Terada N, Kato Y, Fuji Y, Ueda H, Baba T, Ohno S
Department of Anatomy, Yamanashi Medical University, Japan.
J Electron Microsc (Tokyo). 1998;47(1):67-72. doi: 10.1093/oxfordjournals.jmicro.a023560.
The purpose of this study is to develop a method for stabilizing erythrocytes under flowing condition in living livers, as revealed by scanning electron microscopy (SEM). After the procedure of the 'in vivo cryotechnique', both freeze-substitution and subsequent t-butyl alcohol freeze-drying methods were used for preparing SEM specimens. By freeze-fracturing with a scalpel in liquid nitrogen before the freeze-substitution, better preserved surface tissues were obtained for examination. Erythrocytes in hepatic sinusoids were clearly detected without plasma components by the freeze-substitution method, and well preserved in parts where they were flowing with their original shapes. Some were accumulated in sinusoids, especially injunctioning areas of sinusoidal networks, as compared with those in narrow lumens between hepatocyte plates. Shapes of such erythrocytes were various, locating along endothelial cells. After stopping the blood supply into livers by artificial cardiac arrest, their shapes were dramatically changed into biconcaves and they became aggregated side by side to be packed in the sinusoids. The three-dimensional shapes of flowing erythrocytes in hepatic sinusoids were demonstrated for the first time by the 'in vivo cryotechnique' combined with SEM.
本研究的目的是开发一种在活体肝脏流动条件下稳定红细胞的方法，这通过扫描电子显微镜（SEM）得以揭示。在采用“体内冷冻技术”的操作流程后，冷冻置换法和随后的叔丁醇冷冻干燥法都被用于制备SEM标本。通过在冷冻置换前于液氮中用手术刀进行冷冻断裂，可获得保存更好的表面组织用于检查。通过冷冻置换法能清晰检测到肝血窦中的红细胞，且无血浆成分，并且在它们以原始形状流动的部位保存良好。与肝细胞板之间狭窄管腔中的红细胞相比，一些红细胞聚集在血窦中，尤其是在血窦网络的交汇处。这些红细胞形状各异，沿着内皮细胞分布。在通过人工心脏骤停停止肝脏血液供应后，它们的形状急剧变为双凹形，并并排聚集在一起填充在血窦中。首次通过结合SEM的“体内冷冻技术”展示了肝血窦中流动红细胞的三维形状。
