Tempol对大鼠任意皮瓣成活影响的实验研究

EFFECTS OF Tempol ON SURVIVAL OF RANDOM PATTERN SKIN FLAPS IN RATS

目的探讨新型小分子氧自由基清除剂Tempol对大鼠背部超长任意皮瓣(long random pattern skin flap,LRPSF)成活及血管生成的影响及机制。方法取成年雄性SD大鼠84只,体质量(300±20)g,随机分为Tempol组及对照组,每组42只。两组参照Mcfarlane皮瓣模型方法,于大鼠背部制备面积为9 cm×3 cm的LRPSF。Tempol组术前15 min及术后7 d内每天腹腔注射Tempol(100 mg/kg),对照组同时间点注射相同体积生理盐水。术后观察各组大鼠成活以及皮瓣成活情况;第7天,测量皮瓣成活率,Micro-CT血管成像观察血管形成情况并测量血管容积和血管总长度,HE染色观察皮瓣组织层次结构及炎性反应程度,免疫组织化学染色观察VEGF表达;术后即刻及第1、3、7天,采用水溶性四氮唑-1法及可见光法测定皮瓣组织中超氧化物歧化酶(superoxide dismutase,SOD)和丙二醛(malondialdehyde,MDA)含量,ELISA法测定炎性因子TNF-α和IL-6含量。结果术后各组大鼠均存活至实验完成,无积血、积液和感染等发生。随时间延长,两组皮瓣远端均发生坏死,但Tempol组坏死程度较对照组轻;且Tempol组血管分布及连续性均优于对照组。术后第7天Tempol组皮瓣成活率、血管容积及血管总长度均显著高于对照组(P<0.05)。术后第7天组织学及免疫组织化学染色观察示,与对照组比较,Tempol组皮肤各层组织结构层次尚清楚,组织炎性反应及炎性细胞浸润较轻,VEGF蛋白染色程度增高。术后即刻两组SOD、MDA以及TNF-α、IL-6含量比较,差异均无统计学意义(P<0.05);术后第1、3、7天,Tempol组SOD含量高于对照组,MDA及TNF-α、IL-6含量明显低于对照组(P<0.05)。结论 Tempol显著提高大鼠背部LRPSF成活率,其机制可能与促进皮瓣组织血管生成、降低氧化应激和减轻炎性反应有关。

Objective To study the effects of the new small molecular oxygen free radical scavenger Tempol on the survival and vasculogenesis of the long random pattern skin flap （LRPSF） and its mechanism. Methods Eighty- four male Sprague Dawley rats were randomly divided into control and Tempol groups （42 rats in each group）. LRPSF of 9 cm×3 cm in size were prepared on the backs of rats in two groups based on the Mcfarlane flap. Rats were administered with Tempol （100 mg/kg） in the Tempo1 group and with normal saline in the control group by intraperitoneal injection at 15 minutes before operation and at 1-7 day after operation. The rat and the skin flap survival conditions were observed after operation; the survival rate of skin flap was measured, and the vascular structure, vascular volume, and total length of blood vessels were analyzed with Micro-CT three-dimensional imaging after 7 days; HE staining was used to observe the structure of the skin flaps and inflammation, immumohistochemical staining to observe vascular endothelial growth factor （VEGF） expression; water-soluble tetrazolium-I method was used to measure the content of superoxide dismutase （SOD） and malondialdehyde （MDA）, and ELISA to detect the expressions of tumor necrosis factor ct （TNF-a） and interleukin 6 （IL-6） after 1, 3, and 7 days. Results All of rats survived after operation, without hemorrhage, edema, and infection. With the extension of time, necrosis occurred in the distal part of the skin flaps in 2 groups, but the necrosis degree of the Tempol group was lower than that of control group; meanwhile, the blood vessel distribution and continuity were better than those of control group. The skin flaps survival rate, vascular volume, and total length of blood vessels of Tempol group were significantly higher than those of control group after 7 days （P〈0.05）. The dearer skin flaps structure, lighter inflammation reaction and inflammation cell infiltration, and higher VEGF staining intensity were observed in the Tempol group than the control group after 7 days. There was no significant difference in SOD, MDA, and TNF-a, and IL-6 contents between the 2 groups at immediate after operation. SOD significantly increased, but MDA, TNF-a, and IL-6 contents significantly decreased in the Tempol group when compared with control group after 1, 3, and 7 days （P〈0.05）. Conclusion Tempol can significantly promote the LRPSF survival rates, its mechanism is closely related to the promotion of vasculogenesis and reduction of oxidative stress and inflammation.