The Protective Effect Of Madopar In Mice Models Of Transient Cerebral Ischemia Reperfusion Injury And The Protective Mechanism

Objective:Ischemic stroke is a serious threat to human health because high incidence,high morbidity,high recurrence rate,high mortality,its complex pathogenesis mainly involves exitotoxicity,oxidative damage,inflammation and apoptosis.ICR male mice are used in this study to establish transient cerebral ischemia reperfusion model.And anti Parkinson's disease drug,Madopar,is administrated by p.o per day before ischemia reperfusion,24 hours and72 hours after ischemia reperfusion,nerve function score,brain water content in the ischemic hemisphere were detected,the changes of the infarct volume,neuronal damage and the activation of microglia in the damaged stratium were detected in this study to confirm the neuronal protective effect of the madopar;and it was observed that changes of oxidative damage,inflammatory cytokine in the Iba-1?COX2?NF-kB of madopar treated ischemia group compared to those in the vehicle ischemia group to explore its neuroprotective mechanism.Further,changes of PI3K/Akt pathway were also checked in this study to confirm the neuroprotective mechanism of madopar.Methods:Adult male ICR mice were randomly divided into 6 groups,6 in each group.The experimental mice were divided into sham group;MCAO 1 and 3 day groups;madopar treated sham group;madopar treated MCAO 1 and 3 day groups.Application of suture method for mice middle cerebral artery occlusion(MCAO)model.Madopar were administrated orally to all madopar treated groups madopar 3 days before MCAO.The all experimental mice were sacrificed at designed time point including 0,24 and 72 hours respectively.Neural behavior scores,brain water content,infarct volume,immunohistochemistry were measured in the present study to confirm the neuroprotective effect and related mechanism of madopar.Results:(1)There are no significantly loss on neurological function both vehicle and madopar sham group;neurological function were significantly improved in the madopar treated MCAO group compared to MCAO group(p<0.05).(2)Brain water content was reduced in madopar treated MCAO 1day group compared with the MCAO 1 day group(p<0.05);in MCAO 3 day group,brain water content was lower treated with madopar than only MCAO group,which suggested that edema was alleviated;and there was no difference between madopar treated MCAO 1 day and 3 day groups about brain water content.(3)The volume of cerebral infarction in MCAO 3 day group was higher than in MCAO 1day group;madopar treated MCAO 1 and 3 day groups reduced the infarct volume after MCAO 1day group and MCAO 3 day group respectively.(4)Immunohistochemistry:In the sham group,the neuron stained by NeuN,which shape was regular and structure was integrity in striatum rules;while the number of neurons in the MCAO group were significantly reduced,and cell morphology were irregular;the number of neurons in treatment with madopar MCAO groups were more than the corresponding MCAO groups,and the morphology and arrangement of cells were relatively intact(p<0.05).Microglial cells stained by Ibal in the sham group were lobulated and body was small;however in MCAO groups,the activated microglia cells were form irregular ameboid,and the phenomenon was more obvious in MCAO 3 day group;pretreated with madopar in MCAO groups the activated microglia cells decreased.(5)Western blot:The expression of Ibal protein was increased in the MCAO groups;but in madopar treated MCAO groups the protein expression was lower than MCAO groups;in addition,the levels of Ibal protein expression was more in MCAO 3 day group than in MCAO 1 day group.COX2 and NF-kB protein expression were same in each group.In sham group,their expression were very little,which were significantly lower than in MCAO groups(P<0.05),and with the passage of time,the expression of protein was more in MCAO 3 day group;however,the expression of the protein in the drug intervention group was significantly fewer than that of the corresponding surgery group(P<0.05).SOD1 and SOD2 protein expression were roughly the same,and in MCAO groups which were significantly decreased compared with the sham group(P<0.05);but significantly increased in madopar pretreated MCAO group than the corresponding MCAO group(P<0.05);and the protein expression of MCAO 1 day group was significantly higher than that of the MCAO 3 day group,there was statistical significantly difference(P<0.05).The expression of Akt protein in the MCAO group was decreased than the sham group,but the difference is not obvious;there were no statistically significant of the protein expression in Madopar intervened MCAO group and the MCAO group also.The expression of p-Akt protein in the MCAO group was significantly reduced compared to the sham group,which was higher in MCAO 1 day group than MCAO 3 day group(P<0.05);for madopar treated groups,the expression of protein were more higher than corresponding group(P<0.05).Conclusion:(1)Madopar can significantly improve behavior score after MCAO in ICR mice,and it is beneficial to the recovery of the neurological deficits,to reduce the degree of cerebral edema,to significantly decrease the infarct volume and to maintain the number of neurons in the striatum.(2)Madopar can inhibit activated microglia after ischemia reperfusion injury in the striatum,decrease the expression of Ibal,NF-kB and COX2 protein.(3)Madopar can enhance the activity of superoxide dismutase,decrease the production of free radicals.(4)Madopar can activate PI3k/Akt pathway and inhibit of apoptosis.The experimental results showed that madopar not only has good resistance to Parkinson's disease,but also has the protective effect on cerebral infarction.The finding plays a certain role in ischemic brain injury secondary to Parkinson's disease prevention,and provides a new treatment for cerebral protection treatment of ischemic cerebrovascular disease after thrombolysis.