Comparison of different histological protocols for the preservation and quantification of the intestinal mucus layer in pigs

Abstract

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.

Keywords: Mucus layer; fixation; goblet cell; histochemistry.; intestine; mucin; pig.

Figure 1.

A) Patchy looking mucus in the jejunum; prefixation: cryopreservation; postfixation: PFA; staining: AB pH2.5-PAS. B) Single mucus patches in the jejunum; fixation: methacarn; staining: AB pH2.5-PAS. C,D) Laminar appearance of the mucus in the colon; prefixation: cryopreservation; postfixation: PFA; staining: AB pH2.5-PAS. E) Detachment of the mucus layer in the colon; fixation: methacarn; staining: AB pH2.5-PAS. F) Slight displacement of the mucus over the epithelial borders and in the digesta; prefixation: cryopreservation; postfixation: NBF; staining: AB pH2.5-PAS.

Figure 2.

Comparison of different postfixations, staining solutions and measuring methods for the depiction of the mucus layer thickness in cryopreserved colonal samples(n=10). A) PFA and NBF postfixation. B) PFA and EtOH postfixation. C) AB staining with pH values 2.5 and 0.5. D) “Integral” and the “10 point”measuring method.

Figure 3.

Correlation analyses of different histomorphological parameters, determined in histological slides of methacarn fixed tissue, and the mucus layer thickness, measured in histological sections of cryopreserved tissue, in the colon of piglets (n=10). A) Relationship between the absolute number of goblet cells (total number of goblet cells per crypt) and the mucus layer thickness (Pearson coefficient: 0.554; P=0.096). B) Relationship between the relative number of goblet cells (goblet cells per 1mm basement membrane of crypts) and the mucus layer thickness (Pearson coefficient: 0.201; P=0.577). C) Relationship between the absolute mucin staining area (total mucin staining area per crypt) and the mucus layer thickness (Pearson coefficient: 0.529; P=0.116). D) Relationship between the relative mucin staining area (mucin staining area in % of total crypt area) and the mucus layer thickness (Pearson coefficient: 0.658; P=0.039).