Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo-city, Yamanashi, Japan.
Histochem Cell Biol. 2010 Feb;133(2):229-39. doi: 10.1007/s00418-009-0663-4. Epub 2009 Dec 1.
Soluble proteins and glycogen particles, which are easily lost upon conventional chemical fixation, have been reported to be better preserved in paraffin-embedded sections by 'cryobiopsy' combined with freeze-substitution fixation (FS). In this study, we examined the distribution of glycogen in living mouse livers under physiologic and pathologic conditions with periodic acid-Schiff (PAS) staining by cryobiopsy. The livers of the fully fed mice showed high PAS-staining intensity in the cytoplasm of all hepatocytes. The PAS-staining intensity gradually decreased away from hepatocytes around portal tracts, depending on treatments with different alpha-amylase concentrations. At 6 or 12 h after fasting, PAS-staining intensity markedly decreased in restricted areas of zone I near the portal tracts. The cryobiopsy was repeatedly performed not only on different mice, but also on individuals. Next, glycogen distributions were evaluated by temporarily clipping of liver tissues of anesthetized mice, followed by recovery of blood circulation. In the liver tissues in which blood was recirculated for 1 h after the 30 min anoxia, PAS staining was still observed in zone II and also in restricted areas of zone I far from the portal tracts. In PAS-unstained hepatocytes, the immunoglobulin-kappa light chain was not detected in the cytoplasm, indicating that cell membrane permeability was retained and that glycogen metabolism was related to the functional state of blood circulation. We propose that the level of consumption or production of glycogen particles could vary in zone I, depending on the distance from the portal tracts. Thus, cryobiopsy combined with FS enabled us to examine time-dependent changes in glycogen distribution in the liver tissues of living mice. This combination might be applicable to the clinical evaluation of human liver tissues.
可溶性蛋白质和糖原颗粒在常规化学固定时容易丢失，据报道，通过“冷冻活检”结合冷冻置换固定（FS）可以更好地保存在石蜡包埋切片中。在这项研究中，我们通过冷冻活检用过碘酸希夫（PAS）染色检查了生理和病理条件下活鼠肝脏中糖原的分布。完全喂食的小鼠肝脏中所有肝细胞的细胞质中 PAS 染色强度较高。PAS 染色强度逐渐远离门脉周围的肝细胞，取决于不同α-淀粉酶浓度的处理。禁食 6 或 12 小时后，靠近门脉区的 I 区受限区域的 PAS 染色强度显著降低。冷冻活检不仅在不同的小鼠上进行，而且在个体上也进行了重复。接下来，通过暂时夹闭麻醉小鼠的肝组织，然后恢复血液循环，评估糖原的分布。在血液循环恢复 1 小时后，肝脏组织在 30 分钟缺氧后的 II 区和远离门脉区的 I 区受限区域仍然观察到 PAS 染色。在 PAS 未染色的肝细胞中，细胞质中未检测到免疫球蛋白 κ 轻链，表明细胞膜通透性得以保留，糖原代谢与血液循环的功能状态有关。我们提出，I 区糖原颗粒的消耗或产生水平可能因距离门脉区的远近而有所不同。因此，冷冻活检结合 FS 使我们能够检查活鼠肝脏组织中糖原分布的时间依赖性变化。这种组合可能适用于人类肝组织的临床评估。
