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

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.