The Effect Of Dl-butyphthalide On Expression Of VEGF,HO-1 Flowing Chronic Cerebral Ischemia In Aged Rat

BackgroundBecause the morbidity and mortality of cerebrovascular is rising continually lately, humans attach more importance to study cerebrovascular. But many studies have focused on acute ischemic brain impairments is insufficiency to chronic cerebral hypoperfusion.But, with the ratio of senile increasing in country, More and more people get into trouble with dementia, which brings social economic and family problem. Now,the study of the chronic cerebral ischemia becomes especially importance.Chronic ischemic brain impairment is a common pathological state ,it complicated in many diseases such as Vascular dementia ,Alzheimer disease ,Binswanger disease ,Arterivenous malformation and so on .Because it is chronic ,the impairment and repairment is more complicated.In the early stages,its cardinal manifestation is cognitive disorder.At last,it can cause eternal or progressive impairment of cognitive and neurological. It is criticality for improving brain impairment to recover bloodstream provision early. Studies have proved that the increasing of blood vessel density after chronic cerebral hypoperfusion is related to amendent and prognosis of patient.So it is very important for the recovery of patient to study the factor of vascular growth .vascular endothlial growth factor(VEGF) is a very specific factor to promote vascular growth. It plays an important regulating effection in physiology vascular generation an pathological vascular generation.it can promote vascular regeneration,increase vascular permeability and protect the neuron. The ischemia and hypoxia of tissue is a stimulant factor for the expression of VEGF,The VEGF plays an imoortant role in the cellur recovery after chronic cerebral ischemia. The endogenous carbon monoxide(CO)can activate guanylate cyclase,regulate the cyclic guanosine,it is a cell messenger which have a great deal of biological functions .Recently ,many studies discover that carbon monoxide(CO) have the effect of endothelial dependence vasodilation to be similar to nitrogen monoxidum (NO),and have the capability of promoting composition of VEGF.Hemeoxygenase(HO) is the key enzyme to creat CO.Till now ,people have discovered three kinds of isoenzyme of H0:H0-l is type induction,HO-2 and HO-3 are type architecture.HO-1 is also called heart shock protein(HSP) and it can be inducted by multiple stress,for example,Heme hypoxia ischemia and injurment. Many studies focused on the its capability of degredating to heme in previous years,the latest study have found that HO-1 have many biological functions in antioxygen antiinflammatory inhibiting platelet aggregation cytoprotection accommodating angiotasis and so on. Recently,the study of the HO/CO system has become a hot topic. Dl-butyphthalide(NBP) was isolated from the seeds of of celery,then was synthesized racemic artificially.NBP is a new drug to cure cerebrovascular disease .In the studyof acute brain ischemic ,NBP was shown to improve brain energy metabolism in mice with complete cerebral ischemic ,and to enhance the rCBF of rats with regional ischemic,and to decrease cerebral infarct volume of rat with MCAO, and to protect rats from ischemic neurological damage,and to improve impairment of learning and memory.But there is very few reports about NBPin the chronic cerebral ischemic. Studies hare proved that chromic cerebral ischemia which is established by occlusiving bilateral carotid artery is am effectual model to study vascular cognitive disorder an estimate the drays of dementia. In this article , by establishing 2V0 model of chronic cerebral hypoperfusion and observing the expressions of VEGF and HO-1 by immunohistochemically,we explore the effection of NBP on revasculari -zation regeneration blocking cell apoptosis and the protection of neurocyte after chronic cerebral ischemia.Materials and methods1. Experiment animal and groupingSelected 80 healthy wistar rats and distributed to 4 groups randomly,regardless of male or female,the age from 12 to 14 months and the weight from 450-550g,Offered by the center of experimental animal of Zhengzhou university henna,20 rats each group. Group A:control group (sham operation plus);group B:ischemia group;group C:ischemic and low dose treatment group;group D:ischemic and high dose treatment group. Cronic cerebral ischemic rat models were established of B C D by permanent occlusiving and snipping bilateral carotid artery .The treatment of the control group is the same to the model groups,but without the snip of bilateral carotid artery. The rats of groups Cand D began to receive daily oral dose of 60mg/kg and 120mg/kg NBP(dissolved into 2ml oil) from the 2 months after the operation,which will last a month.The rats of groups Aand Breceived the same volume of oil,which will last the same time .The standards of successed model are:in the process of the next time ,active ,without secretion of eyes,normal eating and so on,except the rats died after three months.2. manufacturation of modelThe rats were perfused with 10% chloral hydrate for anesthesia,split the center of the neck ,isolate and permanent occlusion and snip of bilateral common carotid arteries,suture skin and breed the rats in virgin circumstance.In rats of control group, bilateral common carotid arteriesonly is only isolated but not be ligated and snipped,other treatments are the same to the rest groups.3. manufacturation of specimenThree months after 2VO,the rats were perfused with 10% chloral hydrate for anesthesia. Cut the head and take out of left brain tissue after perfusing from the heart,then put brain tissue to 4% paraformaldehyde for fixation. Brain tissue is manufactured coronal section for HE and immunohistochemistry staining after dehydration clearing dipping candle and embedding.4. The content of observation(1)The changes of neuron in cortex and hippocampus were observed by HE.(2)The expressions of vascular endothliat growth factor(VEGF) and Hemeoxygenase-1(HO-1) were observed by immunohistochemically.5.Statistical methodThe data was handled with SPSS12.0 static software. The diferece of every two groups was compared with one-way analysis of variance and LSD method,significant level is a=0.05.Result 1.The conditions of experiment animal All the rats were normality before operation. But all the rats were depressed reacted slowly ate little after operation.The next day, the rats of control group recovered significantly, the rats of other groups did not recover obviously .However the operation groups recovered slowly until 3-5 days.A week later,they were normal.2.The results of pathological section with HE staining Only a fewdegenerated and dead neurons were found in cortex and hippocampus,while most neurons were normal in group A.The number of degenerated and dead neurons significantly decreased in group B compared with group A.The number of degenerated and dead neurons was less in group C compared with group B,the normal neuron cells in group C are more than group B. The number of degenerated and dead neurons was less in group D.compared with group C, the normal neuron cells in group D are more than group C.3.The results of immunohistochemical for VEGF The expression of VEGF protein positive mainly in cortex and hippocapus neuron glial cell vascular endothelial cell,endochylema assumes buffy.The number of VEGF protein immunopositive cells were markly increased in group B compairing with group A.The difference between group B and group A was significant (p<0.05).The number of VEGF protein immunopositive cells was markly increased in group C and group D comparing with group B.The difference between group C,D and group B was significant(p<0.005). The number of VEGF protein immunopositive cells was markly increased in group D comparing with group C. The difference between group D and group C was significant(p<0.005).4.The results of immunohistochemical for HO-1 The HO-1 protein mainlyappears in cortex and hippocampus.The HO-1 protein immunopositive cells were markly increased in group B compairing with group A. The difference between group B and group A was significant (p<0.05). .The number of HO-1 protein immunopositive cells was markly increased in group C and group D comparing with group B.The difference between group C,D and group B was significant(p<0.005). The number of HO-1 protein immunopositive cells was markly increased in group D comparing with group C. The difference between group D and group C was significant(p<0.005).Conclusion 1.Chronic cerebral ischemia rat model was established by permanent occulusion and snip of bilateral common carotid arteries.The progress of the model and chronic cerebral ischemia is almost same .Its operation is simple and its reproducibility is good.2.Three months after chronic cerebral ischemic ,neurons of cortex and hippocampus degenerated and died ,the number of neurocyte decreasd.3.NBP can lessen the degeneration death of neurons of cortex and hippocampus,at the same ,it can also promote the expression of VEGF and HO-1 in cortex and hippocampus after Chronic cerebral ischemia.