Effect Of Focal Cerebral Ischemia/Reperfusion On The Cerebral Cortex Neurons In Rats And The Intervention Of SSTF

Objective:In this study,the middle cerebral artery thrombosis model of rats which were intervented by SSTF was established following the modified suture method by Zea Longa,in order to investigate the intervention effect of SSTF to all of them on focal cerebral ischemia-reperfusion injury of cerebral cortex. we recorded the neurologic impairment and the percent infarct volume of cerebral cortex,analyzed the microvesser architecture of cerebral cortex; observed the changes of biochemistry,pathology and some genes related to neuronal apoptosis.Method:200 Sprage-Dawley rats,both male and female,were divided randomly into five groups:every group was 40.rats in group 1(sham) only received vessel separation and 0.1M PBS ig;rats in group 2 received ischemia-reperfusion operation and 0.1M PBS ig;rats in group 3,4 and 5 received ischemia-reperfusion and SSTFⅠ(50mg/kg),SSTFⅡ(100mg/kg) and SSTFⅢ(200mg/kg) ig daily respectively.Every group rats were administered for a week,and then operation was done.Two hours later,we drew out the suture to recover perfusion.We recorded the neurologic impairment according to the 5 score of Zea Longa,measured the infarct volume with the TTC method.Displayed the microvesser of cerebral cortex following the TA-FE method;measured the content of maleic dialdehyde(MDA),the activity of superoxide dismutase(SOD) and lactate dehydrogenase(LDH) with the concentrations of right cerebral cortex; investigated cell apoptosis and the histopathological change by HE and immunohistochemical method;observed the ultrastructure of cortical neurons and blood brain barrier with the transmission electron microscope. All datas were analyzed with One-way ANOVA and LSD test by SPSS 11.5 statistics software,p＜0.05 was regarded to have statistical significance. Results:1 Neurological evaluation of focal cerebral ischemia-reperfusionRats in the sham group can't be seen abnormity.After 2h of occlusion and 24h of reperfusion,the neurological syndromes were showed in the I/R group and the interventional groups by SSTF,especially in the I/R group.Rats displayed Horner's syndrome,forelimb flexion,circling behaviortoward left. SSTFⅡ,Ⅲgroups had significant difference compared to I/R group(P＜0.05);but the neuroprotective effects of SSTF intervention on focal cerebral ischemia-reperfustion in different doses groups had no significant difference(P＞0.05).The neurological deficit evaluation of every group respectively for:sham group:0±0,I/R group:2.80±0.83,SSTFⅠ,Ⅱ,Ⅲgroup:2.00±0.71,1.40±0.54,1.20±1.38.2 The infarction area of cerebral cortexThe infarction size was measured by 2,3,5-triphenytetrazolium chloride (TTC) staining technique.The cerebral slices of the sham group were stained uniform red;the infarction area which was not stained appeared in the frontal-parietal cortex,partial temporal cortex,lateral striatum,hippocampus and so on.Compared to I/R group(8.54±0.76)%,drug-treated groups markly inhibited the infarct size by(6.82±0.60)%(SSTFⅠ,P＜0.01),(5.10±0.74)% (SSTFⅡ,P＜0.01),(4.20±1.34)%(SSTFⅢ,P＜0.01),the results of the two groups(between SSTFⅡandⅢgroup) had no significant difference(P＞0.05).3 Quantitative analysis of microvesser architecture in the cerebral cortexThree visual fields were selected randomly from the frontal-parietal cortex,which were used to analyze the microvesser density(MVD) and microvesser area density(MVA) with Mivnt image analysis system,caculated the number of MVD and MVA respectively.Compared to the I/R group,the number of MVD and MVA in the interventional groups by SSTF increased obviously(P＜0.05),meanwhile,there was statistical difference between SSTFⅡ,Ⅲgroups and SSTFⅠgroup(P＜0.01),but the results of the two groups (between SSTFⅡandⅢgroup) had no significant difference(P＞0.05). 4 The change of biochemical index in the cerebral cortexAfter 2h of occlusion and 24h of reperfusion,in the I/R group,the content of MDA increased sharply and the activity of SOD and LDH decreased obviously.In the interventional groups by SSTF,the content of MDA decreased and the activity of SOD and LDH increased,there was significant difference(P＜0.05～0.01) compared to I/R group.5 Histopathologic changes of cerebral cortex tissue with microscope and electron microscopeThe histopathologic changes with microscope as follows:in the sham group,the cells displayed normal shape,clear margin,clear nucleolus, nuclears were large and circle;in the I/R group,there were malacia,cell fragmentation,liquified and lighter cytoplasm,pyknosis showed triangle or asymmetry;the cells injury in SSTFⅡ,Ⅲreduced,morphology and structure tended to normal,but some abnormal cells were still seen.Under electron microscope,in the sham group,we saw intact cell membrane,clear margin,centered nucleolar,chromatin distributed uniformly, abundant mitochondrions,organelles such as golgi apparatus and endoplasmic reticulums were integrity;the morphology of endotheliocyte cells was normal, basement membranes were integrated.In the I/R group,however,we saw extensive cerebral edema,pycnosis or necrosis neurons,capillaries wrinkled or even closed,the morphology of endotheliocyte cells and membranes were irregular,basement membranes were incomplete.Mitochondrions were swollen and the number decreased,part of nuclear membrane were fusion/disappearance.Compared to I/R group,the results of the interventional groups by SSTF improved,the edema of capillaries reduced,most morphology of cells showed normal,basement membranes were almost integrated,part neurons displayed that nuclears were irregular slightly, heterochromatin got together and were kept to sides.Mitochondrions were swollen slightly and the number increased;golgi apparatus and endoplasmic reticulums tended to normal.The effect of SSTFⅡ,Ⅲgroup was more obvious than SSTFⅠgroup. 6 The expression of Fas,FasL,Hsp70Fas and Hsp70 expressed mainly at cytoplasm and membrane,FasL expressed mainly at membrane,under the microscope,we selected five high power light microscopes to calculate the positive rat of Fas,FasL,Hsp70 in ischemic area.Compared to I/R group,the positive rat of the interventional groups by SSTF dncreased obviously(I/R group:0.047029±0.009163 and 0.050615±0.010453;SSTFⅠgroup:0.038184±0.008293 and 0.038697±0.009886,P＜0.05;SSTFⅡgroup:0.021024±0.004646 and 0.026830±0.006299,P＜0.01;SSTFⅢgroup:0.019851±0.002611 and 0.022099±0.00668,P＜0.01);the positive rate of the interventional groups by SSTF increased apparently(I/Rgroup:0.037565±0.003882,SSTFⅠgroup:0.052303±0.012600,P＜0.05;SSTFⅡgroup:0.068629±0.012943,P＜0.01;SSTFⅢgroup: 0.072681±0.016248,P＜0.01).All datas were analyzed with One-way ANOVA and LSD test by SPSS 11.5 statistics software and expressed with(?)±s.Conclusion:1 The modle of MCAO I/R in rats following the modified suture method by Zea Longa can conctrol the time of ischemia and reperfusion accurately,can model the pathophysiological status of I/R in ischemic cerebrovascular disease of human,can evaluate the choice of the effect of reperfusion and therapeutic time window conveniently.It's a comparatively ideal animal model to study cerebral ischemia-reperfusion injury.2 The interventional groups by SSTF all have protective effect on the cerebral cortex of ischemia-reperfusion injury,in which the effect of SSTFⅡ,Ⅲgroups are better than SSTFⅠgroup,however,the results of the two groups(between SSTFⅡandⅢgroup) had no significant difference, therefore,in order to save the herbs,we think that using SSTFⅡcan reach protective effect.