Histochemical analyses of living mouse liver under different hemodynamic conditions by "in vivo cryotechnique"
Histochemical analyses of living mouse liver under different hemodynamic conditions by "in vivo cryotechnique"
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DOI：
10.1007/s00418-006-0173-6
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发表时间：
2006-09-01
影响因子：
2.3
通讯作者：
Ohno, Shinichi
中科院分区：
文献类型：
--
作者：
Ohno, Nobuhiko;Terada, Nobuo;Ohno, Shinichi
虽然动物肝组织的形态和分子分布已经使用传统的制备方法进行了检查，但结果总是受到灌注固定和组织切除引起的技术伪影的影响。使用活体冷冻技术（IVCT），我们对活体小鼠肝脏进行了组织化学、免疫组织化学和超微结构分析。在IVCT制备的样品中，在血液循环正常的情况下，观察到广泛开放的窦状体和许多流动的红细胞，在缺血或心脏骤停的小鼠中，观察到窦状体塌陷或充血。采用灌注固定的方法人工扩张窦腔，采用浸泡固定和速冻（QF）的方法对切除组织进行塌陷。QF法和IVCT法均能较好地保存肝细胞血清白蛋白和免疫球蛋白G的免疫反应性及周期性酸-希夫染色强度。此外，在组织切除后，血清蛋白迅速转移到肝细胞中，这是在切除后1或5分钟冷冻的QF组织的免疫反应所证明的。在IVCT样本中未观察到易位现象，表明IVCT可用于检测不同病理条件下肝细胞细胞膜通透性。活体小鼠肝脏中可溶性组分的动态形态和免疫分布能够以较高的时间分辨率精确地反映其生理和病理状态。
Although the morphology and molecular distribution in animal liver tissues have been examined using conventional preparation methods, the findings are always affected by the technical artifacts caused by perfusion-fixation and tissue-resection. Using "in vivo cryotechnique" (IVCT), we have examined living mouse livers with histochemical, immunohistochemical and ultrastructural analyses. In samples prepared by IVCT, widely open sinusoids with many flowing erythrocytes were observed under normal blood circulation, and their collapse or blood congestion was seen in ischemic or heart-arrested mice. In contrast, the sinusoidal cavities were artificially dilated by perfusion-fixation, and collapsed by immersion-fixation and quick-freezing (QF) methods of resected tissues. The immunoreactivity of serum albumin and immunoglobulin G and intensity of periodic acid-Schiff-staining in hepatocytes were well preserved with the QF method and IVCT. Furthermore, following tissue resection, serum proteins were rapidly translocated into hepatocytes as demonstrated by immunoreactions on QF tissues frozen 1 or 5 min after resection. Translocation was not observed in IVCT samples, indicating that IVCT could be useful to examine cell membrane permeability of hepatocytes under different pathological conditions. Both dynamic morphology and immunodistribution of soluble components in living mouse livers, reflecting their physiological and pathological states, can be precisely examined by IVCT with higher time-resolution.