Application of "in vivo cryotechnique" to morphological and immunohistochemical analyses of living mouse subepicardial microcirculation under various pathological conditions.

"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.