Röhe Ilen, Hüttner Friedrich Joseph, Plendl Johanna, Drewes Barbara, Zentek Jürgen
Freie Universität Berlin, Department of Veterinary Medicine.
Eur J Histochem. 2018 Feb 5;62(1):2874. doi: 10.4081/ejh.2018.2874.
The histological characterization of the intestinal mucus layer is important for many scientific experiments investigating the interaction between intestinal microbiota, mucosal immune response and intestinal mucus production. The aim of this study was to examine and compare different fixation protocols for displaying and quantifying the intestinal mucus layer in piglets and to test which histomorphological parameters may correlate with the determined mucus layer thickness. Jejunal and colonal tissue samples of weaned piglets (n=10) were either frozen in liquid nitrogen or chemically fixed using methacarn solution. The frozen tissue samples were cryosectioned and subsequently postfixed using three different postfixatives: paraformaldehyde vapor, neutrally buffered formalin solution and ethanol solution. After dehydration, methacarn fixed tissues were embedded in paraffin wax. Both sections of cryopreserved and methacarn fixed tissue samples were stained with Alcian blue (AB)-PAS followed by the microscopically determination of the mucus layer thickness. Different pH values of the Alcian Blue staining solution and two mucus layer thickness measuring methods were compared. In addition, various histomorphological parameters of methacarn fixed tissue samples were evaluated including the number of goblet cells and the mucin staining area. Cryopreservation in combination with chemical postfixation led to mucus preservation in the colon of piglets allowing mucus thickness measurements. Mucus could be only partly preserved in cryosections of the jejunum impeding any quantitative description of the mucus layer thickness. The application of different postfixations, varying pH values of the AB solution and different mucus layer measuring methods led to comparable results regarding the mucus layer thickness. Methacarn fixation proved to be unsuitable for mucus depiction as only mucus patches were found in the jejunum or a detachment of the mucus layer from the epithelium was observed in the colon. Correlation analyses revealed that the proportion of the mucin staining area per crypt area (relative mucin staining) measured in methacarn fixed tissue samples corresponded to the colonal mucus layer thickness determined in cryopreserved tissue samples. In conclusion, the results showed that cryopreservation using liquid nitrogen followed by chemical postfixation and AB-PAS staining led to a reliable mucus preservation allowing a mucus thickness determination in the colon of pigs. Moreover, the detected relative mucin staining area may serve as a suitable histomorphological parameter for the assessment of the intestinal mucus layer thickness. The findings obtained in this study can be used for the implementation of an improved standard for the histological description of the mucus layer in the colon of pigs.
肠道黏液层的组织学特征对于许多研究肠道微生物群、黏膜免疫反应和肠道黏液产生之间相互作用的科学实验至关重要。本研究的目的是检查和比较不同的固定方案，以展示和量化仔猪的肠道黏液层，并测试哪些组织形态学参数可能与所测定的黏液层厚度相关。对断奶仔猪（n = 10）的空肠和结肠组织样本进行液氮冷冻或用甲醇-氯仿溶液进行化学固定。将冷冻的组织样本进行冷冻切片，随后使用三种不同的后固定剂进行后固定：多聚甲醛蒸汽、中性缓冲福尔马林溶液和乙醇溶液。脱水后，用甲醇-氯仿固定的组织样本被包埋在石蜡中。冷冻保存和甲醇-氯仿固定的组织样本切片均用阿尔辛蓝（AB）-过碘酸希夫（PAS）染色，随后在显微镜下测定黏液层厚度。比较了阿尔辛蓝染色溶液的不同pH值和两种黏液层厚度测量方法。此外，还评估了甲醇-氯仿固定的组织样本的各种组织形态学参数，包括杯状细胞数量和黏蛋白染色面积。液氮冷冻保存结合化学后固定可使仔猪结肠中的黏液得以保存，从而能够测量黏液厚度。空肠冷冻切片中的黏液只能部分保存，这妨碍了对黏液层厚度进行任何定量描述。应用不同的后固定方法、AB溶液的不同pH值和不同的黏液层测量方法，在黏液层厚度方面得到了可比的结果。事实证明，甲醇-氯仿固定不适用于黏液描绘，因为在空肠中仅发现黏液斑，或在结肠中观察到黏液层从上皮脱落。相关性分析表明，在甲醇-氯仿固定的组织样本中测得的每个隐窝区域的黏蛋白染色面积比例（相对黏蛋白染色）与冷冻保存的组织样本中测定的结肠黏液层厚度相对应。总之，结果表明，液氮冷冻保存后进行化学后固定和AB-PAS染色可使黏液可靠保存，从而能够测定猪结肠中的黏液厚度。此外，检测到的相对黏蛋白染色面积可作为评估肠道黏液层厚度的合适组织形态学参数。本研究中获得的结果可用于实施改进的猪结肠黏液层组织学描述标准。
