抗细胞间黏附分子-1抗体对大鼠脑缺血再灌注损伤的保护作用

背景 研究发现脑缺血后多形核白细胞( polymorphonuclear leukocyte,PMNL)与微血管内皮细胞( capillary endothelial cell, CEC)间的黏附增多,应用抗细胞间黏附分子-1( intercellular adhesion molecule-1 ICAM-1)抗体可减轻神经元的缺血性损伤.但是目前还没有直接的证据表明抗-ICAM-1抗体可以抑制 PMNL与 CEC间的黏附过程. 目的观察脑缺血再灌注后 PMNL与 CEC间的黏附性变化,探讨应用抗-ICAM-1抗体对脑缺血再灌注后 PMNL与 CEC间黏附性的影响. 设计完全随机设计. 地点和对象实验地点设在第三军医大学大坪医院神经内科实验室.以成年雄性 Wistar大鼠作为实验动物,分为对照组 (n=4)、假手术组 (n=6)、脑缺血再灌注组 (n=10)和治疗组 (n=10). 干预假手术组、脑缺血再灌注组和治疗组大鼠分别于假手术和再灌注 4, 12, 24 h后抽取静脉血 2～ 4 mL.治疗组大鼠于缺血再灌注后 1 h静脉注射抗 ICAM-1抗体( 1 mg/kg).用右旋糖酐沉淀法和 Percoll密度梯度离心法分离出 PMNL,加入培养的 CEC, 1 h后进行微管吸吮检测. 主要观察指标 PMNL与 CEC间的黏附力和黏附应力. 结果脑缺血再灌注 4 h后 PMNL和 CEC间的黏附力和黏附应力明显升高,并于 12 h达到高峰.治疗组大鼠在各时间点的黏附力 4 h (14.3 ± 0.6) N,12 h (16.2± 0.8) N,24 h (13.7± 0.5) N]和黏附应力 4 h (37.2± 16.6) Pa,12 h (38.9± 14.3) Pa,24 h (33.2± 10.1) Pa]都高于假手术组 黏附力 4 h (3.8± 0.3) N,12 h (4.67± 0.2)N,24 h (3.49 ± 0.2) N;黏附应力 4 h (9.7± 1.21) Pa,12 h (10.9± 1.48) Pa,24 h (8.6± 1.39) Pa]( t=2.92～ 38.52,P< 0.01),但明显低于脑缺血再灌注组 黏附力 4 h (17.8± 0.9) N,12 h (24.9± 2.1)N,24 h (15.3 ± 1.2)N;黏附应力 4 h (46.3± 19.8) Pa,12 h (59.7± 20.6) Pa,24 h (38.2± 11.7) Pa](t=-2.82～-13.51,P< 0.01). 结论 ICAM 1可能通过介导脑缺血再灌注后的细胞间黏附过程而参与了神经元的再灌注损伤;抗-ICAM 1抗体可抑制脑缺血再灌注后 PMNL和 CEC间的黏附,为治疗缺血性脑血管病的提拱新的思路.

Effects of anti-intercellular adhesion moleculer-1 antibody on cerebral ischemia-reperfusion injury in rats

BACKGROUND:The adhesion of polymorphonuclear leukocyte(PMNL) and capillary endothelial cell(CEC) increases after cerebral ischemia. Anti-intercellular adhesion molecule-1(ICAM-1) antibody can remarkably relieve the damage of neuron.However,the inhibitory effect of anti-ICAM-1 antibody on the adhesion between PMNL and CEC still lacks direct evidences. OBJECTIVE:To investigate the change of adhesion between PMNL and CEC after cerebral ischemia reperfusion and the effect of anti-ICAM-1 antibody on adhesion between PMNL and CEC. DESIGN:A completely randomized trial. SETTING and PARTICIPANTS:All experiments were performed in the Laboratory of Neurology,Daping Hospital,Third Military Medical University of PLA.Adult male Wistar rats as experimental animals were randomly divided into four groups:normal control group(n=4),sham operation group(n=6),cerebral ischemia reperfusion group(n=10),and anti -ICAM-1 antibody group(treatment group,n=10). INTERVENTIONS:After 4,12,and 24 hours of sham operation and reperfusion,2-4 mL venous blood samples were obtained from rats in sham operation group,ischemia-reperfusion group,and treatment group,respectively.The anti-ICAM-1 antibody was used(1 mg/kg injection of vein) after 1 hour of reperfusion in treatment group.PMNL of venous blood was isolated and CEC was added with dextran sedimentation and Percoll density gradient centrifugation.Micropipette aspiration was performed 1 hour later. MAIN OUTCOME MEASURES:Adhesion force and adhesion stress between PMNL and CEC. RESULTS:Adhesion force and adhesion stress between PMNL and CEC significantly increased after 4 hours of reperfusion,and reached peak after 12 hours.In treatment group,the adhesion force at different time points were as follows: 4 hours,(14.3± 0.6) N;12 hours,(16.2± 0.8) N;and 24 hours,(13.7± 0.5) N.The adhesion stress at different time points were as follows: 4 hours,(37.2± 16.6) Pa;12 hours,(38.9± 14.3) Pa;and 24 hours,(33.2± 10.1) Pa.The results were higher than those in sham operation group4 hours,(3.8± 0.3) N and(9.7± 1.21) Pa;12 hours,(4.67 ± 0.2) N and(10.9± 1.48) Pa;24 hours,(3.49± 0.2) N and(8.6 ± 1.39) Pa](t=2.92-38.52,P< 0.01),but were lower than those in ischemia reperfusion group4 hours,(17.8± 0.9) N and(46.3± 19.8) Pa;12 hours,(24.9± 2.1) N and(59.7± 20.6) Pa;24 hours,(15.3± 1.2) N and(38.2± 11.7) Pa](t=-2.82--13.51,P< 0.01). CONCLUSIONS:ICAM-1 may be involved in reperfusion damage of neuron.Anti ICAM-1 antibody can significantly inhibit the adhesion process after cerebral ischemia-reperfusion and it may be an effective therapeutical approach for ischemic cerebrovascular diseases.