Ussing chambers

Ussing chamber studies were performed to assess intestinal physiology (Clarke, 2009). Briefly, a small piece of intestinal tissue (distal ileum and proximal colon) was collected from individual animals, cut along the mesenteric border, and mounted on Ussing chambers (Physiological Instruments, San Diego, CA), exposing 0.1 cm² of tissue area to circulating oxygenated Ringer’s buffer maintained at 37°C. The Ringer’s buffer was made of (in mM): 115 NaCl, 1.25 CaCl₂, 1.2 MgCl₂, 2.0 KH₂PO₄ and 25 NaHCO₃ at pH 7.35 ± 0.02. Glucose (10 mM) was added to the serosal compartment as a source of energy, which was osmotically balanced with mannitol (10 mM) in the mucosal compartment. Ion transport and permeability across the tissue layer were assessed in real time. Active ion transport was measured by short-circuit current (Isc) at baseline, along with conductance (G) as a marker of paracellular, tight junction permeability. Macromolecular permeability was assessed by mucosal to serosal flux of FITC-labeled dextran (Sigma; 4 kDa), which was measured by sampling every 30 min on the serosal side.