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Objective:To establish right middle cerebral artery occlusion(MCAO)model in rats and to investigate the mechanism underlying motor function regulation by transcranial alternating current stimulation(tACS)intervention.Furthermore,to dynamically observe the effects of electroacupuncture combined with tACS on neurological defi-cit scores(NDS),cerebral blood flow,inflammatory-cell apoptosis gene of MCAO model rats and to explore the mechanism of cerebral and neural regulation on motor function rehabilitation after cerebral ischemia reperfusion. Method:Forty SD rats were randomly divided into sham-operation group(S group),model group(M group),electroacupuncture group(EA group),transcranial alternating current stimulation group(T group)and electroacupuncture combined with transcranial alternating current stimulation group(EA+T group).After 2 h ischemia-reperfusion,EA group was given bilateral Qu chi(LI 11)and Zu san li(ST 36)electroacupuncture under anesthesia.Right Ml was selected for tACS in T group.EA+T group was treated with EA and tACS to-gether.S and M group were treated with anesthesia for 30min per time,for 7 days.The data from before modling(B)to after modling(D7)were recorded,including neurological deficit score and blood flow of the right middle cerebral artery by laser doppler flowmetry.RT-PCR was used to analyze inflammation-cell apopto-sis gene expression at D7. Result:Neurological deficit score:at 2h,D1,M group,EA group,T group and EA+T group increased signifi-cantly compared with other time(P＜0.05).At D3,D5,D7,S group decreased significantly compared with oth-er time(P＜0.05),while M group increased significantly compared with other group(P＜0.05).EA group,T group and EA+T group were significantly different in all times(P＜0.05).At 2h,D1,D3,D5,D7,M group increased significantly compared with B.At D1,D3,D5,D7,NDS decreased significantly compared with that at 2h(P＜0.05).Blood flow:EA+T group and S group decreased at 2h,increased at D1 and decreased at D3.EA group increased at D3.However,M group decreased significantly compared with other group at D1,D3 and D5(P＜0.05).RT-PCR:motor cortex △Ct analysis:Caspase 12 in EA+T group decreased significantly compared with T group(P＜0.05),IL-1β and NLRPla in EA group and T group decreased significantly com-pared with those in S group(P＜0.05).2-△△Ct analysis in ischemic region:ATF4 in M group increased significant-ly compared with that in other groups(P＜0.05);Bcl 2 in M group increased significantly compared with that in S group,EA group and EA+T group(P＜0.05);Bax,Caspase 12,C-fos in M group increased significantly compared with those in S Group(P＜0.05). Conclusion:Electroacupuncture combined with tACS can modulate the inflammatory response and inhibit cell apoptosis through regulating ATF4、Bcl-2、Bax、Caspase 12、C-fos and can be a new strategy for the treatment of ischemic stroke.
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Objective:To explore the evaluation of dual-parameter three dimension arterial spin labelling(3D-ASL)perfusion imaging on blood-supply situation of patients with chronic middle cerebral artery occlusion(CMCAO)and the relationship between that and cerebral infarction area.Methods:A total of 112 patients with unilateral CMCAO admitted to Handan Central Hospital from April 2019 to December 2021 were selected,and all of them were divided into a compensatory group(50 cases)with anterior cerebral artery(ACA)leptomeningeal anastomoses(LMA)and an uncompensated group(62 cases)according to the results of digital subtraction angiography(DSA)examination.The results of diffusion weighted imaging(DWI),magnetic resonance angiography(MRA)and dual-parameter 3D-ASL detection were respectively analyzed,and the clinical data,3D-ASL parameters and the incidence of cerebral infarction between the two groups were compared.The influence factors of compensation were further analyzed.The receiver operating characteristics(ROC)curve of LMA diagnostic value of CMCAO patients was drawn according to cerebral blood flow values[post label delay(PLD)=1.5 s,2.5 s)].The 3D-ASL parameters of patients with different cerebral infarction areas were compared,and the relationship between 3D-ASL parameters and cerebral infarction area was compared.Results:The apparent diffusion coefficient(ADC)at the side of lesion of CMCAO patients was(0.31±0.10),and cerebral blood flow values at 1.5s and 2.5s were respectively(25.67±4.25)and(54.09±4.49),which were significantly lower than those at the side of healthy,and the differences were statistically significant(t=27.591,34.210,3.913,P<0.05),respectively.The differences of cerebral blood flow values(1.5s and 2.5s)between compensatory group and uncompensated group were significant(t=5.584,4.090,P<0.05),respectively.The results of logistic regression analysis showed that age,stroke,cerebral infarction area and cerebral blood flow values(1.5 s and 2.5 s)were influencing factors on LMA compensation of CMCAO patients(OR=4.187,6.604,0.482,5.681,5.807,P<0.05),respectively.The ROC values showed that the area under curve(AUC)of 3D-ASL were respectively 0.720 and 0.812 in diagnosing LMA when PLD were respectively 1.5s and 2.5s.The proportion of normal and lacunar infarctions in the compensatory group was significantly higher than that in the uncompensated group,while the proportions of middle and small infarction,and large area infarctions of the compensatory group were significantly lower than those of the uncompensated group,and the difference was statistically significant(t=28.062,P<0.05).The difference in cerebral blood flow values(1.5s)among patients with different infarct areas was statistically significant(t=0.202,P<0.05).The cerebral blood flow value(1.5s)of 3D-ASL was negatively correlated with the area of cerebral infarction(r=-0.261,P<0.05).Conclusion:Dual parameter 3D-ASL can non-invasively and visually assess the compensatory status of LMA of patients with unilateral CMCAO.The blood flow perfusion of middle cerebral artery(MCA)at the side of lesion is related to the area of cerebral infarction.When the PLD is 1.5s,the sensitive response can be conducted on this,so as to provide objective and reliable basis for clinical diagnosis and treatment and curative effect.
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Aim To investigate the dynamic time-course changes in neuronal cytoskeleton after acute ischemia and reperfusion in rats. Methods Reperfusion was performedin rats by blocking the middle cerebralarteryfor 90 min, then therats wereobserved and collected at different time points. The brain damage wasobserved by Nissl staining,and neurobehavioural function was evaluated with neurological deficit score and forelimb placement test. The cellular changes in the alternations of cytoskeletal elements including microtubule associated protein 2 (MAP2) and neurofilament heavy chain (NF-H) were observed by immunohistochemistry staining and Western blot. Impaired axons, dendrites and cytoskeletal alternations were detected by electron microscope. Results Brain damage and neurobehavioural function were gradually aggravated with the prolongation of reperfusion. Brain damage appeared earlier and more severe in striatum than in cortex. Moreover, decreased MAP2-related and increased NF-H-related immunoreactive intensities were found in the ischemic areas. Impaired cytoskeletal arrangement and reduced dense were indicated. Damaged cytoskeletal components such as microtubules and neurofilament arrangement, decreased axonal filament density, and swelled dendrites were observed after cerebral ischemia reperfusion by ultrastructural observations. Conclusions Different brain regions have diverse tolerance to ischemia-reperfusion injury. Major elements of neuronal cytoskeleton show dynamic responses to ischemia and reperfusion, which may further contribute to brain damage and neurological impairment following MCAO and reperfusion.
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Aim To screen and study the expression of long non-coding RNA (IncRNA) in rats with middle cerebral artery occlusion (MCAO) with MCAO treated with Tao Hong Si Wu decoction (THSWD) and determine the possible molecular mechanism of THSWD in treating MCAO rats. Methods Three cerebral hemisphere tissue were obtained from the control group, MCAO group and MCAO + THSWD group. RNA sequencing technology was used to identify IncRNA gene expression in the three groups. THSWD-regulated IncRNA genes were identified, and then a THSWD-regu-lated IncRNA-mRNA network was constructed. MCODE plug-in units were used to identify the modules of IncRNA-mRNA networks. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) were used to analyze the enriched biological functions and signaling pathways. Cis- and trans-regulatory genes for THSWD-regulated IncRNAs were identified. Reverse transcription real-time quantitative pol-ymerase chain reaction (RT-qPCR) was used to verify IncRNAs. Molecular docking was used to identify IncRNA-mRNA network targets and pathway-associated proteins. Results In MCAO rats, THSWD regulated a total of 302 IncRNAs. Bioinformatics analysis suggested that some core IncRNAs might play an important role in the treatment of MCAO rats with THSWD, and we further found that THSWD might also treat MCAO rats through multiple pathways such as IncRNA-mRNA network and network-enriched complement and coagulation cascades. The results of molecular docking showed that the active compounds gallic acid and a-mygdalin of THSWD had a certain binding ability to protein targets. Conclusions THSWD can protect the brain injury of MCAO rats through IncRNA, which may provide new insights for the treatment of ischemic stroke with THSWD.
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BACKGROUND:The treatment of post-stroke dysphagia with Lipopharyngeal Qibi Formula has achieved good efficacy,and 5-hydroxytryptamine in peripheral serum and neurotransmitters in the nucleus tractus solitarius are closely related to swallowing.Therefore,this study was conducted to explore the modulatory effects of peripheral serum and nucleus tractus solitarius neurotransmitters in swallowing by using modern medical experimental methods such as molecular biology,thereby developing new ideas for the exploration of their mechanisms. OBJECTIVE:To verify the therapeutic effect of Lipopharyngeal Qibi Formula on post-stroke dysphagia and to investigate its mechanism of action. METHODS:Thirty-eight Sprague-Dawley rats were randomly divided into model group(n=14),treatment group(n=14)and sham-operated group(n=10).Animals in the model and treatment groups were modeled by reperfusion after 90 minutes of transient cerebral ischemia by wire bolus method.At 6 hours after modeling,neurological function was scored,and rats with a score of 2 were selected for subsequent experiments.The treatment group was given compound Lipopharyngeal Qibi Formula by gavage starting from the 2nd day after modeling and the remaining two groups were given normal saline by gavage.Changes in body mass,24-hour food and water intake were recorded on days 2,7,14 and 30.The swallowing initiation response time and the number of swallows were detected using a biosignal collector and a tonic transducer on days 14 and 30.After the swallowing test,the ischemic area of the brain in each group was measured by TTC staining.The expression of 5-hydroxytryptamine in the nucleus tractus solitarius of the medulla oblongata was measured by immunohistochemistry.The mRNA and protein expression levels of BCL-2 and BAX in the insula,premotor cortex,cingulate cortex and thalamus of rats in each group were measured by RT-PCR and Western blot,respectively. RESULTS AND CONCLUSION:Compared with the sham-operated group,the body mass,24-hour food intake and water intake were reduced,the swallow initiation response time was prolonged,and the number of swallows was reduced in the treatment and model groups at day 14 of gavage(P＜0.05).Compared with the model group,the body mass,24-hour food intake and water intake of rats were increased in the treatment group at day 30 of gavage(P＜0.05),but were still lower than those in the sham-operated group.Compared with the model group,the swallow initiation reaction time was shortened and the number of swallows increased in the treatment group,but the number of swallows was still significantly lower than that in the sham-operated group(P＜0.05).Cerebral ischemia area was reduced in the treatment group compared with the model group,and the number of 5-hydroxytryptamine-positive cells in the nucleus tractus solitarius of the medulla oblongata was increased in the treatment group compared with the model group,but it was still significantly lower than that in the sham-operated group(P＜0.05).Compared with the model group,the expression of BCL-2 mRNA and protein in the insula,cingulate cortex and thalamus of rats in the treatment group were significantly increased,the expression of BAX mRNA and protein were significantly decreased,and the BCL-2/BAX ratio was significantly increased(P＜0.05).To conclude,the Chinese herbal compound Lipopharyngeal Qibi Formula could improve the number of swallows and swallowing initiation response time,as well as 24-hour food intake,body mass and other swallowing-related indexes in rats with post-stroke dysphagia.The mechanism of action may be achieved by improving the area of cerebral ischemia,inhibiting the apoptosis of neuronal cells in the insula,cingulate cortex and thalamus of rats,thus improving the regulation of the higher centers on the medulla oblongata swallowing center,and regulating the level of 5-hydroxytryptamine in the nucleus tractus solitarius.
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OBJECTIVE To study the intervention effect and mechanism of Zhongfeng yure decoction on ischemic stroke model rats. METHODS Totally 85 rats were randomly divided into sham operation group （normal saline， n＝15）， model control group （normal saline， n＝18）， Nimodipine tablet group （positive control， 10.8 mg/kg， n＝18）， high-dose group of Zhongfeng yure decoction （20.52 g/kg， n＝17） and low-dose group of Zhongfeng yure decoction （5.13 g/kg， n＝17）， respectively. After 7 days of preventive continuous administration （once a day）， except for the sham operation group， the rats’ middle cerebral artery occlusion （MCAO） model was established by the modified suture method in other groups. After modeling， the rats in each group continued to be administered for 3 days. During experiment， general condition of the rats was observed， and the neurological function score was performed. After the last administration， the organ index was calculated， the cerebral infarction area and pathological changes of brain tissue were observed. The levels of tumor necrosis factor-α （TNF-α） and interleukin 6 （IL-6） in brain tissue and serum， and the average optical density value of caspase-3 and phosphorylated protein kinase B（p-AKT） protein in brain tissue were detected. RESULTS Three days after modeling， compared with sham operation group， the neurological function score， in brain tissue index， spleen tissue index， proportion of cerebral infarction area， the levels of TNF-α and IL-6 in brain tissue and serum， and the average optical density value of caspase-3 protein in brain tissue were significantly increased in the model control group （P＜0.05 or P＜0.01）； karyopyknosis， diffuse edema and other lesions appeared in brain tissue. Compared with the model control group， the above indexes in each administration group were improved to varying degrees. Among them， there were significant regression in brain tissue index， spleen tissue index， proportion of cerebral infarction area， TNF-α level in brain tissue and serum， and the average optical density values of caspase-3 protein and p-AKT protein in brain tissue of rats in high-dose group of Zhongfeng yure decoction （P＜0.05 or P＜0.01）. CONCLUSIONS Zhongfeng yure decoction has a certain intervention and therapeutic effect on MCAO model rats. The mechanism may be to reduce the secretion of inflammatory factors TNF-α and IL-6， down-regulate the expression of caspase-3 protein in ischemic brain tissue， up-regulate the expression of p-AKT protein， so as to protect the neurons.
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OBJECTIVES@#Restoration of blood circulation within "time window" is the principal treating goal for treating acute ischemic stroke. Previous studies revealed that delayed recanalization might cause serious ischemia/reperfusion injury. However, plenty of evidences showed delayed recanalization improved neurological outcomes in acute ischemic stroke. This study aims to explore the role of delayed recanalization on blood-brain barrier (BBB) in the penumbra (surrounding ischemic core) and neurological outcomes after middle cerebral artery occlusion (MCAO).@*METHODS@#Recanalization was performed on the 3rd day after MCAO. BBB disruption was tested by Western blotting, Evans blue dye, and immunofluorescence staining. Infarct volume and neurological outcomes were evaluated on the 7th day after MCAO. The expression of fibroblast growth factor 21 (FGF21), fibroblast growth factor receptor 1 (FGFR1), phosphatidylinositol-3-kinase (PI3K), and serine/threonine kinase (Akt) in the penumbra were observed by immunofluorescence staining and/or Western blotting.@*RESULTS@#The extraversion of Evans blue, IgG, and albumin increased surrounding ischemic core after MCAO, but significantly decreased after recanalization. The expression of Claudin-5, Occludin, and zona occludens 1 (ZO-1) decreased surrounding ischemic core after MCAO, but significantly increased after recanalization. Infarct volume reduced and neurological outcomes improved following recanalization (on the 7th day after MCAO). The expressions of Claudin-5, Occludin, and ZO-1 decreased surrounding ischemic core following MCAO, which were up-regulated corresponding to the increases of FGF21, p-FGFR1, PI3K, and p-Akt after recanalization. Intra-cerebroventricular injection of FGFR1 inhibitor SU5402 down-regulated the expression of PI3K, p-Akt, Occludin, Claudin-5, and ZO-1 in the penumbra, which weakened the beneficial effects of recanalization on neurological outcomes after MCAO.@*CONCLUSIONS@#Delayed recanalization on the 3rd day after MCAO increases endogenous FGF21 in the penumbra and activates FGFR1/PI3K/Akt pathway, which attenuates BBB disruption in the penumbra and improves neurobehavior in MCAO rats.
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Animais , Ratos , Barreira Hematoencefálica/metabolismo , Isquemia Encefálica , Claudina-5/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , AVC Isquêmico/metabolismo , Ocludina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Traumatismo por Reperfusão/metabolismoRESUMO
OBJECTIVE@#The present study aimed to evaluate the therapeutic effect and explore the underlying mechanisms of Longxue Tongluo Capsule (LTC) on ischemic stroke rats.@*METHODS@#Twenty-six rats were randomly divided into four groups, including sham group, sham + LTC group, MCAO group, and MCAO + LTC group. Ischemic stroke rats were simulated by middle cerebral artery occlusion (MCAO), and LTC treatment group were orally administrated with 300 mg/kg of LTC once daily for seven consecutive days. LTC therapy was validated in terms of neurobehavioral abnormality evaluation, cerebral infarct area, and histological assessments. The plasma metabolome comparisons amongst different groups were conducted by UHPLC-Q Exactive MS in combination with subsequent multivariate statistical analysis, aiming to finding the molecules in respond to the surgery or LTC treatment.@*RESULTS@#Intragastric administration of LTC significantly decreased not only the neurobehavioral abnormality scores but also the cerebral infarct area of MCAO rats. The interstitial edema, atrophy, and pyknosis of glial and neuronal cells occurred in the infarcted area, core area, and marginal area of cerebral cortex were improved after LTC treatment. A total of 13 potential biomarkers were observed, and Youden index of 11 biomarkers such as LysoPC, SM, and PE were more than 0.7, which were involved in neuroprotective process. The correlation and pathway analysis showed that LTC was beneficial to ischemic stroke rats via regulating glycerophospholipid and sphingolipid metabolism, together with nicotinate and nicotinamide metabolism. Heatmap and ternary analysis indicated the synergistic effect of carbohydrates and lipids may be induced by flavonoid intake from LTC.@*CONCLUSION@#The present study could provide evidence that metabolomics, as systematic approach, revealed its capacity to evaluate the holistic efficacy of TCM, and investigate the molecular mechanism underlying the clinical treatment of LTC on ischemic stroke.
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ObjectiveTo investigate the mechanism of Huazhuo Jiedu Huoxue Tongluo prescription in alleviating cerebral ischemia-reperfusion injury via regulating nerve cell autophagy based on c-Jun N-terminal kinase（JNK）signaling pathway . MethodSixty SD rats were randomly divided into 6 groups： sham group， middle cerebral artery occlusion/reperfusion （MCAO/R） group （model group）， Huazhuo Jiedu Huoxue Tongluo prescription group ［traditional Chinese medicine （TCM） group（25.0 g·kg-1）］， JNK inhibitor SP600125 （SP） group（5 mg·kg-1）， TCM+SP group and JNK agonist Anisomycin （Ani） group（15 mg·kg-1）. After 24 h of modeling， TCM group and TCM+SP group were given TCM decoction （ig） for 3 consecutive days， and the other groups were given equal volume of normal saline （ig）. Neurological deficit was evaluated by neurological function score and cerebral infarct volume was determined by 2，3，5-triphenyltetrazole chloride （TTC） staining. Hematoxylin-eosin （HE） staining and Nissl staining were used to observe the structural changes of brain tissue and the damage of neurons， respectively. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay （TUNEL） was performed to detect cell apoptosis. The ultrastructure of autophagosomes was observed by transmission electron microscope. Western blot was employed to detect the protein expressions of microtubule-associated protein 1 light chain 3A/B （LC3A/B）， autophagy related 5 （Atg5）， the ortholog of yeast Atg6 （Beclin1）， p62， B-cell lymphoma 2 （Bcl-2）， JNK， phosphorylated （p）-JNK and c-Jun in brain tissue. The mRNA expressions of LC3A/B， Beclin1， p62， Atg5， Bcl-2， JNK and c-Jun were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the sham group， the model group had elevated neurological deficit score （P<0.05）， enlarged cerebral infarct volume （P<0.05）and typical infarction manifestations formed in hippocampal region and its surrounding brain tissue. In addition， there were a large number of neuronal cell degeneration， necrosis， liquefaction， nucleus pyknosis and deep staining， and inflammatory cell infiltration in the cortex in the model group， and severe swelling of mitochondria， lysosomes， autophagosomes and autophagolysosomes were clearly seen under electron microscope. TUNEL positive cells were increased （P<0.05）， and cell apoptosis was severe. The nuclear membrane and nucleolus of neurons in brain tissue were blurred with discontinuous processes， and Nissl bodies in cytoplasm were stained light with reduced number. Western blot revealed that the model group had up-regulated protein expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK and c-Jun in brain tissue （P<0.05）， while down-regulated protein expressions of p62 and Bcl-2 （P<0.05）as compared with the sham group. Real-time PCR indicated that the mRNA expressions of LC3A/B， Beclin1， Atg5， JNK and c-Jun in the model group were higher （P<0.05） while the mRNA expressions of p62 and Bcl-2 were lower （P<0.05） than those in the sham group. Compared with the conditions in model group， the neurological deficit scores of TCM， SP and TCM+SP groups were lowered （P<0.05）， and the cerebral infarct volume was reduced （P<0.05）， with improved pathological status of brain tissue， especially in the TCM group. Furthermore， there were abundant and basically normal mitochondrial cristae， slightly dilated endoplasmic reticulum， slightly swollen golgi apparatus， slightly fused nuclear membrane， and few visible lysosomes， autophagosomes and autophagolysosomes. TUNEL positive cells were decreased （P<0.05）， displaying reduced apoptosis， especially in the TCM group. The nucleolus and nuclear membrane of neurons in brain tissue were discernible， and Nissl bodies in cytoplasm was reduced to a certain degree as compared with those in the model group. Western blot showed a decrease in the protein expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK and c-Jun in the TCM group ，the SP group，and the TCM+SP group（P<0.05），while an increase in the protein expressions of p62 in the TCM group and SP group（P<0.05），and an increase in the protein expressions of Bcl-2 in the TCM group and TCM+SP group. By Real-time PCR， the mRNA expressions of LC3A， LC3B， Beclin1， Atg5， JNK and c-Jun had a down-regulation（P<0.05） while the mRNA expression of p62 a up-regulation in the TCM group ，the SP group，and the TCM+SP group（P<0.05），and the mRNA expression of Bcl-2 a up-regulation in the TCM group and the TCM+SP group（P<0.05）.Scores of the Ani group were raised （P<0.05）， and infarct volume was increased significantly， with aggravated neuronal cell necrosis and obvious inflammatory infiltration. Moreover， there were neuronal nuclear membrane fusion with abnormal protrusion， increased heterochromatin aggregation in edge， severe mitochondrial swelling， endoplasmic reticulum expansion， increased lysosomes， increased intracytoplasmic vesicles， and visible autophagosomes and autophagolysosomes. TUNEL positive cells were increased （P<0.05）， representing severe apoptosis. The number of Nissl bodies dropped with light staining， and the nucleolus and nuclear membrane were blurred. Real-time PCR found that the mNRA expressions of Atg5， c-Jun， JNK were up-regulated （P<0.05），while Beclin1， p62， Bcl-2 were were down-regulated in the Ani group （P<0.05）. Compared with the TCM group and SP group，the protein expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK， c-Jun were decreased，and p62， Bcl-2 were increased in the Ani group（P<0.05）. Compared with the TCM group，the mRNA expressions of JNK mRNA had a down-regulation in the SP group and TCM+SP group，while LC3A， LC3B， Atg5， c-Jun， JNK had an up-regulation（P<0.05） and Bcl-2 had a down-regulation in the Ani group（P<0.05）. Compared with the SP group，the mRNA expressions of Atg5， c-Jun， JNK had an up-regulation（P<0.05）， and Beclin1， p62， Bcl-2 had a down-regulation in the Ani group（P<0.05）. ConclusionHuazhuo Jiedu Huoxue Tongluo prescription significantly up-regulates the protein and mRNA expressions of LC3A/B， Beclin1， Atg5， JNK， p-JNK and c-Jun， and down-regulates the protein and mRNA expressions of p62 and Bcl-2， suggesting that the prescription can inhibit autophagy through JNK signaling pathway to reduce ischemia/reperfusion injury in rats.
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Objective To explore the relationship between non-high density lipoprotein cholesterol(non-HDL-C)level and leptomeningeal collateral circulation in patients with acute middle cerebral artery occlusion.Methods A total of 85 patients with first-onset acute cerebral infarction with middle cerebral artery M1 segment occlusion were enrolled.According to the results of DSA,LMC circulation was assessed by American Society of Interventional and Therapeutic Neuroradiology/Society of Interventional Radiology Collateral Circulation Assess-ment System.All patients were assigned to better LMC circulation group(score 2～4,n = 30)and worse LMC circulation group(score 0～1,n = 55),and the levels of non-HDL-C were compared between the two groups.Results The levels of LDL-C and non-HDL-C in worse LMC circulation group were significantly higher than those of the better LMC circulation group(P = 0.026,P = 0.010).non-HDL-C was an independent risk factor for the worse LMC circulation(OR = 3.019,95%CI:1.053～8.658,P = 0.04).LMC circulatory score of patients was negatively correlated with the levels of non-HDL-C level(r =-0.228,P = 0.036).The AUC of non-HDL-C predicted for the worse LMC circulation was 0.638(95%CI:0.521～0.755,P = 0.036).Conclusions non-HDL-C in patients with acute cerebral infarction was significantly related to worse LMC circulation,and was a risk factor for worse LMC circulation.It is suggested that the higher expression of non-HDL-C could be used to predict worse LMC circulation as a serological indicator.
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To study the cognitive effects of diterpene ginkgolides (DG), transient middle cerebral artery occlusion (tMCAO)-induced rats were established. tMCAO-rats induced by suture method were divided into sham operation group, solvent control group, NBP group, DG group. The animal experiments in the present study were performed in accordance with the Ethical Guidelines of the Laboratory Animal Welfare Ethical Committee of Peking Union Medical College (00000646, 00000635). The effects of DG on tMCAO rats were evaluated by neurological severity score, cerebral infarction volume measurement, step-down and Morris water maze test. In the acute tMCAO rat model, 100 mg·kg-1 DG improved the neural score and infarction volume. In the chronic tMCAO rat model, DG 100 mg·kg-1 significantly improved the survival rate of tMCAO-induced rats. The Morris water maze results showed 100 mg·kg-1 DG decreased the latency of tMCAO-induced rats to find the platform, while the effect was weaker than the NBP. However, DG 30 mg·kg-1 did not show a significant effect. In conclusion, DG exerted a therapeutic effect on transient middle cerebral artery occlusion.
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To date, there have been three common methods for sampling the cerebral ischemic border zone in a rat model of transient middle cerebral artery occlusion (tMCAO): the "two o'clock method", the "diagonal method", and the "parallel line method". However, these methods have their own advantages and limitations. Here, we propose a modified technique (the "rectangular method") for sampling the ischemic border zone. A rat tMCAO model was prepared under the support of a compact small animal anesthesia machine. Cerebral blood flow was monitored by high-resolution laser Doppler to control the quality of modeling, and 2,3,5-triphenyl tetrazolium chloride (TTC) staining was used for cerebral infarction location assessment. Superoxide dismutase 2 (SOD2), cysteinyl aspartate specific proteinase (caspase)-3, caspase-9, and heat shock protein 70 (HSP70) were used to verify the reliability and reproducibility of the rectangular method. The expression of biomarkers (SOD2, caspase-3, caspase-9, and HSP70) in the traditional (two o'clock method after TTC staining) and modified (rectangular method) groups were increased. There were no significant differences between the groups. The rectangular method proposed herein is based on a modification of the diagonal method and parallel line method, which could provide a directly observable infarct borderline and a sufficient sampling area for subsequent experimental operations regardless of the cerebral infarct location. The assessed biomarkers (SOD2, caspase-3, caspase-9, and HSP70) demonstrated the reliability and reproducibility of the rectangular method, which may facilitate inter-laboratory comparisons.
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Objective:To explore the possible mechanism of exacerbation of anxiety-like behavior in db/db mice after distal middle cerebral artery occlusion (dMCAO).Methods:The db/db mice was used to establish a type 2 diabetes mellitus model. Meanwhile, heterozygous db/+ mice and C57 wild-type (WT) mice were chosen as double control groups. Then a permanent distal middle cerebral artery occlusion model was employed as an acute ischemic stroke model. The blood glucose levels before and post-dMCAO surgery on day1, day3, and day5 were detected. The brain tissue loss at 35 days after stroke was measured by immunofluorescent staining of MAP2. The open-field test was performed to estimate anxiety-like behavior and general motor and exploring ability of the animals. Axons and myelin were immunostained with non-phosphorylated neurofilaments (SMI32) and myelin basic protein (MBP), respectively, to evaluate differences in white matter integrity in WT, db/+ and db/db mice 35 days after stroke. The correlation between SMI32/MBP and open field test parameters (time in center and corner) was analyzed. Flow cytometry was employed to detect the amount of T cells and B cells, including regulatory T cells (Tregs) in the brain tissue.Results:Blood glucose levels in db/db mice were significantly higher than db/+ mice and WT mice in both sham and dMCAO groups ( P<0.01). There was no significant difference in brain tissue loss 35 days post-stroke among db/db mice, db/+ mice, and WT mice. In the open field test, there were significant differences in the total distance of db/db mice, db/+ and WT mice in the sham and dMCAO groups. Db/db mice shorter than db/+ mice ( P<0.05), WT mice ( P<0.01), and db/+ mice shorter than WT mice ( P<0.05). There were significant time differences in the center among db/db, db/+, and WT mice in sham and dMCAO groups. In both the sham and dMCAO groups, db/db mice spent less time in the center area of the open field than WT mice ( P<0.01). In the sham group, db/+ mice spent less time in the center area than WT mice ( P<0.05). In dMCAO group, db/db mice spent less time in the center area than db/+mice ( P<0.05), and db/+ mice spent less time in the center area than WT mice ( P<0.01). For the time in the corner, in both the sham and dMCAO groups, db/db mice and db/+ mice consumed more time than WT mice ( P<0.01 or <0.05). In the dMCAO group, db/db mice spent more time in the corner than db/+ mice ( P<0.05). Referring to white matter injury, an increased SMI32/MBP ratio in EC area and CTX area (data was not shown in this article) after dMCAO in db/db, db/+ and WT mice were detected. In EC area, db/db mice have a higher SMI32 ratio than db/+ mice and WT mice: 4.24 ± 0.37 vs. 1.96 ± 0.37, 1.80 ± 0.36, both have significant differences ( P<0.01). For db/db mice and WT mice, the SMI32/MBP ratio negatively correlates with time in center and positive correlation with time in the corner. Three days after dMCAO, the total cells of CD 3+ T cells, CD 8+ cells, Tregs, in db/db mice group have significantly decreased compared to WT group: 4 079 ± 1 345 vs. 70 055 ± 3 374, 141.30 ± 28.36 vs. 2 714.00 ± 463.20, 148.00 ± 61.15 vs. 3 007.00 ± 639.90 ( P<0.01), while B cell has no change between two groups. Conclusions:By comparing the severity of anxiety-like behavior of db/db mice, the severity of white matter injury, and the number of T cells and B cells in brain tissue after dMCAO, immune-mediated brain white matter injury may aggravate db/db mice′s post-dMCAO anxiety-like behavior. Due to the gene dose effect, db/+ mice are not suitable as a control group for db/db mice in animal experiments involving anxiety-like behavior assessment.
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ObjectiveOn the basis of determining the protective effect of berberine （BBR） on cerebral ischemia， crucial transcription factors （TFs） of BBR against cerebral ischemia was identified by using transcriptome and proteome sequencing. MethodThe model of middle cerebral artery occlusion （MCAO） was established by thread embolization. The sham operation group， model group， low-dose group of BBR （dose of 37.5 mg·kg-1·d-1） and high-dose group of BBR （75 mg·kg-1·d-1） were set up. The rats were killed after continuous intragastric administration for 7 days. The pharmacodynamics was evaluated by Longa score and cerebral infarction rate， and the expressions of inflammatory cytokines， such as interleukin （IL）-1β， tumor necrosis factor （TNF）-α and monocyte chemotactic protein-1 （MCP-1） were measured by enzyme-linked immunosorbent assay （ELISA）. Then， RNA-Seq technique was used to detect the differentially expressed genes （DEGs） before and after BBR intervention， and DAVID 6.8 was used for enrichment analysis of DEGs. CatTFREs technique was used to detect differential TFs before and after BBR intervention， and DAVID 6.8 and STRING 11.0 were used for enrichment analysis and TFs association analysis. Finally， by integrating the activity of TFs and the changes of downstream target genes， crucial TFs were identified and the related regulatory network was constructed by Cytoscape 3.7.1. ResultCompared with the sham operation group， the neurological impairment was significant in the model group （P<0.01）， and compared with the model group， the low and high dose BBR groups could significantly reduce the neurological function damage （P<0.01） and decrease the rate of cerebral infarction （P<0.01）. Transcriptome data analysis showed that BBR was involved in the recovery process after cerebral ischemia mainly by affecting cell adhesion， brain development， neuron migration， calcium signaling pathway， cyclic adenosine monophosphate （cAMP） signaling pathway， inflammatory response and other related functions and signaling pathways. Proteomic data analysis showed that the differentially expressed TFs after BBR intervention interfered with cerebral ischemia mainly by regulating cell differentiation， immune system process， cell proliferation and other biological processes. In addition， integration analysis of TFs and DEGs revealed that transcription factor CP2-like 1 （TFCP2L1）， nuclear factor erythroid-2 like 1 （NFE2L1）， neurogenic differentiation protein 6 （NeuroD6） and POU domain， class 2， transcription factor 1 （POU2F1） were crucial TFs against cerebral ischemia-reperfusion injury mediated by BBR. ConclusionBBR has obvious protective effect on cerebral ischemia-reperfusion injury and its crucial TFs include TFCP2L1， NFE2L1， NeuroD6 and POU2F1.
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@#Objective To analyze the clinical and imaging characteristics of cerebral infarction patients with unilateral middle cerebral artery occlusion (MCAO),and to explore the pathogenesis of cerebral infarction and the related factors of hemorrhage transformation in different parts of MCAO.Methods A total of 159 MCAO patients with acute cerebral infarction were divided into proximal MCAO group and distal MCAO group according to the location of occlusion.The clinical and imaging characteristics of the two groups were compared,and the independent risk factors of MCAO hemorrhagic transformation (HT) in different locations were analyzed by a multivariate logistics regression analysis method.Results The proportion of hypertension and hyperlipidemia in proximal MCAO group was higher than that in distal MCAO group (P<0.05),the proportion of atrial fibrillation was significantly lower than that of distal MCAO group (P<0.01).The proportion of atherosclerotic type in proximal MCAO group was higher than that in distal MCAO group(P<0.01).The proportion of infarction involving basal ganglia and hemorrhage transformation (HT) in distal MCAO group was higher than that in proximal MCAO group (P<0.05),binary logistic regression analysis showed that different occlusion location of MCA and whether basal ganglia was involved were independent risk factors for hemorrhage transformation after infarction (P<0.05).Conclusion Proximal MCAO cerebral infarction is mostly caused by atherosclerotic aorta,while distal MCAO cerebral infarction is mostly caused by cardiogenic embolism.Patients with distal MCAO infarction involving basal ganglia are prone to hemorrhage transformation,which may be related to reperfusion injury.
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[Abstract] Objective To investigate the role of dihydromyricetin (DHM) in the treatment of ischemic stroke in rats, and to explore the effect of DHM on the expression of inflammasome. Methods The middle cerebral artery occlusion (MCAO) model was induced by endovascular suture method. The therapeutic effect and mechanism of DHM were investigated by Longa score, TTC staining, Nissl staining, immunohistochemical staining and Western bloting. Results After DHM treatment, the motor capacity of MCAO rats was significantly improved, the infarct volume was significantly reduced, the brain structure and neuron morphology were improved, and the expressions of nod-like receptor protein-3 (NLRP3) and interleukin-1(IL-1) decreased significantly. Conclusion DHM can down-regulate the expression of NLRP3 and thus reduces the cerebral infarction volume and improves neurobehavioral performance in MCAO rats.
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In this study, we performed high-throughput sequencing technology methylated RNA immunoprecipitation sequencing (MeRIP-seq), transcriptome sequencing (RNA-seq) and bioinformatics to analyze the differentially m6A-methylated and differentially expressed profile of circular RNA (circRNA) in middle cerebral artery occlusion/reperfusion (MCAO/R) model, which provided some scientific evidences for revealing the relationship between RNA epigenetic modification and cerebral ischemia reperfusion injury. The neurological deficit scores of mice were evaluated by the Longa score standard. TTC staining was used to detect cerebral infarction volumes, and dot blot was used for the quantification of m
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Objective:To investigate the evaluation value of one-stop whole-brain dynamic volume CT angiography and CT perfusion imaging (CTA-CTP) in the cynomolgus monkeys models of middle cerebral artery occlusion (MCAO).Methods:Ten adult cynomolgus monkeys were selected and examined by head and neck CTA-CTP and craniocerebral MRI to rule out craniocerebral space-occupying lesions or cerebrovascular malformation. Under guidance of digital substraction angiography (DSA), the right femoral artery was dissected and monkey autologous thrombosis was injected into the right middle cerebral artery (MCA) through microcatheter to prepare MCAO models. Whole brain DSA was performed intraoperatively to observe whether the model was successfully prepared, and head and neck CTA-CTP was performed 24 h and 7 d after modeling to determine the locations and brain blood flow changes of ischemic lesions. The monkeys were sacrificed 8 d after modeling, and the brain tissues were stained with 2,3,5-triphenyltetrazolium chloride (TTC).Results:Among the 10 cynomolgus monkeys, one was excluded because of preoperative cerebrovascular malformation, and one died of cerebral hernia caused by cerebral hemorrhage during the experiment. The remaining 8 MCAO models were successfully prepared. Intraoperative DSA orthography showed unclear M1 segment and distal branch of MCA. Brain CT scan 24 h and 7 d after modeling showed obvious cerebral ischemic lesions in the right MCA blood supply area, and the infarct extent 7 d after surgery was more obvious than that 24 h after surgery. CTA examination showed obvious blood flow interruption imaging in the in M1 segment of MCA on the right side, the distal vessels were not clearly displayed and the distal branches of the infarct side 7 d after surgery were obvious decreased as compared with those 24 h after surgery. CTP scan showed that the cerebral blood volume of the right cerebrum was obviously reduced as compared with that of the left cerebrum, which was consistent with the blood supply area of MCA; and the infarct cores and penumbra areas 7 d after surgery were obvious increased as compared with those 24 h after surgery. TTC staining showed that the ischemic lesions of the brain tissue on the slices were gray and involved multiple layers, and the range was roughly consistent with the infarction sites shown by DSA and CT imaging.Conclusion:One-stop whole brain dynamic volume CTA-CTP has good evaluation value in imaging findings in MCAO animal models.
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Objective:To explore the function of epidermal growth factor 8 in the polarization of microglia after ischemic brain injury and its mechanism.Methods:Forty C57BL/6 mice were randomly divided into a sham operation group, an ischemia group, a recombinant mouse milk fat globule epidermal growth factor 8 (rmMFG-E8) group and an rmMFG-E8 + colivelin TFA group, each of 10. The middle cerebral artery occlusion and reperfusion model (tMCAO) was established in all except the sham operation group. Right after the modelling the mice in the rmMFG-E8 group were immediately injected with 2μL of 0.4μg/μL of rmMFG-E8 into the ventricle contralateral to the cerebral infarction. The rmMFG-E8+ Colivelin TFA group was injected with the same dose of rmMFG-E8 plus 2μL of 5pmol/μL colivelin TFA. On the 1st, 3rd, 5th and 7th day after the modelling, neurological functioning was documented using behavioral tests. The volume proportion of the cerebral infarction was observed after tissue staining on the 7th day after the operation. The gene expression levels of M1 polarization marker-induced nitric oxide synthase (iNOS), M2 polarization marker arginase-1 (Arg1) and mouse chitinase-like molecule 3 (YM1) in the microglia were detected using real-time fluorescence quantitative polymerase chain reactions. The protein expression levels of MFG-E8, phosphorylated signal transduction and transcription factor 3 (p-STAT3) and cytokine signal transduction inhibitor-3 (SOCS3) were determined using western blotting.Results:The behavior tests revealed significant differences between the sham operation group and the other groups on all four days. Compared with the sham operation group, the average expression of MFG-E8 gene and its protein, Arg1 and Ym1, and the SOCS3/GAPDH protein ratio had decreased significantly in the ischemic group, while the average expression of iNOS and the p-STAT3/STAT3 protein ratio had increased significantly. On the 7th day after the modelling, compared with the ischemic group, the infarct volume was significantly smaller in the rmMFG-E8 group. The average expression of iNOS and the average p-STAT3/STAT3 ratio in the rmMFG-E8+ colivelin TFA group had increased significantly compared with the rmMFG-E8 group, while the average expression of Arg1 and Ym1, and the SOCS3/GAPDH ratio were significantly lower.Conclusion:MFG-E8 promotes the polarization of M2-type microglia after cerebral ischemia through STAT3 signaling, promoting the recovery of neurological functioning.
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This study aims to establish the high-performance liquid chromatography(HPLC) fingerprints of different batches of Notoginseng Radix et Rhizoma, determine their pharmacodynamic indexes of promoting blood circulation, and explore the spectrum-effect relationship between the chemical components of Notoginseng Radix et Rhizoma and the efficacy of promoting blood circulation. Firstly, the HPLC fingerprints of different batches of Notoginseng Radix et Rhizoma were established. Then, the pharmacodynamic indexes were determined after the capillary coagulation experiment and the cerebral ischemia-reperfusion in rats, including capillary coagulation time, percentage of cerebral ischemic area, cerebral water loss rate, and brain-body index. Afterward, the partial least-squares method was used to explore the spectrum-effect relationship between the chemical components of Notoginseng Radix et Rhizoma and the pharmacodynamic indexes. The results showed that this study successfully established the HPLC fingerprints of different batches of Notoginseng Radix et Rhizoma, found 23 common peaks, and identified 12 of them, all of which were saponins. The method was proved stable and reliable. Both the capillary coagulation experiment and the middle cerebral artery occlusion(MCAO)-induced cerebral ischemia-reperfusion experiment on rats revealed that there were obvious differences in the pharmacodynamic indexes of different batches of Notoginseng Radix et Rhizoma. The relationships between 23 common components of Notoginseng Radix et Rhizoma in different batches and the pharmacodynamic indexes were discussed by means of spectrum-effect correlation analysis, of which 17 components had positive effects while 6 components had negative effects on the pharmacodynamic indexes. This study provides a certain reference basis for the clinical rational use and quality control of Notoginseng Radix et Rhizoma.