, is influenced by gene polymorphisms. To explore the association between IS and Apolipoprotein E (APOE) gene polymorphisms, a case-control study containing 513 IS patients and 514 controls without IS was conducted in a Northwest China Han population. MassARRAY iPLEX system was applied to determine the APOE polymorphisms according to the alleles of two single nucleotide polymorphisms (SNPs) of APOE, rs429358, and rs7412. The results showed that rs429358 and rs7412 were in Hardy-Weinberg equilibrium (HWE) in both cases and controls groups. APOE ε4 allele, ε4/ε4 genotype, and ε4-containing genotypes were associated with IS. According to the results of Trial of ORG 10172 in Acute Stroke Treatment (TOAST) classification system, APOE ε2 allele, ε4 allele, and ε4/ε4 genotype were associated with large artery atherosclerosis IS subtypes. In addition, the results also indicated that the ε4 allele related to undetermined IS and ε4/ε4 genotype was related to small vessel disease IS. Compared with subjects with non-ε4-containing genotypes, the total cholesterol (TC) and low-density lipoprotein (LDL) level in blood and the proportion of cardiopath history were higher in all subjects with ε4-containing genotypes. Besides, the triacylglycerides (TG) level in blood was higher in controls with ε4-containing genotypes. In conclusion, in a Northwest China Han population, APOE ε4 allele was associated with blood lipid level. The TC and LDL levels were the independent risk factors for IS. APOE was a risk gene for IS, but not independent, especially for large artery atherosclerosis IS.

[Effect of Scalp-acupuncture Treatment on Levels of Serum High-sensitivity C-reactive Protein, and Pro-inflammatory Cytokines in Patients with Acute Cerebral Infarction].

OBJECTIVE: To observe the influence of scalp acupuncture on levels of high-sensitivity C-reactive protein (hs-CRP), TNF-α, IL-6, and IL-1ß levels in patients with acute cerebral infarction (ACl), so as to investigate its mechanism underlying improvement of ACI. METHODS: A total of 61 patients with ACI were randomly allocated to scalp acupuncture group (n = 31) and control (medication) group (n = 30). The patients of the control group were routinely treated by administration of Aspirin, Danhong injection, Cytidine Diphosphate for neurotrophy, blood pressure-control and blood-fat lowering medicines, etc., while those of the scalp acupuncture group were treated by routine treatment with the medicines mentioned above plus daily scalp acupuncture stimulation of bilateral Dingnieqianxiexian [MS 6, penetrative needling from Qianding (GV 21) to Xuanli (GB 6)] and Dingniehouxiexian [MS 7, from Baihui (GV 20) to Qubin (GB 7)]. The treatment was conducted once daily for 7 days. Serum hs-CRP, TNF-α, IL-6, and IL-1ß contents were assayed by using enzyme linked immunosorbnent assay (ELISA). The therapeutic effects of scalp acupuncture were evaluated by using clinical neurological disfunction scale (NDS, 0-45 points for consciousness, gazing, facial palsy, speech, myodynamia, walking-ability). RESULTS: (1) Of the 30 and 31 cases in the control and scalp acupuncture groups, 5 (16.7%) and 8 (25.8%) were basically controlled, 9 (30.0%) and 16 (51.6%) experienced remarkable improvement in their symptoms, 12 (40.0%) and 6 (19.4%) were improved, 4 (13.3%) and 1(3.2%) failed, with the effective rates being 86.7% and 96.8%, respectively. The increased levels of serum hs-CRP, TNF-α, IL-6 and IL-1ß in ACl patients were reversed on the 3rd and 7th day after scalp acupuncture treatment (P < 0.05, P < 0.01). (2) A positive correlation existed between the NDS score and the serum levels of hs-CRP (r = 0.497, P < 0.01). (3) NDS scores were obviously decreased in both groups on the 7th day after the treatment compared with their baseline data (P < 0.05, P < 0.01). CONCLUSION: Scalp acupuncture treatment can improve the ACI patients' clinical symptoms, probably by reducing ACl induced inflammatory reactions.

[Effect of Scalp-acupuncture Stimulation on Neurological Function and Expression of ASIC 1 a and ASIC 2 b of Hippocampal CA 1 Region in Cerebral Ischemia Rats].

OBJECTIVE: To observe the influence of scalp-acupuncture on the expression of acid-sensing ion channels (ASICs) 1 a and 2 b of hippocampal CA 1 region in cerebral ischemia (CI) rats, so as to investigate its mechanism underlying improvement of ischemic stroke. METHODS: Thirty-two male SD rats were randomly allocated to normal control, model, scalp-acupuncture and Amiloride group (n=8 in each group). The model of focal CI was established by middle cerebral artery occlusion (MCAO). Scalp acupuncture stimulation was applied to bilateral Dingnieqianxiexian (MS 6) and Dingniehouxiexian (MS 7), once daily for 7 days. Rats of the Amiloride group were fed with Amiloride solution, twice a day for 7 days, and those of the normal control and model groups were grabbled and fixed in the same way with the acupuncture and Amiloride groups. The neurological deficit score was given according to Longa's method. The expression of hippocampal ASIC 1 a and ASIC 2 b was detected by immunohistochemistry, and the Ca2+ concentration in the hippocampal tissue assayed using flowing cytometry. RESULTS: After the intervention, the neurological deficit score of both the scalp-acupuncture and Amiloride groups were significantly decreased in comparison with pre-treatment (P<0.01), and the effect of scalp-acupuncture was markedly superior to that of Amiloride in reducing neurological deficit score (P<0.05). The expression of ASIC 1 a and ASIC 2 b in the hippocampal CA 1 region and hip-pocampal Ca2+ concentration were significantly up-regulated in the model group compared with the normal control group (P<0.01), and obviously down-regulated in both scalp-acupuncture and Amiloride groups (P<0.01, P<0.05),without significant differences between the two treatment groups in the ASIC 1 a and ASIC 2 b expression and Ca2+ concentration (P>0.05). CONCLUSIONS: Scalp-acupuncture stimulation can improve neurological function in CI rats, which may be related to its effects in suppressing the increased expression of hippocampal ASIC 1 a and ASIC 2 b proteins and in reducing calcium overload in hip-pocampal neurocytes.

Atorvastatin calcium in combination with methylprednisolone for the treatment of multiple sclerosis relapse.

This study aimed to investigate the efficacy of combined atorvastatin calcium and methylprednisolone for the treatment of multiple sclerosis relapse. Patients with multiple sclerosis (MS) at the relapse phase were randomized to receive either combined treatment of atorvastatin calcium and methylprednisolone (n = 19) or methylprednisolone alone (n = 19). Expanded Disability Status Scale (EDSS) was administered at baseline, 1 week, 2 weeks, 4 weeks, 3 months, and 6 months after treatment initiation. The number and volume of brain lesions were evaluated using magnetic resonance imaging at baseline and 6 months. The levels of IL-13, IL-35, IFN-γ, and IL-10 in the cerebrospinal fluid were examined using the enzyme-linked immunosorbent assay method. There was no significant difference in EDSS scores at 1, 2, and 4 weeks. At 3 and 6 months, the combined treatment group showed significantly lower EDSS scores than the monotherapy group (P < 0.05). The number and volume of brain lesions in the combined treatment group were significantly lower than the monotherapy group at 6 months (P < 0.001). The mean time to relapse was significantly extended in the combined treatment group than the monotherapy group (P < 0.001). At 2 and 4 weeks, the combined treatment group had significantly higher levels of IL-13, IL-35, and IL-10 in the cerebrospinal fluid than the monotherapy group (P < 0.05), but significantly lower level of IFN-γ (P < 0.001). The levels of IL-13 and IL-10 in the combined treatment group were positively correlated with EDSS scores (r = 0.632, P = 0.001; r = 0.731, P = 0.002). Combined treatment with atorvastatin calcium and methylprednisolone can improve the outcomes of MS relapse compared with glucocorticosteroid alone.

Two new neuroprotective phenolic compounds from Gastrodia elata.

Two new phenolic compounds 4-(4'-hydroxybenzyl) phenyl glucoside (gastrodin B, 1) and 1'-hydroxymethyl-phenyl 4-hydroxy-3-(4″-hydroxybenzyl) benzyl ether (gastrol B, 2) were isolated from the rhizomes of Gastrodia elata. Their structures were elucidated on the basis of spectroscopic data and chemical reaction. All compounds exhibited potent neuroprotective activity against H2O2-induced PC12 cell damage.