| Ischemic stroke,induced by deprivation of blood flow to regions of the brain resulting in oxygen and glucose supply deficiency,is a leading cause of death and adult long-term disability worldwide.Importantly,with an aging population due to the increased life expectancy,the incidence and prevalence of stroke are predicted to rise even more,and stroke has become one of the most important health issues in the world.Despite the administration of intravenous thrombolysis is currently approved for acute ischemic stroke,its clinical application is still limited due to the narrow therapeutic window and safety concerns.As a result,most stroke patients will have residual neurological deficits.Therefore,this underscores the need to develop neurorestorative agents to reduce the degree of brain damage or restore brain function after stroke in parallel with advanced reperfusion strategies.Despite the fact that the central nervous system(CNS)has a limited repair capacity,some degree of spontaneous recovery from brain ischemia invariably occurs.This repair process involves angiogenesis,neurogenesis,and axonal sprouting and synaptogenesis.Neurogenesis has become a topic of concern,since it was reported that the brain has the capability to generate new neurons in areas called neurogenic niches.In a variety of mammals,including humans,the subventricular zone(SVZ)of the lateral ventricle and the subgranular zone(SGZ)of the hippocampal dentate gyrus(DG)in the adult brain are the two main neurogenic niches containing adult neural stem cells(NSCs)that proliferate,migrate and differentiate into mature neurons.After ischemic stroke,angiogenesis is an important process for forming new brain microvessels,which improve tissue microperfusion around the ischemic boundary region.And it was shown to affect neurogenesis and neuroblast migration in the adult ischemic brain.Meanwhile,cerebral ischemia induces neurogenesis in the SVZ,and the newly generated neurons migrate toward the cortical ischemic boundary where angiogenesis occurs,and during migration neuroblasts are closely associated with cerebral vessels.Recent experimental evidence has raised the possibility that neurological functional improvement after stroke may be induced through neuronal replacement by endogenous NSCs.Therefore,a promising field of investigation is focused on triggering and stimulating the self-repair system to replace dead neurons following an ischemic attack.The canonical Wnt/β-Catenin signaling pathway exerts roles during development of the mammalian central nervous system and continues to regulate diverse processes throughout adulthood,including maintenance and self-renewal of the stem cell pool,progenitor cell proliferation and adult neurogenesis.Considerable recent attention has focused on the critical effect of Wnt/β-Catenin signaling on hippocampal neurogenesis,while increasing studies suggest it also plays important roles in proliferation of neural progenitor cells and neurogenesis in the SVZ as well as in hippocampus.A growing body of evidence highlights the involvement of Wnt/β-Catenin signaling in stroke-induced neurogenesis in the SVZ of adult brain,thus participating in neuronal repair after stroke.Therefore,targeting Wnt/β-Catenin signaling activity promoting neurogenesis may be a novel approach in regenerative therapies to tissue repair and functional recovery.DL-3-n-butylphthalide(dl-NBP)is a chemically synthetic chiral compound containing L-and D-isomers of butylphthalide based on L-3-nbutylphthalide(l-NBP),which was originally extracted as a pure component from the seeds of Apium graveolens.NBP was approved by the State Food and Drug Administration of China(SFDA)of China at 2005 as a novel agent for the treatment of ischemic stroke in clinic.Clinical trials showed that NBP could significantly improve neurological and behavioral outcomes and cognitive function of patients with ischemic stroke,with a good safety and tolerability.Many basic studies have confirmed that NBP is a potentially beneficial and promising drug for treatment of ischemic stroke with multiple actions both in vitro and in vivo,including suppressing platelet aggregation and thrombosis,protecting mitochondrial function,regulating energy metabolism,decreasing neuronal apoptosis,reducing oxidative damage,down-regulating autophagy activity,inhibiting inflammation,and protecting the blood-brain barrier,all of which are associated with ischemic pathophysiological processes.Besides,the application of NBP could promote central angiogenesis and improve cerebral microcirculation in rodent models of stroke.However,there is still a paucity of data in previous studies regarding the exact role of NBP on neurogenesis in ischemic stroke.Adult male C57BL/6 mice were used and subjected to occlusion of distal branches of middle cerebral artery(dMCAO).In the present study,we designed to investigate whether dl-NBP improved neurological recovery and promoted endogenous neurogenesis in SVZ at the late phase of cerebral ischemia and whether the Wnt/β-Catenin pathway and neurotrophic factors are involved.Part one Effect of NBP on neurological recovery,neuroprotection and angiogenesis in experimental cerebral infarction miceObjective: To observe the neuroprotective and angiogenic effects of NBP on cerebral ischemic mice by measuring the neurological deficits,infarct volume,brain atrophy volume,cerebral blood flow and blood vessel density.Methods: Adult male C57BL/6 mice were used and subjected to dMCAO.Experiment 1: All the mice were randomly assigned to the following groups: Sham group(Sham): animals received sham operation and equal volume of 0.5% Tween-80;Vehicle group(Vehicle): animals received dMCAO and equal volume of 0.05% Tween-80;NBP low dose group(NBPL): animals received dMCAO and treated with 10 mg/kg NBP;NBP middle dose group(NBP-M): animals received dMCAO and treated with 20 mg/kg NBP;NBP high dose group(NBP-H): animals received dMCAO and treated with 40 mg/kg.NBP was administered intraperitoneally once daily until sacrificed or for 13 consecutive days beginning with the first day after surgery.We assessed neurologic deficits with Rota-Rod test and modified neurological severity score(mNSS)at base,3,7,14,21 and 28 days after stroke,in parallel with body weight changes.Experiment 2: All the mice were randomly assigned to the following groups: Vehicle group(Vehicle)and NBP middle dose group(NBP).We calculated infarct volume with TTC staining at 7 days,measured brain atrophy volume with HE staining at 28 days,examined cerebral blood flow(CBF)using a laser speckle imaging at baseline,0,7,14 and 28 days after stroke.Experiment 3: All the mice were randomly assigned to the following groups: Sham group(Sham),Sham + NBP group(Sham + NBP),Vehicle group(Vehicle)and NBP middle dose group(NBP).A fluorescent CD-31 staining technique was used for analyzing the microvessel density in the peri-ischemic cortex at 14 days after stroke.Results:1 In the Rota-Rod test,three dosages of NBP significantly improved neurologic functional recovery compared with Vehicle group on day 7(P < 0.01).However,afterwards only NBP-M group showed significant difference at 14,21 and 28 days(P < 0.01).Neurologic deficit as assessed by mNSS was not significantly different between Vehicle and NBP groups on day 3,7 after dMCAO.However,starting from 14 to 28 days after stroke,the neurologic deficit scores of mice treated with 20 mg/kg NBP were significantly lower than those of vehicle-treated mice(*P < 0.05,**P < 0.01).However mice in NBP-L and NBP-H groups did not show any functional difference at any time points.The body weight of mice in different groups that underwent surgery decreased rapidly during the first 3 days and then slowly recovered to baseline level at 14 or 21 days.However,the difference among the five groups was not statistically significant(P > 0.05).In view of the fact that the NBP-M group obviously improved the neurological deficit symptoms,we chose 20 mg/kg NBP to carry on the following experiment.2 After TTC staining,brain infarct volume was significantly smaller in NBP-treated mice than in vehicle-treated mice on day 7 after d MCAO(P < 0.05).After HE staining,there was no significant difference in atrophy volume between Vehicle group and NBP group on day 28 after dMCAO(P > 0.05).3 Cerebral perfusion on the contralateral side showed a slight decrease(about 83%)after dMCAO,but on the ipsilateral side,there was a significant decrease in CBF flux to about 38% from the baseline(P < 0.001).The CBF in the right MCA territory gradually recovered over time after dMCAO in both groups,reaching maximal recovery 14 days after dMCAO.And,NBP mice showed significantly higher CBF compared to vehicle mice during the course of recovery up to 14 d after dMCAO(P < 0.05).No significant difference in perfusion was seen in vehicle and NBP-treated mice at 0,7 and 28 days after stroke.CD31 immuno-fluorescence staining showed that microvessel density was significantly enhanced in the NBP-treated mice compared to vehicletreated mice at 14 days after dMCAO in the peri-ischemic cortex(P < 0.01).Part two Effect of NBP on neuronal proliferation in experimental cerebral infarction mice and its related mechanismsObjective: To evaluate the effect of NBP on neuronal proliferation and Wnt/β-Catenin pathway in the mouse model of focal cerebral ischemia by measuring the expression of neural stem cell markers and Wnt/β-Catenin pathway-related proteins.Methods: Adult male C57BL/6 mice were used and subjected to dMCAO.Experiment 1: The mice were randomly assigned to the following groups: Sham group(Sham),Sham + NBP group(Sham + NBP),Vehicle group(Vehicle)and NBP group(NBP).We used Brd U+ and Brd U+/Nestin+ as indicators to evaluate the effect of NBP on neuronal proliferation at 7,14 days after stroke.The role of Wnt signaling in therapeutic effects of NBP was measured by qRT-PCR and Western blot at 7 days after stroke.Experiment 2: The mice were randomly assigned to the following groups: Vehicle group(Vehicle),NBP group(NBP),DKK1 group(DKK1): animals treated with DKK1;DKK1 + NBP group(DKK1 + NBP): animals treated with DKK1 and 20 mg/kg NBP.The recombinant human DKK1 protein(rhDKK1)were used here to block canonical Wnt signaling.DKK1 was dissolved in sterile PBS at the concentration of 0.1 μg/μl and one microliter of the solutions was administrated into the ipsilateral cerebral ventricles at 1 and 4 days after ischemia through intracerebroventricular(i.c.v.)injection under anesthesia.We used Brd U+ and BrdU+/Nestin+ as indicators to evaluate the effect of NBP on neuronal proliferation at 7,14 days after stroke.The role of Wnt signaling in therapeutic effects of NBP was measured by qRT-PCR and Western blot at 7 days after stroke.Results:1 Focal ischemia significantly increased the number of Brd U and BrdU/Nestin-positive cells in the ipsilateral SVZ at 7 and 14 days postischemia as compared to sham-operated mice.After systemic administration of 20 mg/kg NBP,the number of Brd U and BrdU/Nestin-immunoreactive cells in the ipsilateral SVZ was significantly higher than that in vehicle-treated animals(P < 0.01).2 qRT-PCR analysis showed NBP treatment significantly enhanced the expression of the Wnt pathway genes such as Wnt-3a,β-Catenin,nuclear transcription factor(TCF-4)and the Wnt target gene(Cyclin D1),inhibited the gene expression of GSK-3β on day 7(*P < 0.05,**P < 0.01).Western blot analysis showed that NBP significantly enhanced the protein expression of Wnt-3a,TCF-4 and Cyclin D1,the ratio of p-GSK-3β/GSK-3β and decreased the ratio of p-β-Catenin/β-Catenin(P < 0.05).3 In the dMCAO mouse brain,qRT-PCR and Western blot showed that intracerebroventricular injection of DKK1 induced a significant effect of reducing the Wnt downstream signaling molecule β-Catenin(*P < 0.05,**P < 0.01).We showed that inhibition of Wnt signaling pathway by DKK1 caused decreased neuronal proliferation at 7 days after stroke(P < 0.05).However,after NBP treatment in DKK1 group,the number of BrdU and BrdU/Nestin-positive cells significantly enhanced(P < 0.01).4 qRT-PCR analysis showed that intracerebroventricular injection of DKK1 caused significantly enhanced GSK-3β,decreased β-Catenin,TCF-4 and Cyclin D1 gene levels involved in NSCs proliferation,while it was abolished by DKK1 + NBP treatment(▲P < 0.05,▲▲P < 0.01).Furthermore,Western blot analysis revealed that DKK1 treatment significantly decreased the ratio of p-GSK-3β/GSK-3β,reduced transcription factor and downstream target genes of the Wnt/β-Catenin pathway such as TCF-4 and Cyclin D1,and increased the ratio p-β-Catenin/β-Catenin,however DKK1 + NBP treatment recovered it(▲P < 0.05,▲▲P < 0.01).These results suggest that NBP is involved in the activation of the Wnt pathway,leading to increased neuronal proliferation.Part three Effect of NBP on neuronal migration and differentiation inexperimental cerebral infarction mice and its related mechanismsObjective: To evaluate the effect of NBP on neuronal migration and differentiation and neurotrophic factors in the mouse model of focal cerebral ischemia by measuring the expression of markers of stem cell migration and maturation as well as neurotrophin factorsMethods: Adult male C57BL/6 mice were used and subjected to dMCAO.Experiment 1: The mice were randomly assigned to the following groups: Sham group(Sham),Sham + NBP group(Sham + NBP),Vehicle group(Vehicle)and NBP group(NBP).We used DCX+ and BrdU+/DCX+ as indicators to evaluate the effect of NBP on neuronal migration at 14 days after stroke.The role of VEGF,BDNF and SDF-1 in therapeutic effects of NBP was measured by qRT-PCR,Western blot and immunofluorescence staining at 14 days after stroke.We used BrdU+/NeuN+ and Brd U+/GFAP+ as indicators to evaluate the effect of NBP on neuronal differentiation at 28 days after stroke.Results:1 Large numbers of DCX-positive cells were interspersed in the white matter between ipsilateral SVZ and ischemic cortex after ischemia whereas in the sham-operated group,DCX-positive cells were mainly located in SVZ.And treatment with 20 mg/kg NBP promoted more DCX-positive cells moving along corpus callosum from the SVZ toward the ischemic cortex(P < 0.01).Importantly,when analyzed separately,the density of DCX+ cells in each zone of three areas was higher in NBP than in vehicle-treated mice(P < 0.05).There were significantly more BrdU+/DCX+ cells in the vehicle mice than in the Sham and Sham + NBP mice.Moreover,the number of BrdU+/DCX+ cells was significantly increased in NBP group compared to Vehicle group(P < 0.01).Also,DCX-positive cells were located in the vicinity of CD31-labeled blood vessels along corpus callosum during migration at 14 days post-ischemia.2 qRT-PCR analysis showed that gene expression of VEGF and BDNF was significantly higher in NBP-treated mice than in vehicle-treated mice at 14 days after stroke(P < 0.05).No significance of SDF-1 gene level was found between Vehicle and NBP group(P > 0.05).Consistent with the results of qRT-PCR,Western blot results also further confirmed that protein expression of BDNF and VEGF was low in sham-operated mice,and that it was higher in NBP-treated dMCAO mice than in vehicle-treated dMCAO mice on day 14(P < 0.05).In the case of SDF-1 expression,there was no significant difference between Vehicle and NBP group.Immunofluorescence staining showed that the number of,VEGF and BDNF-positive cortical neurons in the ischemic penumbra was greater in the NBP-treated dMCAO mice than in the vehicle-treated mice(P < 0.05).Immunofluorescent double labeling showed that VEGF and BDNF seperately colocalized in CD31 positive cells in the peri-ischemic cortex,suggesting that these neurotrophic factors are partly derived from cerebral microvessels.3 No BrdU/Neu N-positive cells were detected in sham-operated mice.In ischemic mice that received NBP administration,there was a much greater increase in the number of NeuN/BrdU-positive cells compared with Vehicle group(P < 0.001).However,no difference of BrdU/GFAP-positive cells was found between vehicle and NBP-treated mice(P > 0.05).The integrated densities of GFAP positive cells in the peri-lesional cortex were significantly increased in Vehicle group compared to the sham-operated group(P < 0.001).However,it was not changed by NBP treatment in comparison with Vehicle group(P > 0.05).Conclusions:1 Adult male C57BL/6 mice were successfully established by dMCAO.We found that 20 mg/kg NBP administered intraperitoneally beginning with the first day after surgery significantly reduced the neurological deficits in mice and reduced infarct volume on day 7,suggesting that 20 mg/kg NBP exerted neuroprotective effect and significantly improved neurological function recovery in the chronic phase of cerebral ischemic mice.2 NBP treatment significantly increased the microvessel density in the ischemic penumbra region and improved the cerebral blood flow perfusion,suggesting that NBP has the effect of promoting angiogenesis.3 NBP treatment promoted post-stroke neuroral proliferation in SVZ via activation of Wnt/β-Catenin signaling pathway,recruited more endogenous neuroblast to migrate to injured region via increased expression of neurotrophic factors,and promoted differentiation into mature neurons,thereby improving neurological function recovery in the chronic phase of cerebral ischemic mice. |
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