采用夹闭髂外动静脉联合缚扎止血带法构建巴马小型猪肢体缺血再灌注损伤模型

Construction of a Bama miniature pig limb ischemia-reperfusion injury model using the method of occluding the iliac artery and vein combined with tourniquet ligation

目的建立简便有效的巴马小型猪肢体缺血再灌注模型。方法将12只巴马小型猪采用随机数字表法分为对照组(Sham)和肢体缺血再灌注损伤组(LI/RI),每组6只。Sham组仅暴露血管,LI/RI组夹闭血管并缚扎止血带模拟损伤。比较两组的氧化应激、炎性反应和组织损伤指标,采用两因素重复测量方差分析和t检验。结果7 d后成功构建模型。LI/RI组血清肌酸激酶(CK)、乳酸脱氢酶(LDH)、丙二醛(MDA)、肿瘤坏死因子-α(TNF-α)、肌酸激酶MB同工酶(CK-MB)、谷草转氨酶(AST)、谷丙转氨酶(ALT)指标整体高于Sham组(F=8.554,P<0.01;F=9.167,P<0.01;F=3.639,P<0.05;F=10.750,P<0.01;F=100.336,P<0.01;F=118.641,P<0.01;F=5.219,P<0.05),血清超氧化物歧化酶(SOD)指标整体低于Sham组(F=2.471,P<0.05)。LI/RI组肌肉MDA、TNF-α,心肌MDA指标和肝脏MDA、TNF-α、IL-6指标高于Sham组[(1.93±0.60)nmol/mg prot比(0.95±0.24)nmol/mg prot,t=-4.666,P<0.01;(75.61±44.17)pg/ml比(20.65±8.91)pg/ml,t=-2.648,P<0.05,(2.56±1.03)nmol/mg prot比(1.20±0.40)nmol/mg prot,t=-3.192,P<0.05;(2.88±0.76)nmol/mg prot比(0.80±0.28)nmol/mg prot,t=-6.218,P<0.05;(1080.79±256.78)pg/ml比(441.08±135.50)pg/ml,t=-4.995,P<0.01;(209.39±94.61)pg/ml比(37.18±32.79)pg/ml,t=-3.479,P<0.05],LI/RI组肌肉、心肌SOD指标低于Sham组[(87.64±15.13)U/mg prot比(114.56±10.41)U/mg prot,t=3.006,P<0.05;(752.26±118.97)U/mg prot比(939.73±90.79)U/mg prot,t=2.777,P<0.05]。LI/RI组的光镜下骨骼肌、心脏和肝脏出现明显损伤。结论6 h夹闭血管联合止血带可成功建立LI/RI模型,引起肌肉损伤、全身氧化应激、炎性反应及心脏、肝脏损伤。

ObjectiveTo establish a simple and effective limb ischemia-reperfusion injury(LI/RI)model in Bama miniature pigs.MethodsTotally,12 Bama miniature pigs were randomly divided into a control group(Sham,n=6)and a LI/RI group(n=6),with 6 pigs in each group.The Sham group underwent exposure of blood vessels only,nd the LI/RI group had blood vessels clamped and a tourniquet applied to simulate injury.Oxidative stress,inflammatory response,and tissue injury indicators were compared between the two groups using two-way repeated measures ANOVA and t-tests.ResultsAfter 7 days,the model was successfully established.The LI/RI group showed significantly higher levels of serum creatine kinase(CK),lactate dehydrogenase(LDH),malondialdehyde(MDA),tumor necrosis factor-alpha(TNF-α),creatine kinase-MB(CK-MB),aspartate aminotransferase(AST),and alanine aminotransferase(ALT)than the Sham group(F=8.554,9.167,3.639,10.750,100.336,118.641,5.219,P<0.05),and lower levels of superoxide dismutase(SOD)(F=2.471,P<0.05).The LI/RI group had higher levels of muscle MDA,TNF-α,myocardial MDA,and hepatic MDA,TNF-α,IL-6 than the Sham group[(1.93±0.60)nmol/mg prot vs.(0.95±0.24)nmol/mg prot,t=-4.666,P<0.01;(75.61±44.17)pg/ml vs.(20.65±8.91)pg/ml,t=-2.648,P<0.05;(2.56±1.03)nmol/mg prot vs.(1.2±0.40)nmol/mg prot,t=-3.192,P<0.05;(2.88±0.76)nmol/mg prot vs.(0.80±0.28)nmol/mg prot,t=-6.218,P<0.05;(1080.79±256.78)pg/ml vs.(441.08±135.50)pg/ml,t=-4.995,P<0.01;(209.39±94.61)pg/ml vs.(37.18±32.79)pg/ml,t=-3.479,P<0.05],and lower levels of muscle and myocardial SOD than the Sham group[(87.64±15.13)U/mg prot vs.(114.56±10.41)U/mg prot,t=3.006,P<0.05;(752.26±118.97)U/mg prot vs.(939.73±90.79)U/mg prot,t=2.777,P<0.05].Histological examination revealed more severe damage to the skeletal muscle,heart,and liver in the LI/RI group.ConclusionThe 6-h vascular clamping combined with a tourniquet can successfully establish the LI/RI model,causing muscle injury,systemic oxidative stress,inflammatory response,and damage to the heart and liver.