The Study Of Mechanism Of Hypothermia Postcondition Affecting On The VEGF Expression And Anti-neuron Apoptosis After Focal Cerebral Ischemia In Tree Shrews

OBJECTIVE:To observe the changes of the neurons microenvironment,as weU as the effect of infarction area resulted fi'om hypothermia postcondition after thrombotic cerebral ischemia in Tree Shrews,to study effect on hypothermia postcondition cerebral ischemia.We explore the effect of VEGFmRNA,VEGF protein and its receptor on cerebral ischemia and hypothermic postcondition,and exposes the mechanism of VEGF expression and modulate on hypothermia postcondition.METHODS:Cerebral ischemia was induced by photochemical reaction.The temperature of Tree Shrews' brain was lowered down to 31±0.5℃for 1 h by local temperature reduction method at 6h after ischemia,which was classified as the hypothermia postcondition group; the foreign VEGF antibody was instantly injected into cerebellomedullary cister at 6h after ischemia,which was classified as VEGF antibody injection group.When these two procedures were all executed,it would be classified as antibody injection+hypothermia postcondition group.The changes of neurons injury,infarction area and hippocampal microenvironment were studied in every group by TUNEL stain,HE stain,TTC and microdialysis in hippocampus;The changes of VEGFrnRNA,VEGF protein and receptor were investigated in hypothermic postcondition group through RT-PCK and ELISA technique. RESULTS:The contents of VEGFmRNA,VEGF receptor(VEGFR-I\VEGFR-2),neurons necrosis and apoptosis in hippocampus CA1 and cortex,hippocampal microenvironment and cortical infarction area had markedly difference between every group.The result of VEGFmRNA indicated that the contents of VEGFmRNA in hippocampus was above cortex (P＜0.05).hypothermia postcondition increased significantly the contents of VEGFmRNA in cortex at 24h after ischemia(P＜0.05),whereas decreased at 72h.But the contents of VEGFmRNA obviously rose at 72h in VEGF antibody injection group(P＜0.05).The results of VEGFmRNA in ischemic hippocampus displayed that the contents of mRNA of hypothermia postcondition group were less than that ischemia control group(P＜0.05),and this change lasted at 72h,while VEGF antibody injection could markedly decrease the contents of VEGFmRNA in contra lateral to ischemia.The results of VEGFmRNA in hippocampus and cortex demonstrated that the contents of VEGFmRNA apparently increased in theses two regions(P＜0.05),while decreased with time(P＜0.05);It rascended markedly in HPhC group at 24h after ischemia(P＜0.05),but descended at 72h and had no difference with control group(P＞0.05).The VEGF antibody injection obviously inhibited increased VEGF resulted from HPC and ischemia.TTC stain showed that the infarction area enlarged to 80%in VEGF antibody group(P＜0.05),while in HPC group the infarction area markedly reduced to19.67%.The analysis of microdialysis in hippocampus suggested that in extracellular fluid the sodium concentration decreased markedly(P＜0.05),potassium concentration increased(P＜0.05) at 24h,calcium concentration decreased markedly (P＜0.05),while HPC helped to inhibit these changes(P＜0.05),intensified these at 72h (P＜0.05).HE stain showed that numbers of neuron necrosis in hippocampus CA1 region were increased(P＜0.05) after ischemia,.but it was decreased at 24h in HPC group(P＜0.05) while increased at 72h.The numbers of neuron necrosis rose at 24h,72h in VEGF antibody injection.Ischemia also made the numbers of cortical neuron necrosis to rise obviously (P＜0.05) as well as in contra lateral(P＜0.05).But in contra lateral,except VEGF antibody group,neuron necrosis only occurred at 72h.HPC could apparently alleviated neuron necrosis at 24h while deteriorated at 72h.TUNEL stain indicated that the neuron apoptosis in ischemic hippocampus CA1 region increased markedly at 24h after ischemia(P＜0.05) while decreased with time,but the neuron apoptosis increased at 72h in HPC group.The neuron apoptosis in HPC+VEGF antibody group were less than in HPC group(P＜0.05).Whereas in cortex,the neuron apoptosis decreased obviously in HPC group,but increased with time (P＜0.05).the neuron apoptosis in ischemia group gradually decreased(P＜0.05).ELISA investigation indicated that the contents of VEGFR-1 rose obviously(P＜0.05) after ischemia, and ischemic lateral was less than contra lateral(P＜0.05),at the same time,the contents of VEGFR-1 was at its peak at 24h after ischemia.The contents of VEGFR-2 were markedly increased in cortex and hippocampus(P＜0.05)and continuously increased with time(P＜0.05). HPC could obviously increase the contents of VEGFR-2(P＜0.05).CONLUSION:①The instant protective effect of HPC can inhibit the neuronal apoptosis and necrosis by upregulating the expression of VEGF after thrombotic cerebral ischemia in Tree Shrews.Its mechanism maybe involved in that VEGF increase,improve the neuron microenvironment and inhibit neuronal apoptosis through multiple pathways,protect neurons from necrosis and apoptosis in hippocampus CA1 region and so on.②The relationship between VEGF protein and VEGFmRNA indicated that over expression of VEGF at ischemic early stage may stimulate mRNA transcription to decrease in cytoplasm through negative feedback,resulting in the reduction in VEGF contents at later stage.③Inhibition of VEGF function may reduce the neuroprotective effect in cerebral ischemia after HPC,HPC confirmed the neuroprotective effect throught VEGF upregulation.