# Mapping Intestinal Paracellular Perm Eability in Mice: Regional and Cellular Variability Under Physiological and Stimulated Conditions

## TL;DR

This study maps how substances pass through the intestinal walls in mice under normal and stressed conditions, revealing cell-specific permeability patterns.

## Contribution

A new method using fluorescent dyes and confocal microscopy was developed to detect paracellular permeability in mouse intestines.

## Key findings

- FM dye selectively labels specific intestinal cell types like Goblet cells and enteroendocrine cells.
- Chronic stress increases paracellular permeability in the colon, detectable with FM staining.
- Lipid-rich diets increase permeability around jejunal enterocytes.

## Abstract

Intestinal paracellular permeability was analyzed ex vivo by incubation of tissue segments at 0°C with the fluorescent dyes FM1‐43FX (FM) or TRITC‐dextran 3 kDa lysine‐fixable (TD3L) and confocal microscopy in (i) healthy mice and (ii) mice submitted to chronic stress or lipid diets. In the small intestine of healthy mice, FM staining was restricted to the apical surface of enterocytes but fully penetrated around Goblet cells, enteroendocrine cells, tuft cells, and apoptotic cells. The same cell types were similarly labeled in the colon when located on the tissue surface but not within the crypts. TD3L exhibited a comparable labeling pattern but also showed moderate staining of the basolateral surface of enterocytes at the tips of small intestinal villi, and also substantial penetration around colonic epithelial cells at the surface or top of crypts. The study reveals patterns of permeability likely corresponding to the “leak” pathway of paracellular transport through the intestinal epithelium, because transcellular endocytosis is blocked at 0°C. This pathway is found around specific cell populations involved in the luminal detection of food, antigens, microbes, or their secretions. These trigger immune, neural, and tissue responses that maintain intestinal homeostasis. Chronic stress induced by glucocorticoid exposure increased FITC‐dextran 4 kDa permeability in vivo. Using FM, increased paracellular permeability was also detected ex vivo and selectively localized in the colon of stressed mice. A single oral administration of a lipid‐rich food also increased ex vivo permeability around jejunal enterocytes. Pathophysiological increases in paracellular permeability are therefore detectable using the FM methodology.

This study analyzed the paracellular permeability of the intestines of healthy mice at a basal level. A new detection method involving tissue incubation for 5 min with luminal fluorescent dyes, such as FM1‐43FX (FM), which bind to accessible plasma (or external) cell membranes, was developed. Using confocal microscopy, we identified certain epithelial cells, such as goblet cells, as being naturally permeable to FM, while others were impermeable. This selective passage may belong to the leak intestinal paracellular pathway.