依达拉奉对深低温冻存大鼠断肢再植后缺血再灌注损伤骨骼肌细胞膜及线粒体的保护效应

背景:大量的重要器官因为无法长期保存而失去临床应用价值,经深低温冻存的组织或器官,随着血流的恢复,同样会发生缺血再灌注损伤,其中骨骼肌是最易受累的组织。目的:观察依达拉奉对深低温冻存大鼠肢体再植后缺血再灌注损伤骨骼肌细胞膜及线粒体的保护作用。设计:随机对照动物实验。单位:南方医科大学基础医学院,山东省立医院手足外科。材料:实验于2006-04/11在山东省立医院低温实验室(省级)完成。选取健康成年雄性Wistar大鼠36只,由山东大学医学院实验动物中心提供,按随机数字表法分为3组,对照组、冷冻组和依达拉奉组各12只。方法:对照组只暴露股动、静脉,不离断肢体;其余两组制作深低温保存断肢再植缺血再灌注损伤动物模型,均截断右后肢,经深低温冻存前处理,然后移入液氮容器内保存1个月;实验时复温、常规洗脱液灌洗后行断肢再植,恢复肢体血供4h后取材(其中依达拉奉组洗脱液中含依达拉奉0.5mg/kg)。选取相同时点骨骼肌标本,制备骨骼肌细胞膜并提取骨骼肌线粒体,测定各组骨骼肌细胞膜荧光偏振度(反应细胞膜脂区流动性)、线粒体丙二醛含量、超氧化物歧化酶活性及呼吸控制率,并对骨骼肌线粒体超微结构行透射电镜观察。主要观察指标:①各组大鼠骨骼肌细胞膜荧光偏振度、线粒体丙二醛含量、超氧化物歧化酶活性和呼吸控制率。②各组大鼠骨骼肌线粒体超微结构。结果:36只大鼠全部进入结果分析,无脱失。①各组大鼠骨骼肌线粒体超氧化物歧化酶活性及呼吸控制率:依达拉奉组高于冷冻组(P均<0.05)。②各组大鼠骨骼肌细胞膜荧光偏振度和骨骼肌线粒体丙二醛含量:依达拉奉组低于冷冻组(P均<0.05)。③各组大鼠骨骼肌线粒体超微结构:依达拉奉组骨骼肌损伤程度明显轻于冷冻组。结论:依达拉奉能减轻骨骼肌缺血再灌注损伤,对骨骼肌细胞膜及线粒体有保护作用。其作用可能与依达拉奉直接抑制羟自由基、提高骨骼肌超氧化物歧化酶活性、减少丙二醛的产生,使细胞进行正常的氧化磷酸化有关。

Protective influence of edaravone on cellular membrane and mitochondria of replanted rat extremities following ischemia/reperfusion injury due to cryopreservation and rewarming

BACKGROUND: A lot of important organs are worthless for clinical application because they are hard to store for a long time. In addition, tissues or organs which are dealt with cryopreservation also attack ischemia/reperfusion injury with the recovery of blood flow; especially, skeletal muscle is the most involved tissue.OBJECTIVE: To observe the protective influence of edaravone on cellular membrane and mitochondria of replanted rat extremities following ischemia/reperfusion injury due to cryopreservation and rewarming.DESIGN: Randomized contrast animal study.SETTING: Basic Medical College of Southern Medical University; Department of Hand and Foot Surgery, Shandong Provincial Hospital.MATERIALS: The experiment was carried out in the Cryopreservation Laboratory, Shandong Provincial Hospital from April to November 2006. A total of 36 healthy adult male Wistar rats were provided by Experimental Animal Center of Medical College of Shandong University. All rats were randomly divided into control group, cryopreservation group and edaravone group with 12 in each group.METHODS: Femoral artery and vein of rats in control group were exposured, but extremities were not blocked. Rats in other two groups were used to establish ischemia/reperfusion injury models of replanted extremities. Before cryopreservation, their right hindlimbs were cut off and maintained in liquid nitrogen container for 1 month. After the operation mentioned above, the broken limbs were rewarmed, perfused with routine eluant and replanted. Four hours later, blood supply of extremities was recirculated and the samples were selected. Eluant in edaravone group contained 0.5 mg/kg edaravone. Samples of skeletal muscle were selected at the same time point to establish cellular membrane and extract mitochondria. Furthermore, fluorescence polarization of cellular membrane (reflecting liquidity in cellular membrane lipid area), malondialdehyde (MDA) content of mitochondria, superoxide dismutase (SOD) activity and respiratory controlling rate were measured; meanwhile, mitochondrial ultrastructure of skeletal muscle was observed under transmission electron microscope.MAIN OUTCOME MEASURES:①Fluorescence polarization of cellular membrane, MDA content of mitochondria, SOD activity and respiratory controlling rate of skeletal muscle; ②mitochondrial ultrastructure of skeletal muscle.RESULTS: All 36 rats were involved in the final analysis without any loss. ①SOD activity and respiratory controlling rate of mitochondria in skeletal muscle: The values of these two items were higher in edaravone group that those in cryopreservation group (P＜0.05).②Fluorescence polarization of cellular membrane and MDA content of mitochondria in skeletal muscle: The values of these two items were lower in edaravone group than those in cryopreservation group (P＜0.05). ③Mitochondrial ultrastructure of skeletal muscle: Injured degree of skeletal muscle was milder in edaravone group than that in cryopreservation group.CONCLUSION: Edaravone can relieve ischemia/reperfusion injury of skeletal muscle and protect cellular membrane and mitochondria of skeletal muscle. Its mechanism may be related to directly inhibiting hydroxy free radicals, increasing SOD activity of skeletal muscle, reducing generation of MDA and promoting normal oxidative phosphorylation.