Effects Of Enriched Environment Combine Recombinant Adenovirus Vector Containing HIF-1α Gene On Glucose Metabolism In Ischemic Penumbra In Cerebral Ischemia-reperfusion Injury Rats

Objective:To observe the effects of enriched environment combined with recombinant adenovirus hypoxia inducible factor-1ɑ(HIF-1ɑ)gene on glucose metabolism in ischemic penumbra in cerebral ischemia-reperfusion injury rats,and to explore the brain protective mechanism of the combined effects.Methods:According to the random number table method,Adult male SD rats were randomly divided into: Sham group,Cerebral Ischemia and Reperfusion(CIR)group,recombinant Adenovirus vector(Ad)group,and recombinant Adenovirus vector containing HIF-1α gene therapy(Ad HIF-1α)group,Enriched Environment intervention(EE)group,Enriched Environment combined with recombinant Adenovirus HIF-1α gene therapy(EE+Ad HIF-1α)group.The rat model of middle cerebral artery occlusion(MCAO)was established by modified Longa method and fed for 28 days.Aseptic normal saline,Ad and Ad HIF-1α were injected into the ischemic lateral ventricles of rats in each group,respectively.Ad and Ad HIF-1α carried Green fluorescent protein(GFP)as tracer markers.Neurological function assessment was performed on the rats at 1d,14 d and 28 d after surgery,respectively.(1)Enriched Environment alone treatment part: Real-time quantitative PCR(RT-q PCR)and Western Blot were used to detect the m RNAs and protein expression of HIF-1α,glucose transporter 1(GLUT1),6-phosphofructo-2-kinase/Fructose-2,6-bisphosphatase-3(PFKFB3)after 1day and 28 days of cerebral ischemia.(2)Ad HIF-1α gene alone treatment part: On the 14 th day after operation,the brain tissues of rats in Ad group and Ad HIF-1α group were made into frozen sections,and the expression of GFP was observed under fluorescence microscope to determine the transfection.2,3,5-Triphenyltetrazolium chloride(TTC)staining was used to calculate the cerebral infarct volume on day 14 after surgery operation.28 days after operation,the protein expression of HIF-1α,GLUT1 and PFKFB3 were detected by Western Blot.(3)Enriched Environment combined with Ad HIF-1α gene therapy part: a modified neurological severity score(m NSS)assessment was performed before modeling and on days 1,14,and 28 after modeling intervention.On the 28 th day after operation,the following experiments were carried out: Hematoxylin-eosin(HE)staining was used to observe the pathomorphological changes of ischemic brain tissue.The number of Nisse bodies in neurons was observed by Nisse staining and TUNEL staining was used to detect apoptosis in ischemic lateral brain tissue.Immunohistochemical method was used to analyze the expressions of HIF-1α,GLUT1 and PFKFB3 in ischemic penumbra.The level of ATP,ADP and AMP in ischemic penumbra was measured by high performance liquid chromatography(HPLC)and the EC value was calculated.Results:(1)Enriched Environment treatment alone part: Compared with the Sham group,the expression of HIF-1α,GLUT1,PFKFB3 m RNAs and protein in model group and Enriched Environment group were significantly higher(P<0.05)of the 1st day after operation.On the 28 th day after operation,the expression of HIF-1α,GLUT1 and PFKFB3 m RNAs and protein in the Enriched Environment group were higher than those in the model group,and the differences were statistically significant(P<0.05).(2)Ad HIF-1α gene alone treatment part: On the 14 th day after operation,GFP expression was observed in brain tissue of Ad group and Ad HIF-1α group under fluorescence microscope.GFP is mainly distributed in the lateral ventricle of rats.TTC staining showed that the cerebral infarction volume of Ad HIF-1α group was lower than that of CIR group and Ad group after 14 days of cerebral ischemia(P<0.05).On the 28 th day after operation,compared with the CIR and Ad groups,the protein contents of HIF-1α,GLUT1 and PFKFB3 in the Ad HIF-1α group were higher(P<0.05).(3)Enriched Environment combined with Ad HIF-1α gene therapy part:MNSS:The m NSS score of rats in Sham group was 0 at each time point,and the neurological function was good and the behavior was not abnormal.The rats in CIR group,Ad group,Ad HIF-1α group,EE group and EE+Ad HIF-1α group showed signs of neurological deficit such as turning in circles or dumping to one side on the first day after operation,and the score was higher than that in Sham group(P<0.05).Compared with CIR and Ad groups,the m NSS decreased in the Ad HIF-1α group,EE group and EE+Ad HIF-1α group since 14 days after operation(P<0.05).On the 28 th day after operation,the scores of EE+Ad HIF-1α group were lower than those of Ad HIF-1α group and EE group respectively(P<0.05).HE : In Sham group,the structure of brain tissue was clear,the nerve cells were arranged regularly,the nucleus was in the middle,and the staining of nucleus and cytoplasm was uniform.On the28 th day after operation,there were obvious infarcted areas in CIR group and Ad group,such as cell edema,neuronal loss,nuclear pyknosis and marginalization,loose tissue and so on.The ischemic penumbra was obviously compared with the surrounding normal brain tissue.The pathological changes such as cell edema,neuronal loss,nuclear pyknosis and vacuoles were decreased in Ad HIF-1α group,EE group and EE+Ad HIF-1α group,and the pathological changes such as cell edema in EE+Ad HIF-1α group were significantly better than those in Ad HIF-1α group and EE group.Nissl staining: on the 28 th day after operation,compared with Sham group,the number of Nissl bodies in CIR group and Ad group decreased,and compared with CIR group and Ad group,the number of Nissl bodies in Ad HIF-1α group,EE group and EE+Ad HIF-1α group increased,and the number of Nissl bodies in EE+Ad HIF-1αgroup was higher than that in Ad HIF-1α group and EE group(P<0.05).TUNEL :No apoptotic cells were detected in Sham group,but apoptotic neurons were detected in the ischemic subcortex of the other groups.The numbers of apoptotic neurons in EE group,Ad HIF-1α group and EE+Ad HIF-1α group were less than that in CIR group and Ad group,and the numbers of apoptotic neurons in EE+Ad HIF-1α group were less than that in Ad HIF-1α group and EE group(P<0.05).Immunohistochemistry and HPLC:on the 28 th day after operation,compared with CIR and Ad groups,the protein expression of GLUT1 and PFKFB3 in Ad HIF-1α group,EE group and EE+Ad HIF-1αgroup increased,ATP level and EC value in ischemic penumbra increased,and EE+Ad HIF-1α group was higher than Ad HIF-1α group and EE group(P<0.05).Conclusions:(1)Long-term Enriched Environment intervention can improve the neurological function of rats with focal cerebral ischemia-reperfusion injury,and one of the mechanisms may be related to the improvement of glucose metabolism in the ischemic penumbra by up-regulating the expression of HIF-lα and its downstream genes GLUT1 and PFKFB3.(2)Exogenous Ad HIF-lα treatment can inhibit neuronal apoptosis,promote nerve cell survival and improve neurological dysfunction in rats with focal cerebral ischemia-reperfusion injury.One of the mechanism may be related to the up-regulated expression of glucose metabolism related proteins GLUT1 and PFKFB3.(3)Enriched Environment combined with exogenous Ad HIF-1α therapy can exert more neuro-cerebral protective effect than single intervention in rats with focal cerebral ischemia reperfusion injury,which may be related to the fact that Enriched Environment can promote the expression of HIF-1α and its downstream genes to some extent.