Shi Liye, Li Zilong, Zhai Xiaoyue, Ning Bin, Yang Bei, Qi Guoxian
Department of Cardiovascular Medicine, The First Hospital of China Medical University, Shenyang, China.
Appl Immunohistochem Mol Morphol. 2012 May;20(3):304-17. doi: 10.1097/PAI.0b013e318225a2a0.
"In vivo cryotechnique" (IVCT), which involves immediately cryofixing cells and tissues of living animals in situ, can display more native morphology in vivo and eliminate artificial changes in conventional preparations. However, the technical characteristics of IVCT are not known for the practical examination of subepicardial microcirculation of beating heart tissue. Histological sections of subepicardial area were prepared using IVCT and conventional fixation methods: quick freezing, immersion fixation, or perfusion fixation followed by alcohol dehydration, respectively from healthy mice. In addition, changes of erythrocyte shape, T-tubule, and microvasculature in mouse heart from a variety of models (acute increase of left ventricular afterload, myocardial ischemia, and cardiac arrest) were examined by IVCT. With IVCT, flowing erythrocytes, blood flow, microvasculature, and myocyte structure could be well preserved without artificial change of erythrocyte shape and translocation of serum proteins as displayed in conventional preparation samples. Furthermore, in various pathological models prepared by IVCT, T-tubules with albumin immuno-positive staining were arranged in a disorderly way and were decreased in volume in samples of acute increase of left ventricular afterload (IVCT-LAA). This was more evident in acute regional myocardial ischemia (IVCT-IC) and less evident in heart arrest (IVCT-HA). In addition, the leakage of serum proteins from microvasculature into myocyte was found only in IVCT-IC but not in IVCT-LAA and in IVCT-HA. In conclusion, IVCT is a new technique for examining morphology of subepicardial microcirculation without artifacts compared with conventional methods and is a more sensitive fixation technique in detecting pathological changes of the heart.
“体内冷冻技术”（IVCT），即将活体动物的细胞和组织在原位立即冷冻固定，能够在体内展现更多的天然形态，并消除传统标本制作中的人为变化。然而，对于跳动心脏组织的心外膜下微循环的实际检查，IVCT的技术特性尚不清楚。分别采用IVCT和传统固定方法（快速冷冻、浸泡固定或灌注固定后酒精脱水），从健康小鼠制备心外膜下区域的组织切片。此外，通过IVCT检查了多种模型（左心室后负荷急性增加、心肌缺血和心脏骤停）小鼠心脏中红细胞形态、T小管和微血管的变化。使用IVCT时，流动的红细胞、血流、微血管和心肌细胞结构能够得到良好保存，不会像传统标本那样出现红细胞形态的人为改变和血清蛋白的移位。此外，在IVCT制备的各种病理模型中，左心室后负荷急性增加（IVCT-LAA）样本中白蛋白免疫阳性染色的T小管排列紊乱且体积减小。这在急性区域性心肌缺血（IVCT-IC）中更为明显，在心脏骤停（IVCT-HA）中则不太明显。此外，仅在IVCT-IC中发现微血管中的血清蛋白漏入心肌细胞，而在IVCT-LAA和IVCT-HA中未发现。总之，与传统方法相比，IVCT是一种用于检查心外膜下微循环形态且无伪像的新技术，并且是检测心脏病理变化更敏感的固定技术。
