The Research Of Enriched Environment Combine Recombinant Adenovirus Vector Containing HIF-1? Gene On The Neuroprotective Effects And Mechanisms To Rats Of Focal Cerebral Ischemia Reperfusion

Objective: To observe the effects of enriched environment combine recombinant adenovirus vector containing HIF-1? gene on nerve functional recovery of rats of focal cerebral ischemia-reperfusion injury,and explore the possible mechanisms.Methods: The adult male SD rats were randomly according to the random number table method divided into: Normal group,Sham group,cerebral ischemic and reperfusion group(CIR group),recombinant adenovirus vector group(Ad group),recombinant adenovirus vector containing HIF-1? gene group(AdHIF-1? group),enriched environment intervention group(EE group)and EE+AdHIF-1? group,each group was divided into 1d,14 d and 28 d subgroups of cerebral ischemia 1h according to the different reperfusion time,and each group of 18 rats at each time point.The model of focal cerebral ischemia reperfusion was prepared by the method of line suppository,and reperfusion followed 1 hours after middle cerebral artery occlusion(MCAO).The sterile physiological saline,Ad and AdHIF-1? were injected into the ischemia side ventricle of rats,Ad and AdHIF-1? were carried with green fluorescent protein as the tracer tag.The neurological deficit scores were evaluated and the expression of green fluorescent protein(GFP)were observed under fluorescent microscope,the moisture content of brain tissue were measured by dry and wet weight,pathological brain damage were observed by performing Hematoxylin and Eosin(HE)staining,TUNEL staining was used to detect the changes of apoptosis in peripheral cerebral infarction,immunohistochemical staining were used to detect the expression of Bcl-2 and Bax protein in ischemic lateral brain tissues,and the expression of BDNF protein in the hippocampus CA1 region by immunofluorescent staining at 1d,14 d,and 28 d after reperfusion.After 28 d of therapy,Morris water maze tests were conducted to assess the learning and memory abilities of rats for consecutive 5d,we recorded the avoidance latency of rats in all groups at the first 4ds,then we recorded the number of crossing platforms of rats at the 5d.Results: 1.Ad group and AdHIF-1a group were observed a faint GFP expression at 1d of cerebral ischemia reperfusionrats under fluorescence microscope,the fluorescence signal was the strongest at 14 d,then weakened and the expression time lasted about 4 weeks,GFP was observed to be expressed in bilateral lateral ventricle,choroid plexus and ependymal epithelium.2.The rats in Normal group and Sham group had good neurological function,but in the CIR group,Ad group,AdHIF-1? group,EE group and EE+AdHIF-1? group the rats showed signs of movement disorders,such as the rotation of the rotating circle or the one side of the group,nerve function score significantly higher than the Normal group and Sham group(P<0.05).However,the score of the AdHIF-1? group,EE group and EE+ AdHIF-1? group were lower than that of CIR and Ad group(P<0.05),and the improvement of EE+ AdHIF-1? group was more obvious than that of AdHIF-1? group and EE group(all P<0.05).3.There was no change of the brain water content of rats in Normal and Sham groups.The brain water content of rats in CIR group,Ad group,the AdHIF-1? group,EE group and EE+AdHIF-1? group increased significantly compared with Normal and Sham group(P<0.05),but both the AdHIF-1? and EE+AdHIF-1? group was less than the CIR group,the Ad group and EE group(P<0.05)at 1d of cerebral ischemia reperfusionrats.Moreover,the brain water contents of rats in the AdHIF-1? group,EE group and EE+AdHIF-1? group were lower than those in the CIR group and Ad group,and the EE+AdHIF-1? group was significantly lower than the AdHIF-1? group and the EE group 14 d and 28d(all P<0.05).4.The brain structure of rats in Normal group and Sham group was observed under the light microscope,and the nerve cells were closely aligned at all time points.At 1d,cell edema was found in CIR group,Ad group and AdHIF-1? group,and the cells were loose.The nerve cells in the center of ischemia were different in size,abnormal in morphology and disorder,and the cerebral tissue was vacuolated and reticulated.With the extension of reperfusion time,the nucleus of the infarcted central area was retracted,the nucleolus disappeared,the number of neurons decreased,and the remaining cells had no complete structure.At 28 d,the AdHIF-1? group,EE group and EE+AdHIF-1? group were observed to significantly reduce the pathological changes in the ischemic side of the brain tissue,and the loss of neurons was relieved,and the ischemic side tissues and cell morphology of rats in EE+AdHIF-1? group were close to normal.5.There was no apoptotic cell was detected in Normal and Sham groups.At 1d after reperfusion,a large number of apoptotic cells were detected in the CIR group,Ad group and AdHIF-1? group,EE group and EE+AdHIF-1? group(P<0.05),and the apoptotic cells in the AdHIF-1? group and EE+AdHIF-1? group were less than those in the CIR group,Ad group and EE group(P<0.05).The apoptotic cells in AdHIF-1? group,EE group and EE+ AdHIF-1? group were less than those of the CIR group and Ad group(P<0.05),and the expression in EE+AdHIF-1? group was significantly less than that of the AdHIF-1? group and the EE group at the other time points(P<0.05).6.Bcl-2 and Bax positive expression were not detected in the cerebral cortex of rats in Normal group and Sham group.Compared with Normal group and Sham group,the positive expression of Bcl-2,Bax in CIR group,Ad group,AdHIF-1? group,EE group and EE+AdHIF-1? group increased significantly(P<0.05).In each group,a large number of Bcl-2 positive protein was expressed at 1d,the expression of Bcl-2 positive protein in AdHIF-1? group and EE+AdHIF-1? group were more than that in CIR group,Ad group and EE group(P<0.05).The expression of Bcl-2 positive protein in EE group,AdHIF-1? group and EE+AdHIF-1? group were more than that in CIR and Ad group(P<0.05),and the EE+AdHIF-1? group was the highest at 14 d and 28d(P<0.05).Bax positive protein was detected in each group at 1d,and the number of Bax positive protein in the AdHIF-1? group and EE+AdHIF-1? group was significantly lower than that in the CIR group,Ad group and EE group(P<0.05).With the extension of the reperfusion time,the expression of Bax was gradually decreased,and the expression in EE group,the AdHIF-1? group and EE+AdHIF-1? group were less than that in the CIR group and Ad group at the other time points(P<0.05).7.There was a little BDNF positive protein expressed in Normal and Sham groups,the expression of BDNF were significantly increased in CIR group,Ad group,AdHIF-1? group,EE group and EE+AdHIF-1? group compared with Normal group and Sham group at 1d(P<0.05).The number of BDNF positive protein was reduced in CIR group,Ad group,AdHIF-1? group,EE group and EE+AdHIF-1? group at 14 d and 28 d,but the positive expression of BDNF protein in EE group and EE+AdHIF-1? group was significantly higher than that in CIR group,Ad group and AdHIF-1? group(P<0.05).8.Compared with Normal group and Sham group,the avoidance latency period of rats in the CIR group,Ad group,AdHIF-1? group,EE group and EE+AdHIF-1? group was prolonged,and the number of crossing platforms was reduced(all P<0.05).Moreover,the avoidance incubation period of rats in AdHIF-1? group,EE group and EE+ AdHIF-1? group was shorter than that of CIR and Ad group and increased across the platform,and both of the results in EE+AdHIF-1? group were better than the AdHIF-1? group and EE group(all P<0.05).There was no statistically significant difference between Normal group and Sham group(P>0.05),which applied to the Ad group and the CIR group(P>0.05).Conclusions: 1.There were much Bcl-2 protein and Bax apoptosis in rats brain tissue after cerebral ischemia reperfusion injury,which promoted cell apoptosis caused,brain edema and pathological morphological changes,reduced the movement and the ability of learning and memory of rats.2.It was not obvious that rats with focal cerebral ischemia and reperfusion injury had an early effect on environmental intervention.In addition to inhibiting apoptosis,it may also be related to promoting the expression of BDNF protein.3.The role of AdHIF-1? in brain protection may be related to the promotion of anti-apoptotic protein bcl-2 and inhibition of expression of protein Bax.There was no significant correlation between the expression of BDNF protein and AdHIF-1?.4.Enriched environment and Ad HIF-1? gene can respectively inhibit apoptosis,reduce ischemia brain tissue pathological damage,improve nerve dysfunction in rats,and the curative effect when they combined is superior to the individual treatment.