| Ischemic stroke is the leading cause of death and disability in adults worldwide.So far,the only drug approved by the FDA for the treatment of ischemic stroke is still recombinant tissue plasminogen activator(rtPA),but its use is limited to the 4.5 h narrow time window and the risk of intracranial hemorrhage.Most neuroprotective drugs find a mismatch between preclinical research and clinical trials,partly because of the lack of observation of the chronic brain repair process.Therefore,it is necessary to find an effective strategy to promote the recovery of long-term neurological function after ischemic stroke,not just the protection of neuronal damage in the hyperacute phase after ischemia.Stroke mainly causes neuronal damage in the brain gray matter,and nerve fibers damage in the white matter.After cerebral ischemic stroke,the brain starts to initiate a long and complex self-repair process,which mainly includes angiogenesis,neurogenesis,axonal sprouting and synapse regeneration.In the recovery process after injury,the nerve filaments in the white matter are rewired to promote the improvement of neurological function after ischemic stroke.Previous studies in the field have mainly focused on the protection of neurons in the gray matter.However,studies have found that the degree of gray matter damage is not always consistent with the degree of behavioral function impairment.The improvement of white matter function has been shown to be highly correlated with the recovery of neurological function after ischemic stroke.Therefore,more and more researches focus on intervention axonal plasticity in the white matter after ischemic stroke.Sonic Hedgehog(Shh),a secreted glycoprotein,is an endogenous agonist of the Hedgehog pathway.Shh initiates Shh signaling by binding to its cell surface receptor Patched-1(Ptch-1).When Shh protein is present,Ptch-1 is inactivated and releases its suppression of Smo.The transcription factor Gli-1 is activated and transferred to the nucleus,thereby inducing the transcription of Gli-1 targeted genes.Our recent studies have proved that the endogenous Hedgehog pathway is activated after ischemic stroke,which promotes the neurogenesis process in the subventricular region in animals,and promotes the growth of neurites of neurons in vitro.At the same time,the activation of Shh signaling pathway promotes axon regeneration after ischemic stroke.Therefore,Shh and its downstream signaling molecules can be used as potential therapeutic targets to stimulate axonal plasticity after ischemic stroke.Cerebroprotein hydrolysate(CH)is a mixture of low-molecular-weight neuropeptides derived from purified pig brain tissue.CH can easily penetrate biological membranes and has the ability to penetrate the blood-brain barrier,which makes CH an effective choice for clinical applications.In vivo,CH has been proven to promote neurogeneration,improve the long-term neurological function after ischemic stroke,reduce neuroinflammation,and inhibit the formation of oxygen free radicals.Importantly,several randomized clinical trials for patients also revealed the therapeutic value of CH in the field of ischemic stroke.However,the effect of CH on post-stroke axon plasticity in white matter remains unclear.In this study,we tried to explore the potential application value of a new cerebroprotein hydrolysate,CH1.We used healthy adult male C57BL/6 mice as the research object,and established a distal middle cerebral artery occlusion(dMCAO)model by electrocoagulation,aiming to evaluate whether cerebroprotein hydrolysate for injection(?)can improve neurological function after ischemic stroke and its related mechanisms.In terms of mechanism,we explored whether CH1 regulates white matter injury after ischemic stroke by activating the shh/ptch/Gli-1 pathway.The research is divided into three parts,and the contents of each part are summarized as follows.Part ?:Effect of cerebroprotein hydrolysate for injection(I)on the recovery of neurological function in mice with focal cerebral ischemic strokeObjective:By observing the neurological behavioral changes of mice with experimental cerebral ischemic stroke,we aim to evaluate the effect of CH1 on the recovery of neurological function after ischemic stroke,and screen the effective dose of CH1.By observing the cerebral infarct volume,cerebral atrophy volume,and Nissl staining and NeuN staining,we aim to evaluate the effect of CH1 on cerebral gray matter damage after ischemic stroke.Method:Using healthy adult male C57BL/6 mice as the research object,dMCAO models were established.dMCAO mice received intraperitoneal injections of CH1(5 mg/kg,10 mg/kg,15 mg/kg dissolved in saline)immediately after the operation until the mice were executed or for 14 consecutive days(once a day).Grouping for Experiment(1):Mice was randomly divided into 4 groups:Vehicle group:The animals received the dMCAO model,and daily intraperitoneal injection of equal volume of normal saline;Cerebroprotein hydrolysate for injection(?)low-dose group(CH1-L group):The animals received the dMCAO model and were given daily 5 mg/kg CH1 intraperitoneal injection;Cerebroprotein hydrolysate for injection(?)medium-dose group(CH1-M group):The animals received the dMCAO model and were given daily 10 mg/kg CH1 intraperitoneal injection;Cerebroprotein hydrolysate for injection(?)high-dose group(CH1-H group):The animals received the dMCAO model and were given 20 mg/kg CH1 intraperitoneal injection daily;Rotarod test,Adhesive removal test and mNSS neurological function score were performed before operation and 3,7,14,21,28 days after operation.Grouping for Experiment(2):Mice randomly divided into 3 groups:Sham group:animals received sham operation and daily intraperitoneal injection of equal volume of normal saline;Vehicle group:Animals received dMCAO model and daily intraperitoneal injection of equal volume of normal saline;CH1 group:The animals received the dMCAO model and were given daily 10 mg/kg CH1 intraperitoneal injection;TTC was used to measure the infarct volume 14 and 28 days after dMCAO models.Cortical width index(CWI)was used to measure brain atrophy on 14 and 28 days after dMCAO models.Immunofluorescence staining was used to evaluate the neuronal survival in the gray matter on 14 and 28 days after dMCAO models.Nissl staining was used to evaluate neuronal activity 14 and 28 days after dMCAO models.Result:1.Cerebroprotein hydrolysate for injection(?)promotes the recovery of neurological function in mice after cerebral infarction and improves behavioral scores.1.1 Rotarod experimentThe Rotarod experiment was used to measure the preoperative and postoperative movement and limb coordination ability of dMCAO mice.The results showed that,at the 3rd day after the dMCAO model,the latency on the rod of each group was significantly reduced,and there was no statistical difference between the groups.From the 7th day,the behavioral function of mice in each group began to gradually recover.Compared with mice in the vehicle group,mice in the CH1-M and CH1-H groups had a significant increase in the latency on the 14th and 28th days after dMCAO model(P<0.05)and the results has statistical difference.The above results indicate that compared with the Vehical group,CH1 administration at a dose of 10 mg/kg can show a significant improvement in neurological function,and this improvement is mainly manifested in the postoperative recovery period.Therefore,in follow-up experiments,we will mainly study the effects of CH1 at 10 mg/kg at 14 and 28 d after dMCAO and its related mechanisms.1.2 mNSS scoreThe mNSS results showed that the scores of the mice in each group were 0 before dMCAO.Meanwhile,all mice had severe neurological deficits 3 days after the cerebral ischemic stroke,and there was no statistical difference between groups.Compared with the vehicle group,the CH1(10 mg/kg)group significantly reduced the neurological deficits of dMCAO mice on the 7th and 14th day after dMCAO surgery(P<0.05).However,CH1 treatment had no effect mNSS score at 28 d after dMCAO.1.3 Adhesive removal testAdhesive removal test was performed on mice before dMCAO and 3,7,14 and 28 days after dMCAO to assess autosensory loss.3 days after dMCAO,there was obvious autosensory loss in the forelimbs innervated by the injured brain area of each mice compared with preoperative behavior,and there was no statistical difference in the contact and removal time in each group of mice.Compared with the vehicle group,the CH1-M group showed short contact time and removal time at 14 and 28 days after dMCAO.The CH1-H group showed a reduction in the contact time 14 days after dMCAO,and a shortened time in the removal time on both 14th and 28th days after dMCAO.The above results indicate that compared with the vehicle group,CH1 administration at a dose of 10 mg/kg show a significant improvement in neurological function,and this improvement is mainly manifested in the postoperative recovery period.Therefore,in the follow-up experiments,we will mainly study the effects of CH1 at 10 mg/kg at 14 and 28 d after dMCAO and its related mechanisms.2.Cerebroprotein hydrolysate for injection(?)can reduce cerebral infarct volume in mice with focal ischemic stroke2.1 Determination of cerebral infarct volumeThe cerebral infarct volume was measured at 14 and 28 d after dMCAO by TTC staining.The results showed that the cortex of the mice in Vehicle and CH1 groups appeared cerebral focal injury at 14 and 28 d after stroke,manifested as a multi-level white infarct area.Compared with the vehicle group,the cerebral infarct volume of CH1 mice was reduced 14 days after dMCAO(P<0.05).However,at 28 d post stroke,the cerebral infarct volume of CH1 mice was comparable with that of the vehicle group(P>0.05).2.2 Determination of brain atrophy volumeCompared with mice in the Vehicle group,the brain atrophy volume of CH1 mice was reduced 14 days after surgery(P<0.05).However,at 28 d post stroke,there was no significant difference in the brain atrophy volume of CH1 mice compared with the vehicle group(P>0.05).Conclusion:1.In this part of the experiment,a permanent distal middle cerebral artery occlusion model in mice was established by electrocoagulation.This model is simple to operate,reproducible,low in mortality,and high in stability.It perfectly simulates ischemic stroke in human.The pathogenesis of ischemic stroke is an ideal model for studying focal cerebral ischemia,and it is especially suitable for the observation of neurological function in the recovery period after ischemic stroke.2.By observing the neurobehavioral and neurohistology in mice,it is found that CH1 can significantly improve the motor nerve function of mice in the subacute and chronic phases after cerebral ischemic stroke.At the same time,CH1 can reduce the cerebral infarct volume and brain atrophy in the chronic phase after cerebral ischemic stroke.In addition,CH1 can penetrate the blood-brain barrier and may become a promising stroke drug.3.In the chronic phase of cerebral ischemia(28 d),CH1 has no effect on the infarct volume and cerebral cortex width,but it has improved neurobehavior.We need to continue to explore the reasons for this inconsistency.Part ?:Cerebroprotein hydrolysate for injection(?)reduces the white matter injury in the chronic phase of focal cerebral ischemic process and promotes white matter repairObjective:Through the above experiments,it can be seen that CH1 can improve the neurological behavioral function of mice at 14 and 28 d after dMCAO.However,histological measurements showed that CH1 only had a significant effect on the cerebral infarction volume and brain atrophy at 14 d after ischemic stroke while CH1 had no improvement on infarction volume and brain atrophy at 28 d after ischemic stroke.Therefore,we aim to explore the reasons for the difference between behavioral function and histological staining at 28 d after ischemic stroke.We speculate whether the improvement of CH1 on the neurological behavioral function of mice in the chronic phase is not caused by affecting gray matter,but by affecting white matter.Therefore,we respectively measured the integrity of gray matter by NeuN staining and Nissl staining,and measured the integrity of white matter by SMI-32/MBP staining.Methods:Using healthy adult male C57BL/6 mice as the research object,dMCAO models was established.dMCAO mice received a medium dose of CH1(10 mg/kg)intraperitoneal injection every day immediately after dMCAO,and until the brain tissues were obtained or for 14 consecutive days(once a day).Experimental grouping:The C57BL/6 mice were divided into 3 groups using a random method:Sham group:mice were intraperitoneally injected with the same amount of normal saline after sham operation;Vehicle group:mice were intraperitoneally injected with the same amount of normal saline after the dMCAO experiments;Cerebroprotein hydrolysate for injection(?)medium-dose group(CH1 group):mice were intraperitoneally injected with the same amount of 10 mg/kg CH1 after the dMCAO experiments.NeuN staining and Nissl staining were performed at 14 and 28 days after dMCAO,and SMI-32,MBP immunofluorescence and BDA antegrade tracing were performed on 28 days after dMCAO.Results:1.CH1 has no effect on the gray matter integrity of mice with 28 days after dMCAO.1.1 NeuN stainingWe used NeuN staining to evaluate the number of remaining neurons in the ischemic penumbra of mice with dMCAO.The results showed that compared with mice in the sham group,ischemic injury caused a significant decrease in the number of remaining neurons,and the results were statistical significance.Compared with mice in the vehicle group,the CH1 group promoted neuronal survival at 14 days after ischemic stroke,but the number of gray matter neurons at 28 days was not statistically significant compared with the vehicle group.1.2 Nissl stainingAfter cerebral ischemia,the cell arrangement is disordered,the number of Nissl bodies is significantly reduced,the interstitial space increases,and many vacuoles are formed in the cells.Compared with the sham group,neurons with abundant Nissl bodies were significantly reduced after focal cerebral ischemia,and the decrease of Nissl bodies was restored by CH1 administration on the 14th day after cerebral ischemia.However,at 28 d after cerebral ischemia,there was still no statistical significance between the vehicle group and the CH1 group.2.CH1 promotes the recovery of white matter integrity in the chronic phase of focal cerebral infarction in mice.We use the ratio of SMI32/MBP to explore the effect of CH1 on the whitematter integrity in the cortex and striatum in the chronic phase of dMCAO mice.The results showed that compared with the Sham group,theSMI-32/MBP ratio in the Vehicle group increased(P<0.05),that is,after cerebral ischemic stroke,severe white matter damage occurred in the cortex and striatum around the infarction.Compared with the Vehicle group,the CH1 group significantly reduced the value of SMI32/MBP in the CTX and STR regions,indicating that CH1 improved white matter integrity in the long-term repair process.3.CH1 enhanced axonal plasticity in mice after ischemic stroke.BDA staining was used to mark the ischemic penumbra area and the axonal elongation from the opposite side of the ischemia.The results showed that at 28 days after focal cerebral ischemia in mice,The area of BDA-positive neurons in the ischemic penumbra area and the number of axons that cross the midline from the contralateral side increased significantly.Compared with the Vehicle group,CH1 further increased the BDA positive area in the penumbra zone and the number of axons crossing the midline,and the results were statistically significant(P<0.05).At the same time,CH1 increased the protein expression level of GAP43 on the 28th day after dMCAO.Conclusions:1.CH1 promotes the recovery of white matter integrity in the chronic phase after focal cerebral ischemia,but has no effect on gray matter integrity.It is suggested that CH1 promotes the recovery of neurological function after cerebral ischemic stroke by improving the integrity of white matter.2.Endogenous axonal regeneration is initiated after focal cerebral ischemia.CH1 can further promote axonal regeneration in the chronic phase and promote the recovery of neurological function after cerebral ischemic stroke.Part ?:Cerebroprotein hydrolysate for injection(1)Promotes white matter repair through shh/Ptch/Gli-1 pathwayObjective:In order to explore the mechanism of CH1,we detected the effect of CH1 on the expression of shh pathway proteins and the nuclear translocation of Gli-1 in the ischemic penumbra area of dMCAO mice.We observed whether the shh pathway is involved in the neuroprotective effect of CH1 on mice with focal cerebral ischemia.In addition,we chose CYC as the inhibitor of the Shh pathway to observe whether the neuroprotection effects of CH1 still exists after the shh pathway is inhibited.Methods:The C57BL/6 mice were divided into 3 groups using a random method:Sham group:mice were intraperitoneally injected with the same amount of normal saline after sham operation;Vehicle group:mice were intraperitoneally injected with the same amount of normal saline after the dMCAO;Cerebroprotein hydrolysate for injection(?)medium-dose group(CH1 group):mice were intraperitoneally injected with the same amount of 10 mg/kg CH1 after dMCAO.Cerebroprotein hydrolysate for injection(?)medium-dose+CYC group(CH1+CYC group):After dMCAO experiments,the mice were intraperitoneally injected with the same amount of 10mg/kg CH1+CYC(1 mg/ml,10 mg/kg).Mice were treated every day for 14 days after ischemic stroke.The expression levels of shh pathway proteins Shh,Ptch-1,Smo,and Gli-1 were detected by western bolt at 28 d after dMCAO,and the nuclear translocation of Gli-1 was detected by immunofluorescence.After the Shh pathway inhibitor CYC were treated,we tested whether the neurological improvement by CH1 is still effective in behavioral tests.Results:1.CH1 promotes axonal regeneration by activating shh/Ptch-1/Gli-1 signaling pathway.We detected the expression level of the shh signaling pathway protein by western blot,and the results showed that the shh signaling pathway was activated after focal cerebral ischemia.Compared with the Vehicle group,CH1 treatment significantly increased the relative expression of shh,Ptch-1 and Gli-1 at 28 d after focal cerebral ischemia.We detected the distribution of Gli-1 in cells by immunofluorescence analysis.The results showed that Gli-1 was translocated from the cytoplasm to the nucleus at 28 d after dMCAO.Compared with the Vehicle group,the CH1 group accumulated more Gli-1 in the nucleus.2.Inhibiting the shh pathway reversed the improvement of neurological function caused by CH1.We added the Shh signaling pathway inhibitor cyclopamine(CYC)to study the role of the Shh signaling pathway in CH1-induced neurological behavioral improvement.The results showed that compared with CH1 group,CYC significantly reduced the expression of Gli-1 and Ptch-1,but did not reduce the expression of shh,indicating that CYC can effectively block Shh signaling pathway.Compared with the CH1 group,the rotating rod time in the CH1+CYC group had a significant reduction in the rotarod experiment.The effect of CYC on blocking CH1 was also observed in the adhensive removal test and the mNSS test.Then,after applying CYC,the expression of GAP43 was detected by Western bolt,and the results showed that the increase of GAP43 expression by CH1 was inhibited.Conclusions:1.This experiment proved that the Shh pathway in the penumbra is activated in ischemic penumbra at 28d after focal cerebral ischemia,and CH1 can further activated the shh pathway.2.CYC,the inhibitor of the Shh pathway,will reverse the protective effect of CH1 on the neurological function of dMCAO mice.It shows that the neuroprotective effect of CH1 after focal cerebral ischemia is at least partially attributed to the activation of the shh pathway. |
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