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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 investigate the application of in vivo cryogenic technique in the evaluation of myocardial ischemia-reperfusion injury in mice. Methods: 36 mice were randomly divided into sham operation group (C group), ischemia group (I group), reperfusion 0 min group (I / R 0 min group), reperfusion 15 min group min group), reperfusion 1 h group (I / R 1 h group), atorvastatin 10 mg · kg-1 2 h before reperfusion (A102 group) and atorvastatin 2 h before reperfusion (A202 group) and 20 mg · kg-1 atorvastatin group (A2012 group) 12 h before reperfusion, atorvastatin 20 mg · kg -1 gavage + 15 min before reperfusion, L-NAME (N-nitro-L-arginine methyl ester) 15 mg · kg -1 tail vein injection group (AL202 group) Mouse. Myocardial ischemia / reperfusion model was established by ligation of the coronary arteries under light microscope. All hearts were harvested by in vivo cryotechnique (IVCT), followed by cryopreservation and paraffin embedding. The sections were stained with HE staining and Albumin immunohistochemical staining , And semi-quantitative analysis of the intensity of Albumin immunohistochemical staining in the stroma. Results: (1) Compared with C group, in group I, the number of erythrocytes in myocardial microvessels obviously decreased or absent, and the myocardial cells swollen, and albumin obviously exuded to the interspace. I / R 0 min group blood flow recovery was acceptable, Albumin no significant leakage to the interstitial and other parts; I / R 15 min group and I / R 1 h group with reperfusion time, myocardial cells and microvascular Erythrocyte morphology gradually swelling, chaos, broken, Albumin significantly increased leakage into the gap. (2) Compared with I / R 1 h group, the myocardial cells in A202 group were clearer, the morphology of erythrocytes in capillaries was normal, and albumin did not leak to the interstitium and other sites. A102 group and A2012 group were similar to those in A202 group, but the morphology of erythrocytes and the boundary of myocardial cells in microvessels and Albumin leakage to stroma were not as good as A202 group. AL202 group showed myocardial cell swelling, microvascular erythrocyte reduction, absent, but microcirculation and albumin leakage is still better than the I / R group. Conclusion: The changes of cardiac microcirculation and microvascular permeability during ischemia and reperfusion in mice can be directly evaluated by IVCT. Atorvastatin 2 h before reperfusion could improve the microcirculation state and microvascular permeability after ischemia-reperfusion.