Mechanism Of Dl-3-N-Butylphthalide(NBP) In Neural Plasticity Recovery After Focal Cerebral Ischemia

Objective:Cerebrovascular disease is the primary cause of death and the leading cause of disability in adults in our country.Surveys have shown that ischemic stroke has several characteristics such as high incidence,high morbidity,high recurrence rate,high mortality and short thrombolysis time window.That is why the treatment and rehabilitation of ischemic stroke has always been the most difficulty in research.Studies show that 80%of patients with ischemic strokes have permanent disability and even 30%of them cannot live on their own.Therefore,the search for effective drugs to promote the rehabilitation and prognosis of ischemic stroke patients and improve the quality of life is the fundamental purpose of treating ischemic stroke.Ischemic stroke can lead to rapid and severe brain damage,but researches data show that brain tissue has the ability to repair itself after injury.This self-repair ability is closely related to neuroplasticity.Neuroplasticity can be observed at multiple scales.Microstructure refers to axons,branches of dendrites,density of dendritic spines,synapse size and number,receptor density,certain brain regions,and neuron structure etc.From the macrostructure,behaviors,environment stimulation,thoughts and emotions may also change in neural plasticity.Neural plasticity is important for the recovery of healthy development,learning,memory and brain injury.Therefore,how to improve the recovery of neuroplasticity after ischemic stroke provides new ideas for clinical treatment.Dl-3-n-butylphthalide（NBP）,also known as NBP,is a new kind of ourself-developed drugs which can treat ischemic stroke.Previous studies have found that NBP can block multiple pathological processes of brain injury caused by ischemic stroke,and has strong brain protection.NBP can significantly reduce the infarct size of cerebral ischemia in experimental animals and improve brain energy metabolism.NBP can promote microcirculation and blood flow in ischemic brain regions.It can also reduce cerebral edema and inhibit nerve cell apoptosis.Continuous treatment of NBP or sequential therapy can significantly improve the neurological impairment.However,the mechanism of NBP on the recovery of neural plasticity after stroke is still unclear.Adult male C57BL/6 mice were used and subjected to occlusion of distal branches of middle cerebral artery（dMCAO）.In our study,we investigated how NBP improved the recovery of neuroplasticity after cerebral ischemia.The study was divided into four parts.The contents of each part are summarized as follows.Part 1 Effect of NBP on Improvement of Neurological Function in Cerebral Infarction miceObjective:To investigate the neuroprotective effects of NBP on cerebral ischemia in mice by measuring Rotarod Test and choose the effective dose.Delayed NBP treatment was given at different time points after cerebral infarction.Neuroprotective effects of NBP on subacute and recovery phases of cerebral infarction were studied by behavioral scoring method.Methods:Healthy adult male C57BL/6 mice,weighing 20-25 g,8-12weeks old,clean grade,were used and subjected to dMCAO.Experiment 1:Select effective dose for NBP treatment:C57BL/6 mice were randomly divided into the following groups:Vehicle group and NBP（10mg/kg/d,20mg/kg/d,40 mg/kg/d）groups.Intraperitoneal injection of NBP was started1 hour after surgery and consecutively injected for 14 days.Rotarod Test of nerve function scores were performed on each group of mice before and after dMCAO.Observation time point were 3 to 5 days,1 week,2 weeks,and 4weeks after dMCAO.And body weight changes were measured at the same time.Experiment 2:To study the neuroprotective effect of NBP on subacute and recovery phases of cerebral infarction:C57BL/6 mice were randomly divided into the following groups:Sham group,Vehicle group,and NBP（20mg/kg/d）group.Intraperitoneal injection of NBP was started at 1 hour,24hours,72 hours and 7days after surgery and consecutively injected for 14days in different groups.Observation time point were 3 to 5 days,1 week,2weeks,4 weeks,6 weeks and 8 weeks or even longer after d MCAO.Body weight changes were measured at the same time.Result:1.NBP had a parabolic dose-related effect on neurological function recovery after cerebral infarction:10 mg/kg/d,20 mg/kg/d,and 40 mg/kg/d were given at 1 hour after cerebral infarction,and continued for 14 days.The behavioral assessment of the Rotarod Test was performed at different time points after cerebral infarction.It was found that at 1 week,2 weeks,and 4weeks after surgery,the three doses of NBP group were significantly different from the Vehicle group（P<0.05）.NBP 10mg/kg/d had no significant effect on the recovery of neurological function at 3-5 days after dMCAO（P>0.05）.The behavioral results at any time point were higher in the NBP 20 mg/kg/d group than in the NBP 10 mg/kg/d group or NBP 40 mg/kg/d group.Deficit symptoms was not affected by body weight changes.So we chose 20 mg/kg/d NBP for follow-up experiments.2.Effects of different time points after cerebral infarction to NBP delayed treatment on neurological recovery.24h-NBP delayed treatment can improve subacute and convalescent neurological function recovery after cerebral infarction:24 hours after cerebral infarction,NBP 20 mg/kg/d was injected for 14 consecutive days.It was found that delayed treatment with 24h-NBP could not improve the recovery of neurological function at 3-5 days and 1 week postoperatively（P>0.05）.At 2weeks,3 weeks,4 weeks,6 weeks,and 8 weeks after dMCAO,behavioral results in 24h-NBP group were significantly improved and neurological recovery was promoted（P<0.05）.Delayed 72h-NBP treatment can improve the recovery of neurological function at decubation after cerebral infarction:intraperitoneal injection of NBP 20 mg/kg/d at 72 hours after cerebral infarction,continuous treatment for14 days.Delayed 72h-NBP treatment did not improve neurological recovery within 4 weeks after dMCAO（P>0.05）.At 4^th,6^th,and 8^th weeks postoperatively,the behavioral results were significantly improved and neurological recovery was promoted（P<0.05）.Delayed 7d-NBP treatment can improve the recovery of neurological function at decubation after cerebral infarction:intraperitoneal injection of NBP 20 mg/kg/d at day 7 after cerebral infarction,continuous treatment for 14days.It was found that the 7d-NBP delayed treatment began to have a statistically significant difference in behavioral results at the 14^thh post-op-erative weeks（P<0.05）.In summary,NBP treatment in hyperacute,acute and subacute phases after cerebral infarction can promote neurological recovery to varying degrees.However,the earlier NBP treatment after cerebral infarction,the better degree of neurological recovery.Part 2 Effect of NBP on Cerebral Blood Flow at Different Stages after Cerebral Infarction.Objective:Using a laser speckle imager to monitor the NBP-treated groups’cerebral cortical blood flow on both sides of the cortex after cerebral infarction in mice.Methods:C57BL/6 mice were randomly divided into the following groups:Vehicle group and NBP 20mg/kg/d group.Intraperitoneal injection of NBP was started at 1 hour after surgery and consecutively injected for 14 days.The dMCAO model was established by electrocautery and the cerebral blood flow（CBF）was monitored with a laser speckle imager.Laser speckle imager was used to monitor cerebral blood flow（CBF）at baseline,immediately after operation,and also on the 7^th,14^th,and 28^th day after dMCAO.It was observed whether NBP could improve both sides of CBP at cerebral cortex in brain mice or not.Result:1.NBP can improve cerebral blood flow in infarcted cerebral cortex at subacute stage after cerebral infarction.A laser speckle imager was used to monitor cortical blood flow in the cerebral infarction side.It was found that NBP could improve CBF in the infarcted cortex at 14 days after surgery（Vehicle vs.NBP group:80.55%±4.36%vs.97.53%±4.46%,P<0.05）.Immediately after surgery,7 days and28 days after dMCAO were no difference in CBF between Vehicle and NBP groups（P>0.05）,and all CBF at these time points were significantly lower than baseline（P<0.05）.This demonstrated that NBP could improve cerebral blood flow in infarcted cortex at subacute stage after cerebral infarction.2.Changes of cerebral cortical blood flow in the contralateral cortex at different periods after cerebral infarction.Cerebral blood flow in the contralateral cortex was measured immediately after surgery.CBF was also reduced by about 18%after cerebral infarction（P<0.05）.At 7 days and 28 days after the operation,CBF of contralateral cortex was still different from the baseline level（P<0.05）.On the14^th day after operation,cerebral cortical blood flow in the contralateral hemisphere reached its maximum recovery level（P>0.05）.It showed that the contralateral cortex was also affected after cerebral infarction.Cerebral blood flow in acute and convalescent periods was significantly decreased.The subacute cortical blood flow was recovered.Combined with changes in blood flow in the infarct cortex,it suggested that the subacute phase after cerebral infarction is the critical period for blood flow recovery.3.NBP can not improve CBF in contralateral cortex after cerebral infarction.After cerebral infarction,the cerebral cortex blood flow in contralateral cortex decreased in the acute and recovery phases and recovered in the subacute phase.However,no significant difference was observed in contralateral cerebral cortical blood flow between Vehicle and NBP groups（P>0.05）.This demonstrated that NBP could not improve cerebral blood flow in the contralateral cortex after cerebral infarctionIn summary,NBP can improve cerebral blood flow in infarcted cerebral cortex after cerebral infarction but does not cause changes in contralateral cortical blood flow.Part 3 Effect of NBP on Dendrites and Dendritic Spinal Plasticity in Central Nervous System after Cerebral InfarctionObjective:To verify the promoting effect of NBP on dendrites and dendritic spinal plasticity,Golgi-stain method was used to observe the growth trend of nerve dendrites,and density,classification,morphology of dendritic spines in different stages at peri-infarction after cerebral infarction.Experiment 1:Observe the growth trend of nerve dendrite at peri-infarction and the number of branches of dendrites:C57BL/6 mice were randomly divided into the following groups:Vehicle group and NBP20mg/kg/d group.Intraperitoneal injection of NBP was started at 1 hour after surgery and consecutively injected for 14 days.After Golgi staining,the growth trend of peri-infarct dendrites at 1 day,3 days,7 days,14 days and 28days after dMCAO was observed.After Golgi staining,the number of 3 levels of branches at infarct cavity and peri-infarct cortex was calculated on the 1^stt day after dMCAO.The number of 3 levels of branches at peri-infarct cortex was calculated on day 3,day 7,day 14,and day 28 after dMCAO.Experiment 2:To observe density,classification,and morphological changes of dendritic spines in cortical Layer V pyramidal neurons:C57BL/6mice were randomly divided into the following groups:Vehicle group and NBP 20mg/kg/d group.Intraperitoneal injection of NBP was started at 1 hour after surgery and consecutively injected for 14 days.Golgi-stain method was used to observe dendritic spine density,classification and morphological changes in of layer V pyramidal neurons in the peri-infarct cortex at 28 days after d MCAO.Result:1.The dendrites of the peri-infarct cortex in NBP-treated mice had a clear tendency to grow toward the infarct cavity in the acute,subacute,and convalescent phases after cerebral infarction.The results of Golgi staining showed that the dendrites in the peri-infarct cortex of both NBP and Vehicle group did not have a tendency to grow to the infarct center on the 1^st and 3^rd days after d MCAO.At 7 days,14 days and 28days after dMCAO,the dendrites of the peri-infarct cortex in NBP-treated mice had a clear tendency to grow toward the infarct cavity compared with the vehicle-treated mice.2.NBP promoted the increase of dendritic branches in different parts of the brain at different stages after cerebral infarction.NBP promoted dendrites recovery after cerebral infarction in hyperacute phaseNBP 20 mg/kg was intraperitoneally injected 1 hour after cerebral infarction in mice and Golgi staining was performed 24 hours after the operation.Compared with the Vehicle group,NBP group significantly increased the first and third level of dendritic branch in infarct cavity at day 1after dMCAO（P<0.05）.The third level of dendritic branches with NBP treatment mice in peri-infarct cortex at day 1 after dMCAO were increased compared with vehicle group（P<0.05）.NBP promoted the recovery of cortical dendrites at peri-infarct cortex in the acute,subacute and convalescent phases after cerebral infarctionDendritic branches of all the three levels in NBP treatment mice at 3 days and 14 days after dMCAO were increased compared with vehicle group（P<0.05）.Dendritic branches of the third level in NBP treatment mice at 7days after dMCAO were increased compared with vehicle group（P<0.05）.The first and third levels of dendritic branches with NBP treatment mice in peri-infarct cortex at day 28 after dMCAO were increased compared with vehicle group（P<0.05）.In summary,NBP promoted the increase of dendritic branches at different parts of cortex at hyperacute,acute,subacute,and convalescent phases after cerebral infarction.NBP can promote dendritic plasticity recovery after cerebral infarction.3.NBP promoted recovery of dendritic spine at recovery phase in peri-infarctin.The effect of NBP on the classification ratio of dendritic spines at recovery phase.Golgi-stain method was used to observe the classification ratio of dendritic spines in layer V pyramidal neurons at 28 days after operation.Compared with the Vehicle group,dendritic spines of layer V pyramidal neurons in the peri-infarct cortex in NBP group were no statistical difference between Mushroom type（M-type）,Thin type（T-type）and Mushroom type plus Thin type（M+T-type）（P>0.05）.This shows that NBP does not promote a certain type of dendritic spines,the classification ratio is roughly the same.The effect of NBP on the percentage of three types of dendritic spines at recovery phase.In terms of dendritic spine density,S-type,M-type,T-type,M+T-type,and total types in NBP group at 28 days were significantly higher than Vehicle group（P<0.05）.The effect of NBP on the dimension of dendritic spines at recovery phase.In terms of dendritic spine dimensions,length and head width of dendritic spines were significantly increased in NBP-treated mice compared with vehicle group（P<0.05）.There was no difference in the neck width between the two groups（P>0.05）.In summary,NBP promoted different types of dendritic spine density and increased dendritic spine length and head width during recovery period after cerebral infarction.It was suggested that NBP can promote plastic recovery of dendritic spines in recovery phase after cerebral infarction.Part 4 The mechanism of NBP promotes axon regeneration in experimental cerebral infarction in miceObjective:To observe the promoting effect of NBP on axons after recovery by morphological staining（BDA and immunofluorescence confocal technique）;To investigate the promoting effect of NBP on axons and the promoting effect of NBP on the growth of GAP43（GAP43）gene in cells.To observe the mechanism of NBP in promoting axonal plasticity,by measuring the changes of GAP43 and phosphorylated GAP43（P-GAP43）protein levels in cortex at different times after dMCAO.Experiment 1:To observe the promoting effect of NBP on axons after recovery by morphological staining（BDA）:C57BL/6 mice were randomly divided into the following groups:Vehicle group and NBP 20mg/kg/d group.Intraperitoneal injection of NBP was started at 1 hour after surgery and consecutively injected for 14 days.BDA cerebral cortical injections were performed on the 14th day after dMCAO.Immunohistochemical staining and immunofluorescence staining of brain tissue were performed to observe axonal regeneration at week 4 after dMCAO.Experiment 2:To observe of the effect of NBP on cortical axons in peri-infarction by immunofluorescence confocal technique.C57BL/6 mice were randomly divided into the following groups:Vehicle group and NBP20mg/kg/d group.Intraperitoneal injection of NBP was started at 1 hour after surgery and consecutively injected for 14 days.Immunofluorescence staining of brain tissue were performed to observe axonal regeneration at week 2 and week 4 after dMCAO.Experiment 3:To investigate the promoting effect of NBP on axons and the promoting effect of NBP on the growth of GAP43（GAP43）gene in cells.Primary cultured cortical neurons were obtained from E15-18 of C57BL/6mice.The expriment was divided into control group,different concentrations of NBP（0.1μM,1μM,10Μm,100μM）group.Neurons were treated with different drugs for 24 h.The effect of NBP on neurite outgrowth of cortical neurons was determinated byβ-Tubulin immunofluorescence staining;The expression of GAP43 was deteched by the immunofluorescence;Using RT-q PCR method to detect the expression levels of GAP43 gene NBP-indeuced neurons;.Experiment 4:To observe the mechanism of NBP in promoting axonal plasticity,by measuring the changes of GAP43 and phosphorylated GAP43（P-GAP43）protein levels in cortex at different times after dMCAO:C57BL/6 mice were randomly divided into the following groups:Sham group;Vehicle group and NBP（20mg/kg/d,40mg/kg/d）groups.Intraperitoneal injection was started at 1 hour after surgery and consecutively injected for 14 days.GAP43 and phosphorylated GAP43（P-GAP43）protein at cerebral cortex was measured by Western Blot in different groups on day 3,7,14 and 28 after d MCAO.Results:1.NBP promotes regeneration of axons from contralateral cortical to peri-infarct cortex during convalescence after cerebral infarction.Through immunohistochemical staining and immunofluorescence staining of brain tissue sections,it was found that neurons axons which ingested BDA were marked brown or red.On the ipsilesional cortex,axon fibers from the contralesional cortex can be seen in peri-infarct cortex.It was suggested that the NBP group promoted the growth of axon fibers from contralesional cortex to peri-infarct cortex.2.NBP promotes axonal plasticity recovery in peri-infarct cortex in subacute and convalescent phase.The results of immunofluorescence confocal staining showed that the growth of axons in the NBP group had a tendency regenerated from peri-infarction to infarct cavity compared with the Vehicle group on the 14th and28th days after dMCAO.3.NBP promote cortical neurite elongation1μM、10μM and 100μM NBP significantly increased neurite outgrowth in primary cortical neurons compared to untreated control（P<0.05）.In addition,NBP 10μM had a remarkable promoting effect.4.Expression of GAP43 in cultured cortical neuronsGAP43 was expressed in primary cortical neurons,suggesting that GAP43 could have biological effects on primary cortical neurons to regulate neurite outgrowth.5.NBP can promote the expression of GAP43 mRNA in the cultured cortical neurons.The expressions of GAP43 genes were significantly increased in NBP-treated group compared with Vehicle-treated group（P<0.05）.NBP may partially affect the neurite outgrowth of cortical neurons by regulating GAP43.6.To observe the mechanism of NBP in promoting axonal plasticity,by measuring the changes of GAP43 and phosphorylated GAP43（P-GAP43）protein levels in cortex at different times after dMCAO:The results of brain tissue protein levels after d MCAO in mice showed that GAP43 was no significant difference among the groups at each time（P>0.05）.The P-GAP43 protein in the NBP 20 mg/kg/d group was significantly increased on the 3^rd,7^th,and 14^th day compared with the Vehicle group（P<0.05）.The P-GAP43 protein in NBP 40 mg/kg/d group was significantly lower than the NBP 20 mg/kg/d group at 3^rd and 7^th days after dMCAO（P<0.05）.There was no significant difference in P-GAP43 between Sham group,Vehicle group and NBP group at day 28（P>0.05）.Conclusion:1.Continuous intraperitoneal injection of 20mg/kg/d NBP at 1 hour after dMCAO could significantly improve the recovery of nerve function in mice,suggesting that NBP has brain protection.2.Delayed NBP treatment can significantly improve the neurological function of mice with dMCAO,suggesting that delayed treatment with NBP still has brain protection.3.NBP can improve cerebral blood flow in infarcted cerebral cortex after cerebral infarction but does not cause changes in contralateral cortical blood flow.4.NBP can significantly increase the number of graded branches of dendrites in infarct cavity at peri-infarct cortex.NBP can promote dendritic spine density,dendritic spine length,and head width in the peri-infarct cortex.NBP can increase the growth of dendrites and axons in the peri-infarct cortex suggesting that NBP has the effect of promoting neuroplasticity.5.By BDA staining,it was found that NBP can promote the growth of axons in the contralateral cortical neurons to the ipsilateral cortex.By immunofluorescence staining,it was found that NBP promoted the growth of axons in the cortical neurons around the peri-infarct cortex.6.It was found that NBP can promote the growth of neuronal processes in cells.GAP43 can be detected in cortical neuron cells by cellular immunof-luorescence.Change of GAP43 in cells can be detected by RT-q PCR in NBP treatment,which suggesting that NBP may partially affect the neurite outgrowth of cortical neurons by regulating GAP43.7.NBP could up-regulate the expression of P-GAP43 protein.But NBP could not change the total expression of GAP43 protein.It was found that NBP may enhance the neural plasticity mechanism by promoting the increase of P-GAP43.