CAJ | 학술논문

目的观察瑞芬太尼对大鼠全脑缺血再灌注损伤后海马神经细胞凋亡和半胱天冬酶(Caspase)-3表达的影响.方法 80只大鼠随机分为假手术(sham)、NS、REM2、REM6和REM20组,每组16只.NS、REM2、REM6和REM20组分别于缺血再灌注前静脉滴注0.9%氯化钠溶液及瑞芬太尼2、6、20μg·kg~(-1)·min~(-1).采用双侧颈总动脉阻断+低血压法建立大鼠短暂性全脑缺血模型.于全脑缺血前30 min由靶控微量注射泵经股静脉注射瑞芬太尼,并于双侧颈总动脉阻断前及开放后10 min进行动脉血血气分析.再灌注后24 h,每组各取8只大鼠,取其新鲜海马组织,采用实时逆转录-聚合酶链反应(RT-PCR)检测各组Caspase-3 mRNA的表达.再灌注后72 h,取剩余大鼠,灌注取脑,制作脑组织切片,采用苏木精伊红(H-E)染色和原位末端标记法(TUNEL法)观察各组大鼠海马退变的神经元数和凋亡细胞数,采用免疫组织化学法检测各组Caspase-3蛋白的表达.结果 ①H-E染色结果:sham组偶可见退变的锥体细胞,缺血再灌注72 h后,NS组及各瑞芬太尼预处理组海马退变的神经元数较sham组显著增加(P值均<0.05),REM6及REM20组海马退变的神经元数较NS组显著减少(P值均<0.05).②TUNEL法检测结果:sham组未见TUNEL阳性细胞,缺血再灌注72 h后,NS组及各瑞芬太尼预处理组海马CAI区的凋亡锥体细胞数较sham组显著增加(P值均<0.05),REM6和REM20组海马CAI区的凋亡锥体细胞数较NS组显著降低(P值均<0.05).③免疫组织化学法检测结果:sham组可见少量Caspase-3免疫阳性细胞,缺血再灌注72 h后,NS组及各瑞芬太尼预处理组海马区Caspase-3蛋白的免疫阳性细胞数较sham组显著增多(P值均<0.05),各瑞芬太尼预处理组Caspase-3蛋白的免疫阳性细胞数显著低于NS组(P值均<0.05),REM6和REM20组Caspase-3蛋白的免疫阳性细胞数显著低于REM2组(P值均<0.05).④实时RT-PCR结果:sham组大鼠海马组织中Caspase-3 mRNA呈低水平表达,缺血再灌注24 h后,NS组及各瑞芬太尼处理组Caspase-3 mRNA的表达水平较sham组显著升高(P值均<0.05),REM6组Caspase-3 mRNA的表达较NS组显著降低(P<0.05).结论瑞芬太尼预处理(以6 μg·kg~(-1)·min~(-1)为佳)对全脑缺血再灌注后的神经元损伤具有一定的保护作用,其机制可能与抑制神经元凋亡及下调Caspase-3基因有关. 目的觀察瑞芬太尼對大鼠全腦缺血再灌註損傷後海馬神經細胞凋亡和半胱天鼕酶(Caspase)-3錶達的影響.方法 80隻大鼠隨機分為假手術(sham)、NS、REM2、REM6和REM20組,每組16隻.NS、REM2、REM6和REM20組分彆于缺血再灌註前靜脈滴註0.9%氯化鈉溶液及瑞芬太尼2、6、20μg·kg~(-1)·min~(-1).採用雙側頸總動脈阻斷+低血壓法建立大鼠短暫性全腦缺血模型.于全腦缺血前30 min由靶控微量註射泵經股靜脈註射瑞芬太尼,併于雙側頸總動脈阻斷前及開放後10 min進行動脈血血氣分析.再灌註後24 h,每組各取8隻大鼠,取其新鮮海馬組織,採用實時逆轉錄-聚閤酶鏈反應(RT-PCR)檢測各組Caspase-3 mRNA的錶達.再灌註後72 h,取剩餘大鼠,灌註取腦,製作腦組織切片,採用囌木精伊紅(H-E)染色和原位末耑標記法(TUNEL法)觀察各組大鼠海馬退變的神經元數和凋亡細胞數,採用免疫組織化學法檢測各組Caspase-3蛋白的錶達.結果 ①H-E染色結果:sham組偶可見退變的錐體細胞,缺血再灌註72 h後,NS組及各瑞芬太尼預處理組海馬退變的神經元數較sham組顯著增加(P值均<0.05),REM6及REM20組海馬退變的神經元數較NS組顯著減少(P值均<0.05).②TUNEL法檢測結果:sham組未見TUNEL暘性細胞,缺血再灌註72 h後,NS組及各瑞芬太尼預處理組海馬CAI區的凋亡錐體細胞數較sham組顯著增加(P值均<0.05),REM6和REM20組海馬CAI區的凋亡錐體細胞數較NS組顯著降低(P值均<0.05).③免疫組織化學法檢測結果:sham組可見少量Caspase-3免疫暘性細胞,缺血再灌註72 h後,NS組及各瑞芬太尼預處理組海馬區Caspase-3蛋白的免疫暘性細胞數較sham組顯著增多(P值均<0.05),各瑞芬太尼預處理組Caspase-3蛋白的免疫暘性細胞數顯著低于NS組(P值均<0.05),REM6和REM20組Caspase-3蛋白的免疫暘性細胞數顯著低于REM2組(P值均<0.05).④實時RT-PCR結果:sham組大鼠海馬組織中Caspase-3 mRNA呈低水平錶達,缺血再灌註24 h後,NS組及各瑞芬太尼處理組Caspase-3 mRNA的錶達水平較sham組顯著升高(P值均<0.05),REM6組Caspase-3 mRNA的錶達較NS組顯著降低(P<0.05).結論瑞芬太尼預處理(以6 μg·kg~(-1)·min~(-1)為佳)對全腦缺血再灌註後的神經元損傷具有一定的保護作用,其機製可能與抑製神經元凋亡及下調Caspase-3基因有關.
목적 관찰서분태니대대서전뇌결혈재관주손상후해마신경세포조망화반광천동매(Caspase)-3표체적영향.방법 80지대서수궤분위가수술(sham)、NS、REM2、REM6화REM20조,매조16지.NS、REM2、REM6화REM20조분별우결혈재관주전정맥적주0.9%록화납용액급서분태니2、6、20μg·kg~(-1)·min~(-1).채용쌍측경총동맥조단+저혈압법건립대서단잠성전뇌결혈모형.우전뇌결혈전30 min유파공미량주사빙경고정맥주사서분태니,병우쌍측경총동맥조단전급개방후10 min진행동맥혈혈기분석.재관주후24 h,매조각취8지대서,취기신선해마조직,채용실시역전록-취합매련반응(RT-PCR)검측각조Caspase-3 mRNA적표체.재관주후72 h,취잉여대서,관주취뇌,제작뇌조직절편,채용소목정이홍(H-E)염색화원위말단표기법(TUNEL법)관찰각조대서해마퇴변적신경원수화조망세포수,채용면역조직화학법검측각조Caspase-3단백적표체.결과 ①H-E염색결과:sham조우가견퇴변적추체세포,결혈재관주72 h후,NS조급각서분태니예처리조해마퇴변적신경원수교sham조현저증가(P치균<0.05),REM6급REM20조해마퇴변적신경원수교NS조현저감소(P치균<0.05).②TUNEL법검측결과:sham조미견TUNEL양성세포,결혈재관주72 h후,NS조급각서분태니예처리조해마CAI구적조망추체세포수교sham조현저증가(P치균<0.05),REM6화REM20조해마CAI구적조망추체세포수교NS조현저강저(P치균<0.05).③면역조직화학법검측결과:sham조가견소량Caspase-3면역양성세포,결혈재관주72 h후,NS조급각서분태니예처리조해마구Caspase-3단백적면역양성세포수교sham조현저증다(P치균<0.05),각서분태니예처리조Caspase-3단백적면역양성세포수현저저우NS조(P치균<0.05),REM6화REM20조Caspase-3단백적면역양성세포수현저저우REM2조(P치균<0.05).④실시RT-PCR결과:sham조대서해마조직중Caspase-3 mRNA정저수평표체,결혈재관주24 h후,NS조급각서분태니처리조Caspase-3 mRNA적표체수평교sham조현저승고(P치균<0.05),REM6조Caspase-3 mRNA적표체교NS조현저강저(P<0.05).결론 서분태니예처리(이6 μg·kg~(-1)·min~(-1)위가)대전뇌결혈재관주후적신경원손상구유일정적보호작용,기궤제가능여억제신경원조망급하조Caspase-3기인유관.
Objective To study the effect of remifentanil preconditioning on neuron apoptosis and Caspase-3 expression after forebrain ischemia/reperfusion (I/R) injury in rats. Methods Eighty male Sprague-Dawlay rats were randomly divided into 5 groups: sham group (n=16) ; control group (NS, n=16) ; REM2 group(remifentanil 2μg·k~(-1)·min~(-1), n=16) ;REM6 group (remifentanil 6 μg·kg~(-1)·min~(-1),n=16); and REM20 group (remifentanil 20 μg·kg~(-1)·min~(-1) , n=16). Transient forebrain ischemia was induced by bilateral common carotid artery (CCA) occlusion for 10 min combined with hypotension. Rats received preconditioning with intravenous NS, remifentanil 2, 6 or 20μg·kg~(-1)·min~(-1) 30 min before occlusion. The right femoral artery was cannulated to collect 0.5 mL blood samples for analysis blood gas 10 min before ischemia and 10 min after ischemia. Half of rats in each group were killed 24 h after reperfusion, and real-time reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the expression of Caspase-3 mRNA in the hippocampus. The rest rats were killed 72 h after reperfusion, and brain paraffin sections were prepared. The total degenerated neurons in hippocampal CA1 were counted btindly under high power field. The number of neuronal apoptosis was assessed by terminal oxynucleotidyl transferase mediated dUTP biotin nick end labeling (TUNEL). The apoptosis-related proteins caspase-3 were analyzed, and immunohistochemical was used to detect caspase-3 protein.Results ①The degenerated neurons in hippocampal CA1 were significantly increased in NS, REM2, REM6, and REM20 groups compared with that in the sham group after 72 hours reperfusion. Degenerated neurons in hippocampal CA1 were significantly reduced in REM6 and REM20 groups compared with that in the NS group (P< 0.05). ② TUNEL showed no positive cells in the sham group. The neuron apoptosis increased significantly in the hippocampus CA1 in REM groups 72 h after reperfusion. The numbers of neuronal apoptosis in hippocampus CAI were significantly decreased in REM6 and REM20 groups compared with that in NS group (P <0. 05). ③Immunohistochemical results showed weak Caspase-3 protein expression in the sham group, and the expression in the hippocampal region was greatly increased in REM groups 72 h after reperfusion. Caspase-3 protein expression was higher in REM2 group than in REM6 and REM20 group (P <0.05) ,with no significant differences found between the latter two groups (P> 0.05). ④ Real-time RT-PCR results indicated that Caspase-3 mRNA expression increased significantly in the hippocampus following forebrain cerebral ischemia-reperfusion. As compared with that in group NS,the expression of caspase-3 mRNA in the hippocampus decreased significantly in group REM6 24 h after reperfusion (P<0.05). Conclusion Remifentanil, especially at 6μg·kg~(-1)·min~(-1), has a neuroprotective effect against forebrain I/R injury, and the mechanisam may be related to inhibition neuronal apoptosis and down-regulation of Caspase-3 expression.