活体冷冻技术在评估小鼠心脏缺血再灌注损伤中的应用

Application of in vivio cryotechnique in the assessment of myocardial ischemia reperfusion injury in mice

目的:探讨活体冷冻技术在评估小鼠心脏缺血再灌注损伤中的应用。方法:将36只小鼠随机分为假手术组(C组)、缺血组(I组)、再灌注0 min组(I/R 0 min组)、再灌注15 min组(I/R 15 min组)、再灌注1 h组(I/R 1 h组)、再灌注前2 h阿托伐他汀10 mg·kg-1灌胃组(A102组)、再灌注前2 h阿托伐他汀20 mg·kg-1灌胃组(A202组)、再灌注前12 h阿托伐他汀20 mg·kg-1灌胃组(A2012组)、再灌注前2 h阿托伐他汀20 mg·kg-1灌胃+再灌注前15 min L-NAME(N-硝基-L-精氨酸甲酯)15 mg·kg-1尾静脉注射组(AL202组)等9组,每组均4只小鼠。于光镜下结扎冠状动脉制备心肌缺血再灌注模型,均以活体冷冻技术(in vivo cryotechnique,IVCT)摘取心脏后行冷冻置换、石蜡包埋,之后切片进行HE染色及Albumin免疫组织化学染色,并对间质中的Albumin免疫组织化学染色强度进行半定量分析。结果:(1)相比C组,I组可见心肌微血管内红细胞明显减少或缺如,心肌细胞肿胀,Albumin向间隙内明显渗出。I/R 0 min组血流恢复情况尚可,Albumin无明显向间质等部位渗漏;I/R 15 min组及I/R 1 h组可见随再灌注时间的延长,心肌细胞及微血管内红细胞形态渐趋肿胀、紊乱、破碎,Albumin向间隙内的渗漏明显增多。(2)与I/R 1 h组相比,A202组心肌细胞界限较清晰,微血管内红细胞形态正常,Albumin无明显向间质等部位渗漏。A102组及A2012组镜下所见与A202组类似,但微血管内红细胞形态及心肌细胞界限、Albumin向间质的渗漏程度等不及A202组。AL202组可见心肌细胞肿胀,微血管内红细胞减少、缺如,但微循环血流及Albumin渗漏情况仍优于I/R组。结论:应用IVCT可直观评估小鼠缺血及再灌注过程中心脏微循环状态及微血管渗透性变化。再灌注前2 h应用阿托他汀强化治疗可改善缺血再灌注心脏微循环状态及微血管渗透性。

Objective： To analyze the state of microcirculation and microvascular permeability during myocardial ischemia reperfusion by using in vivio cryotechnique （IVCT）. Methods： Thirty- six mice were divided into 9 groups randomly ： sham- operated （ C ） group, ischemia （ I ） group, ischemia- reperfusion 0 min （ I/R 0 min ） group,ischemia-reperfusion 15 min （I/R 15 min）group, ischemia-reperfusion 1 h （I/R 1 h）group, atorvastatin 10 mg · kg- 1 gavage 2 h before reperfusion （A102）group, atorvastatin 20 mg · kg-1 gavage 2 h before reperfusion （A202） group, atorvastatin 20 mg· kg-1 gavage 12 h before reperfusion （A2012）group, atorvastatin 20 mg· kg-1 gavage 2 h before reperfusion ＋L-NAME 15 mg · kg-1 tail vein injection 15 min before reperfusion （AL202） group. The myocardial ischemia reperfusion model was prepared under light microscopy. IVCT followed by freeze-substitution fixation was used to prepare the heart sample, and then stained with hematoxylin-eosine （HE） and albumin antibody for light microscopy observation. Results ： （ 1 ） Compared with C group, I and I/R 1 h group showed that the amount of erythrocyte was decreased or absent in microvasculatures, and albumin was obviously immunolocalized in interstitium. （2） Compared with I/R 1 h group, A202 group showed a more clear structure of microenvironment, a better blood flow in microvascular and less leakage of albumin in the blood vessel. A102 group and A2012 group showed a similar effect, but less than A202 group. AL202 group showed a slightly narrow myocardial microvascular and myocardial cell swelling, but the blood flow and leakage of plasma albumin were less than those in I/R group. Conclusion： IVCT could morphofunctionally and immunohistochemically display the circulation and permeability of microvascular in ischemia-reperfusion mouse heart. Pretreatment with atorvastatin 2 h before reperfusion could significantly improve the blood flow state and the plasma albumin leakage of microvascular.