Oral Gavage in Neonatal Mouse Pups and Functional Assessment of Gut Barrier Integrity Using Ussing Chambers

Growing evidence underscores the importance of the first 1,000 days of life in shaping the gut-microbiome axis. This early-life window is critical in the establishment of long-term physiological trajectories and immunological adaptations, potentially influencing susceptibility to diseases linked to dysbiosis and barrier dysfunction. A deeper understanding of the underlying mechanisms requires a comprehensive assessment of key physiological parameters, including paracellular and transcellular permeability of the neonatal gut. Evaluating these parameters is essential to elucidate how early exposures to exogenous molecules may influence gut integrity and long-term health outcomes. Therefore, the first part of this article describes the oral administration of molecules of interest in mouse pups as early as day of life 6, while minimizing stress, risk of injury, and cannibalism. Lubricated, rounded-tip 24-G feeding needles are used to gavage mouse pups weighing a minimum of 2.5 g. The second part outlines the ex vivo assessment of paracellular and transcellular permeability using Ussing chamber assays on colonic samples from pups between DOL-10 and weaning. Pinless sliders adapted for small biopsies are used in combination with Ussing chambers to mount neonatal colonic samples. The paracellular probe FITC-Dextran 4 kDa and the transcellular marker horseradish peroxidase 44 kDa Type VI are added to the apical compartment of the system at the start of the assay. Samples are collected in the basolateral compartment at 0 min, 30 min, 60 min, 90 min, and 120 min to quantify probe passage. The passage of both markers is quantified directly (FITC) and indirectly (HRP) by a plate reader, calculated using standard curves, and expressed as flux.