The Therapy Effect Of HIF-1α To The Cerebral Ischemia Eperfusion Injury Rats

Objective: The aim of the present study was to test the treatment effectof HIF-1α to cerebral infarction based on the results from the experimentintroduced. In the experiment, the vitro recombination technology ofpcDNA3.1－HIF-1α was used to prepare HIF-1α plasmid. We injected HIF-1αplasmid into external carotid artery of the middle cerebral artery (MCA)occlusion reperfusion rats. After that, we use western blot technique to test theprotein expression of HIF-1α and vascular endothelial growth factor (VEGF)in the ischemic cerebral tissue at different time points6h,24h,48h,72h and7d after ischemia reperfusion injury. Under these experiment results, we candemonstrate the protection effect of exogenous HIF-1α in nervous tissueduring cerebral ischemia reperfusion injury.Methods:1In vitro synthetic HIF-1α cDNA cloned into the T vector-pcDNA3.1after gene sequencing, cloned sequence size:2.5kb; identify the recombinantthrough restriction enzyme digestion, amplify in vitro according to theexperiment need.2The experiment model of cerebral ischemia reperfusion (MCAO)preparation:10%chloral hydrate was injected into the abdominal cavity ofmale SD rats (weight280-330g) for intraperitoneal anesthesia. We give therats a median neck incision, and then separated the neck vessels to exposecarotid, internal carotid and external carotid artery and inserted filament intothe internal carotid through the external carotid artery. The filament was usedto make a middle cerebral artery occlusion with end19±0.5mm away fromthe bifurcation of vessels. We removed the filament and injected HIF-1αplasmid to the experimental groups via external carotid artery after2hours,while PBS was injected into control groups. Neurological disfunction scoring was graded by Zea Longa standard.3We choose the rats that neurological disfunction scoring between1to4points, and obtain the cerebral tissue using decollation at different time points:6h,24h,48h,72h and7d. The brain tissue specimens of corresponding timepoints were taken after brain death. We made serial sections (2mm thick)posterior optic chiasm, the slice was stained by TTC to observe the proportionof ischemic area.4We use western blot technique to test the protein expression of HIF-1αand VEGF in the ischemic cerebral tissue at different time points6h,24h,48h,72h and7d after ischemia reperfusion injury.5Statistical analysis: Use SPSS17.0statistical software to conductstatistical analysis of the experimental data. T test was used to comparemeasurement data between groups. P <0.05indicated that there werestatistically significant differences.Results：1We identified pcDNA3.1-HIF-1α plasmid by restriction enzymedigestion. HIF-1α cDNA was amplified in vitro according to the experimentaluse. Cloned sequence size:2.5kb.The sequencing results of RT-PCR wereexactly the same with the Genebank records（GenBank ID：3091）.2The rats＇neurological disfunction was graded by Zea Longa standardat6h，24h，48h，72h and7d after the MCAO. The differences of rats death ratebetween two groups external carotid artery injected with HIF－1α plasmid andthose with PBS are no significant in time points6h，24h，48h，72h and7d. Andthe neurological disfunction scoring at corresponding time points of treatmentgroups and control groups are also not significant different.3Infarct size in TTC stained brain slices of the experimental group andcontrol group were no significant differences at early stage（6h—24h）ofreperfusion.However, after72h, the Infarct size of experimental group issmaller than control group.4We use western blot technique to test the protein expression of HIF-1αand VEGF in the ischemic cerebral tissue, the quantitative analysis showed that the expression of HIF-1α and VEGF in the experimental groups increasedrapidly, peaked at24h and then began to decline. The comparative levels ofHIF-1α expression in cerebral ischemic-reperfusion groups were significantlyhigher than that in sham group (P<0.05).Conclusions：1The recombinant pcDNA3.1-HIF-1α plasmid injected through theexternal carotid-internal carotid artery can be transfected into the ventricularsystem and nerve tissue.2The external carotid—internal carotid artery injection of recombinantplasmid pcDNA3.1-HIF-1α play a neuroprotective role in reducing infarct sizeof rats with cerebral ischemia-reperfusion injury3Gene therapy such as HIF-1α plasmid transfection can reduce theinjuries of vascular endothelial cells in the choroid plexus at the early stage ofcerebral ischemia-reperfusion injury.4The injection of recombination pcDNA3.1-HIF-1α plasmid throughcarotid artery could effectively regulate the expression of the vascularendothelial growth factor.