CAJ | 학술논문

目的观察浅低温联合艾芬地尔对大鼠全脑缺血再灌注后海马神经元存活和凋亡诱导因子(AIF)易位的影响,探讨两者在脑复苏中联合作用的机制.方法 80只雄性SD大鼠按随机数字表法分组(n=16):假手术组(Ⅰ)、模型组(Ⅱ)、艾芬地尔组(Ⅲ)、浅低温组(Ⅳ)和艾芬地尔+浅低温组(Ⅴ).Ⅰ组不给予电刺激和心肺复苏;Ⅱ～Ⅴ组致颤(4 min)建立全脑缺血再灌注模型;Ⅲ和Ⅴ组于再灌注即刻腹腔注射艾芬地尔,其余组注射等容量双蒸水;Ⅳ和Ⅴ组于再灌注即刻乙醇涂擦体表,10 min内将直肠温度降至(32±1)℃,维持4h,其余组维持直肠温度(37±1)℃.HE染色观察海马CA1区锥体细胞病理改变及计数;免疫荧光法检测海马CA1区AIF易位;免疫蛋白印迹法半定量测定AIF易位.结果与Ⅰ组(75.0±3.2)个比,Ⅱ、Ⅲ、Ⅳ和Ⅴ组CA1区锥体细胞计数均减少(P＜0.05);与Ⅱ组(36.0±1.2)个比,Ⅲ组(46.8±1.3)个、Ⅳ组(49.0±2.7)个和Ⅴ组(61.3±2.6)个均增加(P＜0.05),Ⅴ组增加最明显(P＜0.05).与Ⅰ组比,免疫荧光法观察Ⅱ、Ⅲ、Ⅳ和Ⅴ组均出现AIF易位.免疫蛋白印迹法结果显示,与Ⅰ组(0.022±0.003)比,其余各组AIF在细胞核的表达均增加(P＜0.05);与Ⅱ(1.020±0.029)组比,Ⅲ(0.870±0.016)、Ⅳ(0.820±0.050)、Ⅴ(0.550±0.050)组AIF在细胞核的表达均减少(P＜0.05),Ⅴ组减少最明显(P＜0.05).结论浅低温联合艾芬地尔能更有效地减轻全脑缺血再灌注后神经元损伤,其机制可能是减轻AIF易位后启动的促凋亡作用.
목적 관찰천저온연합애분지이대대서전뇌결혈재관주후해마신경원존활화조망유도인자(AIF)역위적영향,탐토량자재뇌복소중연합작용적궤제.방법 80지웅성SD대서안수궤수자표법분조(n=16):가수술조(Ⅰ)、모형조(Ⅱ)、애분지이조(Ⅲ)、천저온조(Ⅳ)화애분지이+천저온조(Ⅴ).Ⅰ조불급여전자격화심폐복소;Ⅱ～Ⅴ조치전(4 min)건립전뇌결혈재관주모형;Ⅲ화Ⅴ조우재관주즉각복강주사애분지이,기여조주사등용량쌍증수;Ⅳ화Ⅴ조우재관주즉각을순도찰체표,10 min내장직장온도강지(32±1)℃,유지4h,기여조유지직장온도(37±1)℃.HE염색관찰해마CA1구추체세포병리개변급계수;면역형광법검측해마CA1구AIF역위;면역단백인적법반정량측정AIF역위.결과 여Ⅰ조(75.0±3.2)개비,Ⅱ、Ⅲ、Ⅳ화Ⅴ조CA1구추체세포계수균감소(P＜0.05);여Ⅱ조(36.0±1.2)개비,Ⅲ조(46.8±1.3)개、Ⅳ조(49.0±2.7)개화Ⅴ조(61.3±2.6)개균증가(P＜0.05),Ⅴ조증가최명현(P＜0.05).여Ⅰ조비,면역형광법관찰Ⅱ、Ⅲ、Ⅳ화Ⅴ조균출현AIF역위.면역단백인적법결과현시,여Ⅰ조(0.022±0.003)비,기여각조AIF재세포핵적표체균증가(P＜0.05);여Ⅱ(1.020±0.029)조비,Ⅲ(0.870±0.016)、Ⅳ(0.820±0.050)、Ⅴ(0.550±0.050)조AIF재세포핵적표체균감소(P＜0.05),Ⅴ조감소최명현(P＜0.05).결론 천저온연합애분지이능경유효지감경전뇌결혈재관주후신경원손상,기궤제가능시감경AIF역위후계동적촉조망작용.
Objective To explore the effects of mild hypothermia combined with ifenprodil on the survival of neuronal and translocation of apoptosis inducing factor (AIF) following global cerebral ischemiareperfusion to understand the mechanism of combination in cerebral resuscitation.Methods Eighty male SD rats were randomly divided into 5 groups of sham (Ⅰ),model (Ⅱ),ifenprodil (Ⅲ),mild hypothermia (Ⅳ) and ifenprodil plus mild hypothermia (Ⅴ) (n =16 each).Group Ⅰ completed all procedures except for ventricular fibrillation (VF) and cardio pulmonary resuscitation (CPR).For groups Ⅱ and Ⅴ,the model of global cerebral ischemia-reperfusion was established and VF induced with transoesophageal cardiac pacing; groups Ⅲ and Ⅴ received by an intraperitoneal injection of ifenprodil immediately after reperfusion and other groups had an equal volume of distilled water.Rectal temperature was cooled down to (32 ± 1) ℃ in groups Ⅳ and Ⅴ by rubbing body surface with ethanol in 10 min after reperfusion and maintained 4 hours continuously while other groups at (37 ± 1)℃.In hippocampal CA1 region at 24 hours after reperfusion,the pathomorphological changes and quantity of pyramidal cells were detected with hematoxylin and eosin staining,nuclear translocation of AIF was shown with immunofluorescence technique and the nuclear expression level of AIF was measured with Western blot.Results Compared with group] (75.0 ± 3.2),the number of pyramidal cells decreased in other groups (P ＜ 0.05) ; compared with group Ⅱ (36.0 ± 1.2),the number increased in group Ⅲ (46.8 ± 1.3),Ⅳ (49.0 ±2.7)and Ⅴ (61.3 ±2.60) (P ＜0.05).In particular,cell count increased significantly in group Ⅴ (P ＜ 0.05).Compared to group Ⅰ,the transloctation of AIF form mitochondria to nucleus was detected in other groups ; compared with group Ⅰ (0.022 ± 0.003),the expression level of AIF in the nucleus was higher in other groups (P ＜ 0.05).Compared with group Ⅱ (1.020 ± 0.029),the expression levels of AIF in groups Ⅲ (0.870 ±0.016),Ⅳ(0.820 ±0.050) and Ⅴ (0.550 ±0.050)were lower (P ＜0.05).And it decreased significantly in group Ⅴ (P ＜ 0.05).Conclusion Mild hypothermia plus ifenprodil may alleviate neuronal damage after global cerebral ischemia/reperfusion injury through mitigating its proapoptotic role after AIF translocation.