Intestinal barrier analysis by assessment of mucins, tight junctions, and α-defensins in healthy C57BL/6J and BALB/cJ mice

Abstract

The intestinal barrier is gaining increasing attention because it is related to intestinal homeostasis and disease. Different parameters have been used in the past to assess intestinal barrier functions in experimental studies; however most of them are poorly defined in healthy mice. Here, we compared a number of barrier markers in healthy mice, established normal values and correlations. In 48 mice (24 C57BL/6J, 24 BALB/cJ background), we measured mucus thickness, and expression of mucin-2, α-defensin-1 and -4, zonula occludens-1, occludin, junctional adhesion molecule-A, claudin-1, 2 and -5. We also analyzed claudin-3 and fatty acid binding protein-2 in urine and plasma, respectively. A higher expression of mucin-2 protein was found in the colon compared to the ileum. In contrast, the α-defensins-1 and -4 were expressed almost exclusively in the ileum. The protein expression of the tight junction molecules claudin-1, occludin and zonula occludens-1 did not differ between colon and ileum, although some differences occurred at the mRNA level. No age- or gender-related differences were found. Differences between C57BL/6J and BALB/cJ mice were found for α-defensin-1 and -4 mRNA expression, and for urine and plasma marker concentrations. The α-defensin-1 mRNA correlated with claudin-5 mRNA, whereas α-defensin-4 mRNA correlated with claudin-3 concentrations in urine. In conclusion, we identified a number of murine intestinal barrier markers requiring tissue analyses or measurable in urine or plasma. We provide normal values for these markers in mice of different genetic background. Such data might be helpful for future animal studies in which the intestinal barrier is of interest.

Mucosubstances in the porcine gastrointestinal tract: Fixation, staining and quantification

“…Cohen and colleagues reported their protocol to be suitable for the demonstration of the natural appearance of mucus in frozen sections. 7 Comparing their to our results as well as to images from literature, 25 reporting to use the protocol of Cohen et al ., the mucins seemed to leak out of the goblet cells, forming fuzzy or foamy staining halos around them. One possibility could be that the samples were not appropriately fixed for the target.…”

Fecal Microbiota Transplantation Prevents Intestinal Injury, Upregulation of Toll-Like Receptors, and 5-Fluorouracil/Oxaliplatin-Induced Toxicity in Colorectal Cancer

“…ZOs are the most important components for the construction of a constitutive barrier of epithelial cells, and they regulate the permeability of the barrier by tightly sealing the cell-cell junctions [34]. Several potential markers of the intestinal barrier, the ileum, or the colon can be assessed in mice, including mucus thickness, mRNA expression, and levels of Muc-2 and junction molecules (JAM-A, ZO-1, occludin, claudin-1, -2, and -5) [35]. Chemotherapeutic drugs could increase intestinal epithelial barrier permeability by reducing tight junction expression in vivo.…”

Polymorphic Immune Mechanisms Regulate Commensal Repertoire

“…To identify innate polymorphic mechanisms operating in the gut, we profiled gene expression (using RNA-seq) in three intestinal compartments from B6 and BALB/c mice populated with the same input microbiota (Figure 4; Tables S2 and S3). Among the polymorphic innate factors, we found a family of antimicrobial peptides, defensins alpha (Defa), which differ in expression between mouse strains and between the regions of the gut (Volynets et al, 2016;Karlsson et al, 2008;Shanahan et al, 2011). Since RNA-seq reads are tested against a reference B6 genome, some genes that are either deleted or too different between B6 and BALB/c mice may not be properly identified.…”