| Section 1 The Endovascular Perforation Model of Subarachnoid Hemorrhage Producing and SAH GradingObjective: The aim of this study is to examine the effects of apoptosis on EBI after SAH in mice.So successfully producing the endovascular perforation model of SAH is the basic of this study. Simultaneously ,the model was also assessed in terms of mortality,neurological scores and grade of SAH at 24 hours.Methods:1 Experimenal animal and groups 30 of the male adult kunming mice(28g-32g weight) were randomly divided into 2 groups: 6 of sham group and 24 of model group.2 Model madeBriefly, animals were anesthetized with an intraperitoneal injection of Chloral hydrate (0.3ml/kg). By using surgical microscope, the right common carotid artery was exposed, and the external carotid artery was isolated and ligated.a blunted 5-0 monofilament nylon suture was introduced into the external carotid artery and advanced through the internal carotid artery (ICA) to the right anterior cerebral artery (ACA) near the anterior communicating artery, where resistance was encountered. The filament was advanced 3mm further to perforate the wall of artery, then immediately withdrawn. In the sham surgery the filaments were advanced without arterial perforation. Rectal temperature was kept constant (37.5±0.5°C) during the operation.3 Severity of SAH Brains were analyzed under light microscopy (x6 magnification) to determine the magnitude of SAH by an observer blinded to each group. Hemorrhage size was graded by 2 characteristics: area of hemorrhage distribution and density of clot formation. Hemorrhage size was scored as follows: 1=SAH extends <1.0 mm from MCA-ACA bifurcation; 2=SAH extends >1.0 mm from bifurcation; and 3=SAH extends >1.0 mm from bifurcation with extension to the contralateral internal carotid artery. Hemorrhage density was scored as follows: 1=brain parenchyma visualized through clot; 2=brain parenchyma not visualized through clot. Hemorrhage grade (2 to 5) was defined as the sum of size and density scores.4 Neurological Scores and MortalityThe neurological scores were evaluated in a blinded fashion at 24 hours post-SAH, based on the scoring system of Garcia et al with modifications. A motor score (0 to 12) was derived from spontaneous activity, symmetry of limb movements, climbing, balance, and coordination, with each scored 0 to 3. A sensory score (1 to 9) was derived from examination of body proprioception and vibrissae, visual, olfactory, and tactile responses to stimulus, with each parameter scored on a scale of 1 to 3. Animals were given a score of 3 to 21(higher scores indicate greater function). Mortality was calculated at 24 hours after SAH.5 Statistical Analysist test was used to compare continuous variables through SPSS13.0 for windows. There was statistically significant if lateral p value < 0.05 .Results:1 general conditionMice of sham group looked well as conditions of preoperation,while model mice were sick and lack of movement.2 Neurological scoreThe neurological scores of the sham group were 21, which was higher than model group(14.8±1.5). There were significant difference between two groups at 24 hours (P<0.05). 3 SAH gradingSAH was distributed in the basal cistern of all SAH mice. SAH grade 2 were 4 mice, grade 3 were 8 and grade 4 were 12.4 MortalityMortality was calculated at 24 hours after SAH. Mice of sham group were none of died, while 9 of model group were died(death rate=37.5%, n=24). Conclusions: Brain swelling was apparent immediately after the puncture of middle cerebral artery . Blood was all over the whole brain at 24 hours after SAH, especially around MCA-ACA bifurcation ipsilateral of operation.Section 2 Edaravone Attenuating Apoptosis of Hippocampal Neurons by Downreguling Expression of ERK1/2,Bax and caspase-3 in early stage of subarachnoid hemorrhage of miceObjective: To investigate the role of the ERK1/2 and the cascades mechanism mediated by it in early stage of subarachnoid hemorrhage of mice. To investigate the influence of edaravone on the changes of p-ERK1/2, Bax, caspase-3 in hippocampus of the mouse model of SAH induced by endovascular perforation of the right anterior cerebral artery, to clarify the edaravone's protective effects in neuronal apoptosis and to explore its molecular mechanism in order to offer theoretic evidences for clinical therapy.Methods:1 Experimenal animal and groupsThe male adult kunming mice(28g-32g weight) were randomly divided into 3 groups: sham group , SAH +NS group and SAH + edaravone group.2 Model and Drug InjectionWe used the endovascular perforation model of SAH as previous.At different time points after SAH , the mice were designated as subgroups 12h, 24h, 48h and 72h. At 30min after SAH immediately, we administered edaravone (3mg/kg) intraperitoneally injected in edaravone group. Then it was administered once every 12h untill the death of the time points . In the SAH+NS group, the equal value of NS was injected at the same time point.3 Neurological score and SAH gradingSee section 1.4 Detection methodIt was estimated the neurological score and the SAH grading at 24,48,72hours. The histopathologic changes were observed by the HE staining method. Western Blot and immunochemistry were used to evaluate the expression level and regional distribution of p-ERK1/2,Bax,caspase-3 in hippocampus after SAH.Apoptosis neurons were detected by Terminal deoxynucleotidyl transferase–mediated uridine 5'-triphosphate-biotin nick end-labeling (TUNEL) method.5 Statistical analysisParametric values were given as mean±SD. Comparisons among multiple groups were performed with a one-way ANOVA(SPSS13.0 for windows). Comparisons between 2 groups were achieved with rank sum test and Student's unpaired t test(SPSS13.0 for windows). Pearson correlation analysis was used to the relationship among multiple factors. A P value of <0.05 was considered statistically significant.Results:1 SAH grading and neurological scoreSAH grade was no difference between groups (SAH+NS vs SAH+ edaravone group,P>0.05). The sham group found no SAH. The neurological scores of the sham group were 21, which was higher than SAH+NS group and SAH+edaravone group. There were significant difference among 3 groups at 24h.2 Pathological changesH-E staining showed a large number of neat, complete form neurons in sham-operated group.The neurons in SAH group were swelling. Some cells diminished and nucleus karyopyknosis and anachromasissized. The neurons of SAH +edaravone group had the same changes compared with the SAH+NS group, while the quantity decreased.3 p-ERK1/2 Protein ExpressionThe production of p-ERK1/2 located in the cytoplasm and nucleus of neural cells and were stained yellow and brown. Compared with the sham group, positive cells increased at 12h after SAH ,then reached its peak at 24h and decreased at 48h. The changes of p-ERK1/2 in the SAH+ edaravone group were similar to the SAH+NS group, but significantly decreased at 24h. Western blot quantification indicated that the sham group has only little expression of p-ERK1/2. There were no changes in each time point. p-ERK1/2 in SAH+NS group began to increase obviously at 24h. Quantification showed decreased expression of p-ERK1/2 in SAH+edaravone group at 12h(2.04±0.04,2.91±0.11)and 24h(2.62±0.14,3.97±0.07) (P<0.05) .4 Bax Protein ExpressionThe production of Bax located in the cytoplasm of neural cells and were stained yellow and brown. Compared with the sham group, positive cells increased at 12h after SAH ,then reached its peak at 24h. The changes of Bax in the SAH+edaravone group were similar to the SAH+NS group, but significantly decreased at 12h and 24h. Western blot quantification indicated that the sham group has only little expression of Bax. There were no changes in each time point. Bax in SAH+NS group began to increase obviously at 24h peak. Quantification showed decreased expression of Bax in SAH+edaravone group at 12h(1.06±0.04,2.35±0.04)and 24h(2.03±0.06,3.97±0.02)(P<0.05).5 caspase-3 Protein ExpressionThe production of caspase-3 located in the cytoplasm of neural cells and were stained yellow and brown. Compared with the sham group, positive cells increased at 24h after SAH ,then reached its peak at 48h and decreased at 72h. The changes of caspase-3 in the SAH+edaravone group were similar to the SAH+NS group, but significantly decreased at 48h and 72h.Western blot quantification indicated that the sham group has only little expression of caspase-3. There were no changes in each time point. Caspase-3 in SAH+NS group began to increase obviously at 24h and peaked at 48h. Quantification showed decreased expression of caspase-3 in SAH+edaravone group at 48h(2.03±0.04,3.97±0.07)and 72 h(1.50±0.02,2.09±0.01)(P<0.05).6 TUNEL StainingAs compared with the sham group, TUNEL positive cells began to increase obviously at 24h and peaked at 72h. The TUNEL positive cells in SAH+edaravone group were almost the same as that of SAH+NS group, but the rate of positive cell obviousely decreased compared with the latter, especially at 24h ( 0.176±0.002 , 0.261±0.002 ),48h ( 0.241±0.002 ,0.336±0.010),72h(0.270±0.005,0.483±0.009)(P<0.05).7 Pearson correlation analysisThe quantification of p-ERK1/2 expression was related to the Bax immunoreactivity at 12h to 48h after SAH (r=0.676,P<0.05) ;The caspase-3 immunoreactivity was related to the Bax immunoreactivity at 12h to 72h after SAH(r=0.612,P<0.05); The caspase-3 immunoreactivity was related to the number of TUNEL positive cell at12h to 72h after SAH (r=0.659,P<0.05) .Conclusions:1p-ERK1/2 plays an important role in promoting apoptosis through Bax and caspase-3 in mice after SAH.2Edaravone can attenuate apoptosis of hippocampal neurons by downregulaion expression of ERK1/2,Bax and caspase-3 in early stage of SAH.Section 3 Edaravone Attenuating Apoptosis of Cerebrovascular Endotheliocyte and Cerebral Vasospasm by Downregulating Expression of VEGF in Early Stage of Subarachnoid Hemorrhage of MiceObjective: To investigate the role of edaravone in regulation of apoptosis of endotheliocyte in vasospastic intracranial arteries, and how to affect the expression of VEGF.Methods: 1 Experimenal animal and groups The male adult kunming mice(28g-32g weight) were randomly divided into 3 groups: sham group , SAH +NS group and SAH + edaravone group.2 Model and Drug InjectionWe used the endovascular perforation model of SAH as previous.At different time points after SAH , the mice were designated as subgroups 12h, 24h, 48h and 72h. At 30min after SAH immediately, we administered edaravone (3mg/kg) intraperitoneally injected in edaravone group. Then it was administered once every 12h untill the death of the time points . In the SAH+NS group, the equal value of NS was injected at the same time point.3 Neurological score and SAH gradingSee section 1.4 Detection methodIt was estimated the neurological score and the SAH grading at 24,48,72hours. The histopathologic changes were observed by the HE staining method. Western Blot and in situ hybridization were used to evaluate the expression level and regional distribution of VEGF and VEGF mRNA in cerebrovascular endotheliocyte after SAH. Apoptosis endotheliocytes were detected by Terminal deoxynucleotidyl transferase–mediated uridine 5'-triphosphate-biotin nick end-labeling (TUNEL) method.5 Statistical analysisParametric values were given as mean±SD. Comparisons among multiple groups were performed with a one-way ANOVA(SPSS13.0 for windows). Comparisons between 2 groups were achieved with rank sun test and Student's unpaired t test(SPSS13.0 for windows). Pearson correlation analysis was used to the relationship among multiple factors. A P value of <0.05 was considered statistically significant.Results:1 SAH grading and neurological scoreSee section 1.2 Pathological changes A main effect was present for vascular diameter24h,48h and 72 hours after SAH or sham surgery (P<0.05). In SAH+NS mice, SAH caused reduction in MCA diameter (53.33±2.52μm). In edaravone -treated mice, SAH caused extention in MCA diameter (88.00±2.65μm).3 VEGF Protein ExpressionWestern blot quantification indicated that the sham group has only little expression of VEGF. There were no changes in each time point. VEGF in SAH+NS group began to increase at 12h and peaked at 24h. Quantification showed decreased expression of VEGF in SAH+edaravone group at 24h(1.73±0.02,2.57±0.04),48h(1.57±0.01,2.50±0.02)(P<0.05).4 VEGF mRNA ExpressionThere was no expression of VEGF mRNA in sham group.VEGF mRNA was significantly increased in endotheliocyte and lateral layer of arteries. In SAH+ edaravone group its expression was significantly decreased at 24h(1.987±0.004,2.275±0.001),48h(1.764±0.001,2.271±0.003)(P<0.05).5 TUNEL Staining in Middle Cerebral ArteryAs compared with the sham group, TUNEL positive cells began to increase obviously at 24h and peaked at 72h. The TUNEL positive cells in SAH+edaravone group were almost the same as that of SAH+NS group, but the number of positive cell obviousely decreased compared with the latter, especially at 24h(0.232±0.019, 0.342±0.010),48h(0.262±0.002, 0.422±0.003),72h (0.372±0.018, 0.555±0.035) (P<0.05).6 Pearson correlation analysisThe quantification of VEGF expression was related to the TUNEL positive cell at 12h to 72h after SAH(r=0.659,P<0.05),while the rate of TUNEL positive cell was related to the severity of cerebral vasospasm (r=0.631,P<0.05).Conclusions: VEGF contributes to early brain injury after SAH by enhancing the activation of the VEGF; Edaravone reduce early brain injury after SAH and apoptosis of cerebral arteries endotheliocyte by inhibiting the activation of the VEGF espression.
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