| BackgroundCerebral atherosclerosis(CAS),which manifests itself clinically through ischemic cerebrovascular disease,would cause brain damage because of cerebral ischemia and hypoxia and develop vascular cognitive impairments characterized by memory and cognitive impairment without immediate prevention and treatment.However,there is no effective treatment for ischemic cerebrovascular disease and vascular dementia caused by diffuse small vessel disease,which results from cerebral atherosclerosis.Ad-HGF,which is a patented human carrying human HGF gene,has entered the stage of clinical phase II and has achieved good evaluation in the treatment of myocardial ischemia and peripheral arterial disease.Previous experiments confirmed that Ad-HGF into the CAS model of the mouse subarachnoid space,can be effectively expressed in the brain tissue.ObjectiveStudy was performed to observe the effects on the memory,cognition and regional blood flow and the function to promote angiogenesis of exogenous Ad-HGF in the CAS rats after being injected into the subarachnoid space.Our study might provide basic data for the safety and efficacy of Ad-HGF in the treatment of ischemic cerebrovascular disease caused by cerebral atherosclerosis.Methods1.Rat model: Aged brain atherosclerosis model was replicated through renal hypertension combined with high fat diet.2.Behavioral evaluation experiment: Rats were divided into normal control group,model group and Ad-HGF group.In 2 weeks the navigation experiment and space exploration experiment were conducted through water maze.After that,immunohistochemistry was performed to detect the expression of Bcl-2 and Bax protein in the hippocampus of the brain tissue.3.Angiogenesis experiment: Rats were divided into normal control group,model group and Ad-HGF10d/20d/30 d group.Laser Doppler Flowmetry(LDF)was adapted to monitor regional blood flow in the brain.Quantitative Real-time PCR and immunofluorescence were performed to detect the level of CD31 and vascular endothelial growth factor mRNA and protein expression in the brain tissue.Results1.The blood pressure and blood lipid level of the rats in the model group were significantly higher than those before the experiment.Plaque can be observed in the end of the internal carotid artery via Hematoxylin-eosin staining.2.Positioning navigation and space exploration experiment: Compared with the normal control group,in the model group the escape latency was obviously longer,the average number of wearing and the duration in the target quadrant decreased,and the difference was significant(P<0.01).Compared with the model group,the latency of the Ad-HGF group was shorter,the average number of wearing and the duration in the target quadrant increased and the difference was significant(P<0.01).3.The expression of Bcl-2 and Bax protein in the hippocampus of the brain through immunohistochemistry: Compared with the model group,the expression of Bcl-2 and Bax protein in Ad-HGF group was significantly different(P<0.01),Bcl-2 protein expression increased,Bax expression decreased,and Bcl-2 / Bax ratio increased.The difference is significant(P<0.01).4.Changes in cerebral blood flow: Comparisons showed that the blood flow volume in the model group is less than that in the normal control group and the Ad-HGF group.The difference was significant(P<0.01).In Ad-HGF group,the blood flow volume gradually increased as time went on,and cerebral blood flow in 20 d and 30 d was obviously increased compared with 10 d.the difference was significant(P<0.05).However,there was no statistical difference between 30 d and 20 d.5.Quantitative Real-time PCR: Exogenous Ad-HGF can up-regulate the expression of CD31 and VEGF mRNA,and the expression level of CD31 and VEGF mRNA increased gradually with the prolongation of time.The mRNA expression of CD31 and VEGF mRNA was the highest in 20 days,which was statistically significant compared with the model group(P<0.05).6.Immunofluorescence: Comparisons between groups showed that the microvessel density(MVD)marked by CD31 and VEGF protein in Ad-HGF group was significantly higher than that in model group and normal group,and the difference was significant(P<0.05).In Ad-HGF group,MVD was the most obvious at 20 d,and the difference between 20 d and 10 d was significant(P<0.05).Conclusions1.Exogenous Ad-HGF can significantly improve the learning and memory ability of CAS model rats by regulating the expression of Bcl-2 and Bax in hippocampus and provide basic experimental data for safety assessment on application of Ad-HGF in the treatment of cerebrovascular disease.2.Exogenous Ad-HGF can promote the angiogenesis and increase the regional cerebral blood supply by up-regulating the expression of CD31 and VEGF in CAS model rats. |
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