| Objective:In this study,the global cerebral ischemia-reperfusion rat model was established by modified Pulsinelli four-vessel occlusion(4VO)method.Nasopharyngeal cavity cooling was instituted continuously to maintain mild hypothermia.The microtialysis solution of hippocampus was collected by microdialysis,its glutamate(Glutamic acid,Glu)content and concentration([Glu]e)were measured.Compared the expression of GLT-1 and the changes of extracellular [Glu]e after instituting GLT-1 specific inhibitor DHK or PI3K/Akt channel specific inhibitor LY294002 in mild hypothermia.To evaluate the effects of PI3K/Akt channel regulate GLT-1 expression on global ischemia reperfusion injury,and provide a reference for the mild hypothermia treatment in clinical.Methods:1 Experimental animal groupingThirty healthy male Wistar rats(provided by Experimental Animal Center,Hebei Medical University).The rats were randomly divided into 5groups(n=6): sham operation group(group Sham/group S),normal temperature ischemia/reperfusion group(group IN),hypothermic ischemia-reperfusion group(Group IH),GLT-1 specific inhibitor group(group DHK),PI3K/Akt kinase specific inhibitor LY294002 group(group LY).2 Animal model preparationThe whole cerebral ischemia-reperfusion model was prepared by modified Pulsinelli four-vessel occlusion(4VO).The hypothermia model of head was prepared by nasopharyngeal cavity cooling method.(1)Sham group(group Sham/group S)(n=6): only exposed bilateral vertebral artery but not burning,only separation of bilateral common carotidartery but not clipping.(2)Normal temperature ischemia reperfusion group(group IN)(n=6):permanent thermal coagulation of bilateral vertebral artery,48 hours after exposure to bilateral common carotid artery threading and clipping bilateral 8min recovery perfusion.(3)hypothermic ischemia-reperfusion group(group IH)(n=6): permanent thermal coagulation of bilateral bilateral vertebral artery,48 hours after separation of bilateral common carotid artery threading,nasopharyngeal cavity cooling method sustained cryogenic liquid,Maintain the hippocampal temperature(33.0±0.5)℃,clamping bilateral carotid artery 8 min after reperfusion.(4)GLT-1 specific inhibitor group(group DHK)(n=6): permanent thermal coagulation of bilateral vertebral artery,48 h after exposuring to bilateral common carotid artery,nasopharyngeal cavity to the target temperature of continuous collection of cells external fluid,clamping the bilateral common carotid artery 30 minutes,the right ventricle slowly injected DHK solution 20 μl(200nmol),clamping bilateral carotid artery 8 min reperfusion.(5)LY294002 group(group LY)(n=6): permanent thermal coagulation of bilateral vertebral artery,48 hours after exposuring to bilateral carotid artery,bilateral carotid artery occlusion 20 min,right ventricular slow injection5 μl LY294002(LY294002 dissolved in DMSO,the concentration was 25mmol/L),clamping bilateral carotid artery 8 min reperfusion.All groups should be monitored for rectal temperature at the same time and maintained at(37±0.5)℃.3 Specimen collection and detection methods3.1 micro-dialysis solution collection and determinationBefore cerebral ischemia 20 min ~ 10 min,recorded as t0;10 min before cerebral ischemia ~ before ischemia,recorded as t1;ischemia immediately ~10 min,recorded as t2;10 min ~ 20 min after ischemia,recorded as t3;20 min~ 30 min after ischemia,recorded as t4;30 min ~ 40 min after ischemia,recorded as t5.The microtialysis solution of each group was collected by microdialysis and sealed in a refrigerator at-80 ℃.The peak area of glutamate standard at different concentrations was determined by high performance liquid chromatography(HPLC).The standard curve was drawn and the standard curve regression Equation to obtain the glutamate content and concentration change in the microdialysis solution.3.2 The expression of GLT-1,Bcl-2,Bax and p Akt in hippocampal CA1 region was detected by immunohistochemistryAfter the collection of microdialysis solution in each group,the head maintained the hypothermia(33±0.5)℃,the rectal temperature was maintained at(37±0.5)℃,and after 2 hours,The expression of Bax,Bcl-2,GLT-1 and p Akt protein was observed by immunohistochemistry.The expression of Bax,Bcl-2,GLT-1 and p Akt protein was observed by immunohistochemistry.Results:1 Comparison of [Glu]e in hippocampal CA1 region in each groupCompared with t0,[Glu]e of t1 and t5 were no statistical significance(P>0.05),and [Glu]e of t2,t3 and t4,were statistically significant(P<0.05).Compared t2,group IN,group DHK,group LY was significantly higher than S group and group IH,there was statistical difference(P<0.05).Compared t3,there was no statistically significant difference between group DHK and LY group(P>0.05).Compared t3,[Glu]e in group S,group IH,group DHK and group LY was lower than in group IN,there was statistically difference(P<0.05).Compared t4,group DHK and group LY was higher than group S,[Glu]e in group IH was lower than in group LY,the difference was statistically significant(P<0.05).2 Immunohistochemical expression of Bax and Bcl-2 in hippocampal CA1 region of each groupCompared with group S,the expression of Bax in group IN,group DHK and group LY increased and the expression of Bcl-2 decreased(P<0.05);andthe expression of Bcl-2 in group IH increased(P<0.05).Compared with group IN,the expression of Bax in group IH,group DHK and group LY decreased,and the expression of Bcl-2 increased(P<0.05).Compared with group IH,the expression of Bax group DHK and group LY increased,and the expression of Bcl-2 decreased(P<0.05);compared group DHK and group LY there were no statistically significant difference(P>0.05).3 Immunohistochemical expression of p Akt in hippocampal CA1 region of each groupCompared with group S,in group IN and group LY the expression of p Akt increased(P<0.05).Compared with group IN,the expression of p Akt in group IH and group DHK increased,and the expression of p Akt decreased in group LY increased(P<0.05).Compared with group IH,the expression of p Akt in group DHK and group LY decreased(P<0.05).Compared with group DHK,the expression of p Akt in group LY decreased(P<0.05).4 Immunohistochemical expression of GLT-1 in hippocampal CA1 region of each groupThe expression of GLT-1 protein was expressed in astrocytes of hippocampal CA1 region.Group S had a small amount of brownish-green GLT-1 protein positive immunized particles.Compared with group S,the expression of GLT-1 in group IN was down-regulated,the expression of GLT-1 in group IH and group DHK was up-regulated(P<0.05).Compared with group IN,GLT-1 expression in IH group,DHK group and LY group was up-regulated(P<0.05).Compared with group IH,GLT-1 expression in group DHK and group LY was down-regulated(P<0.05).Compared with group DHK,GLT-1 expression in LY group was down-regulated(P<0.05).Conclusions:1 Mild hypothermia in brain can effectively alleviate the cerebral ischemia/reperfusion injury.2 Mild hypothermia regulates the expression of GLT-1 in hippocampal CA1 region,which can increased its expression,and decrease the extracellular glutamate concentration,reduce its excitotoxicity function to protect theglobal. |
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