Experimental Study Of Xingnaojing Injection In Protecting Penumbra In Acute Phase Of Cerebral Ischemia

The lesions of acute cerebral infarction were composed of central necrosis area and ischemic penumbra.Cerebral infarction is a common refractory cerebrovascular disease,with the features of high incidence,high morbidity and high mortality rate,high recurrence rate and multiple complications features.The most effective treatment of acute cerebral infarction is thrombolytic therapy,but the thrombolytic therapeutic has strict restrictions of time window(4.5 hours)and exists a certain risk.Ischemic penumbra is a reversible brain injury,which is usually located in the marginal zone of the infarct,but it still maintains normal ionic balance and cellular structure integrity.In the central area of infarction,brain cells died due to complete ischemia.However,there were still some collateral neurons in the penumbra zone,therefore,it is the key to the treatment of acute cerebral infarction.Autophagy and apoptosis are related to each other,they affect the homeostasis of cells,and autophagy and apoptosis can damage the brain tissue.Blood brain barrier(BBB)is a special barrier between blood and brain tissue,it can prevent harmful substances from entering the brain,and can also transmit nutrients.Cerebral ischemia and hypoxia lead to increased permeability of blood brain barrier,leading to secondary brain damage such as brain edema and inflammatory reaction.Therefore,this study through the behavior,pathology,imaging and molecular biology methods,and from the perspectives of apoptosis,autophagy and blood-brain barrier,to study for Xingnaojing Injection on acute cerebral ischemia penumbra injury,and provide the basis for clinical application of Xingnaojing Injection in acute phase of cerebral ischemia.Objective1 Establishing the rat model of middle cerebral artery occlusion(MCAO),after modeling,MRI was detected at different time points,the size of cerebral infarction,the formation of cerebral edema and the changes of infarct volume in rats were observed,and through the imaging related software,the DWI and PWI were used to determine the changes of ischemic penumbra by imaging software.2 Using HE staining and transmission electron microscopy to detect the pathological changes of ischemic penumbra.The expression of apoptosis related proteins(Caspase-3),autophagy related protein(Beclin-1)in brain ischemic penumbra and the expression of Blood brain barrier permeability related protein(ZO-1,Claudin-5)were detected by immunohistochemistry and molecular biology.3 Through regulating cell apoptosis,autophagy and blood-brain barrier tight junction protein expression to explore the pathway and mechanism of traditional Chinese medicine Xingnaojing for protecting the semi dark band,which in order to provide theoretical and experimental basis for the early intervention of ischemic stroke in Chinese medicine under the guidance of the theory of "Disease collaterals-toxin damaging brain collaterals".Method1 A total of 40 healthy male SD rats(SPF)were used in the research,2 of them were used the sham operated group,and the remaining 38 rats were used to establish the model of middle cerebral artery occlusion(MCAO)in rats,the successful model of the rats were randomly divided into model group and Xingnaojing group,after the operation,the patients were given intravenous injection of caudal vein and the mNSS nerve function score was adopted.After modeling,4.5h,24h and 7 days using MRI to detect the following parameters:T1WI,T2WI,PWI,DWI and MRA,so the size of cerebral infarction and brain edema were observed and the loss rate of penumbra was calculated in rats at different time intervals from the view of imaging.2 Using 100 SPF level healthy male SD rats,except the sham operation group,the other according to the preparation method of MCAO model rats were randomly divided into model group and Xingnaojing group,after operation,drugs were administered to mice by caudal vein,2 times one day,after 4.5h,24h and 7days rats were evaluated using the mNSS score.At each time point,25 rats were anesthetized and then opened the chest,inserted into the needle in the left ventricle of the heart,cut off the right atrial appendage,0.9%saline was used to wash the blood vessels and the brain tissue was fixed with 4%paraformaldehyde,with 4%paraformaldehyde for more than 24 hours and then fixed.HE and electron microscopy were used to observe the pathological changes in the penumbra of rat brain,and Immunohistochemical method was used to detect the expression of apoptosis related protein(Caspase-3)and tight junction protein(ZO-1)in brain tissue.3 According to the preparation method of the model,route of administration and the nerve function as,after 4.5h,24h and 7 days,the rats were decapitated at low temperature,removing the redundant tissue then placed the brain tissue of the right half dark zone of the rat in a frozen tube,at last put them in liquid nitrogen.The expression of autophagy related protein(Beclin-1)and the expression of tight junction associated protein(Claudin-5,ZO-1)were detected by Western blot;Western blot results showed that the expression of ZO-1 protein was less in the model group,and Xingnaojing group compared with model group,the expression of ZO-1 protein increased,there was significant difference between the two(P<0.05).Results1 mNSS neurological score found that between MCAO rats after 4.5 hours the treatment group and control group showed no obvious difference of neurological deficit(P>0.05),and after 24 hours and 7 days the neural function of MCAO rats of Xingnaojing group was superior to the model group(P<0.05),and MCAO rats after 24 hours the mNSS score of each group was higher than the groups of 7 days.From the MRI images and related data,XNJI cerebral infarction area was less than the control group in each time period,4.5 hours of penumbra volume Xingnaojing group lower than model group,CBF infarct volume also decreases.According to the comparison of the penumbra loss rate we can be found that the penumbra loss rate of model group was higher than Xingnaojing group,and the difference was statistically significant(P<0.05).2 Pathological changes:HE staining showed that the morphology of the neurons in the sham operation group was complete,the intercellular space was normal,the vascular endothelium was intact,and there was no edema around the blood vessels.In the model group,there were obvious perivascular edema,neuron pyknosis,cell apoptosis and inflammatory cell infiltration,and Xingnaojing group vascular relatively complete,some mild edema,a small amount of neuron shrinkage.Nissl body staining observed the morphological changes of Nissl body in each group.It was showed that the structure of Nissl body in the sham operation group was intact,alkaline plaque or granular,large and many,such as tiger skin pattern,evenly distributed,Nissl body structure of model group damaged and disintegrated into Dust particles,Nissl body morphology in Xingnaojing group was superior to the model group.Under the electron microscope observation of ischemic penumbra in ultrastructure can be found similar pathological changes from HE staining,we can also see the model group had more pyknotic neurons,also can see each time the model group BBB were markedly dilated,and vascular endothelial basement membrane damaged.Compared with the model group,Xingnaojing group in astrocyte edema,nuclear pyknosis,and tight junction integrity was better than the model group.3 Immunohistochemical results:Caspase-3 in the mirror showed Brown granular,mostly scattered in the distribution,and it can also be seen in the normal tissue.No obvious difference of expression of Caspase-3 was seen in the Xingnaojing group and model group after 4.5 hours(P>0.05),after 24 hours and 7 days,the expression of Caspase-3 in model group was much higher than that of Xingnaojing group,there was statistically significant difference(P<0.01).The positive substance of ZO-1 protein was dark brown,with a punctate or massive distribution in the mirror.MCAO rats after 4.5h,24h and 7 days,expression of ZO-1 protein in Xingnaojing group was significantly higher than the model group(P<0.01).4 Western results:There was a large number of Beclin-1 protein expression in the sham operation group,compared with the model group,the expression of Beclin-1 protein in the model group was much less.After 4.5 hours the expression of Beclin-1 protein in Xingnaojing group was higher than in the model group(P<0.05),compared with the model group,the differences were more pronounced in 24 hours(P<0.01);After 4.5 hours the expression of Claudin-5 protein in the Xingnaojing group was higher than the model group(P<0.05),and after 24 hours it showed the same trend,but the difference was not statistically significant(P>0.05).Conclusion1 From the point of view of pathological changes,the sham group vascular endothelial cells,nerve cells and other tissue morphology were normal,model group under the microscope can see perivascular edema,nerve cell necrosis,inflammatory cell infiltration etc.These were consistent with the pathological changes of ischemic cerebral infarction,suggesting that the model can succeed.2 From the overall evaluation of the imaging findings and neurological score we found that in Xingnaojing group cerebral infarction area was less than the model group,and 24 hours the area of cerebral infarction was to reach the maximum.In addition the ratio of penumbral tissue loss of Xingnaojing group was less than that of model group,it meant in the acute stage of cerebral infarction less of the penumbra regions changed into irreversible infarction tissue in Xingnaojing group than the model group,so that we can say Xingnaojing saved a part of the penumbra in a certain extent.3 From the ultrastructure observation of electron microscope penumbra,Xingnaojing group nerve cells morphology,basement membrane and vascular endothelial integrity of BBB was better than that of model group,Xingnaojing injection can protect neurons,decrease the permeability of blood brain barrier,thereby preventing further brain damage.4 From the immunohistochemistry and Western results,Xingnaojing injection can up regulate the expression of autophagy related protein Beclin-1 and apoptosis related protein Caspase-3 to reduce the loss of neurons,and maintain steady-state intracellular environment.In addition Xingnaojing injection can prevent the tight junction associated protein ZO-1 and Claudin-5 protein from lossing,thereby protect the integrity of blood brain barrier,relieve cerebral edema,oxidative stress and secondary brain damage.