应用活体冷冻技术研究活体小鼠心脏微循环

Visualization of Mouse Cardiac Microvascular Blood Flow and Distribution of Serum Protein with in Vivo Cryotechnique

目的:分别用传统组织制备方法与活体冷冻技术(in vivo cryotechnique,IVCT)联合冷冻置换制备方法制备样本,对比研究小鼠心外膜下微循环内血流、红细胞及血清蛋白在心脏的免疫定位分布。方法:实验分三组,活体冷冻组(IVCT)、浸泡固定组(immersion dehydration fixation,IM-DH)及灌流固定组(perfusion dehydration fixation,PF-DH),每组8只小鼠,分别对跳动的小鼠心脏进行活体冷冻技术联合冷冻置换,浸泡固定及灌流固定,制作的组织样本分别进行苏木素-伊红染色、免疫组织化学染色以及双重免疫荧光染色,光镜及共聚焦荧光显微镜观察,对结果进行对比分析。结果:IVCT从组织形态学水平显示微血管内流动状态的血流,即单行排列的顶端指向血流方向的成串的降落伞型红细胞,以及微血管分流和汇合处;血清白蛋白和免疫球蛋白不但通过微血管壁进入细胞间隙,白蛋白还单独分布在心肌细胞间的特殊结构闰盘和心肌细胞表面的T小管。结论:IVCT实现了从免疫组织化学水平显示活体状态小鼠心脏的微循环;心肌细胞间的闰盘及心肌细胞表面的T小管对血清蛋白具有分子大小选择性。

Comparing study Of epicardial microcirculation blood flow, red blood cells and of serum proteins in mouse heart by traditional organization preparation methods and in vivo cryotechnique （IVCT） combined with freeze substitution preparation samples respectively. Methods： Mice were divided into three groups, in vivo cryotechnique （IVCT） combining flee-substitution, immersion fixation group （immersion fixation followed by dehydration, IM-DH） and perfusion fixation group （perfusion fixation followed by dehydration, PF-DH）, each group of 8 mice, respectively. Beating mice heart were suffered by in vivo cryotechnique combined with freeze substitution, immersion fixation and perfusion fixation, respectively. All the tissue samples were hematoxylin-eosin staining, immunohistochemical staining and double immunofluorescence staining, and then detected by light microscopy and confocal fluorescence microscopy. The results were compared and analysed. Results： IVCT displayed microvascular blood flow in morphological level- one-way arrangement erythrocytes shaped parachute and the top points to the direction of blood flow, and microvascular shunt and confluence; serum albumin and immunoglobulin （IgG） not only immunodistributed in microvasculature and interstitium, albumin also immunolocolized in the special structure of myocardial cells-intercalated disc and T tubule. Conelusions： IVCT realized the display ofmurine cardiac microcirculation in vivo from the immunohistochemical level; intercalated disc and T tubules has molecular size selectivity on serum protein.