In vivo measurement of water movement and paracellular flux in anti-CD3–induced diarrhea. (A) An in vivo perfusion system was developed to measure water movement and paracellular flux in a segment of intestine with an intact neurovascular supply. Water movement was determined via changes in the concentration of ferrocyanide in the perfusate, while paracellular flux was measured by the movement of intravenously injected fluorescent-tagged BSA into the perfusate. (B) In control mice, water was absorbed. Consistent with the development of diarrhea, anti-CD3 treatment reversed net water flow, resulting in net secretion. These changes were prevented by injection of a TNF-neutralizing antibody (n = 4). (C) BSA flux into the lumen of a perfused segment of small intestine, a measurement of paracellular macromolecular flux, was increased 340% ± 35% after anti-CD3 injection. Treatment with TNF-neutralizing antibody prevented 59% ± 5% of this anti-CD3–induced increase in BSA flux (n = 4). (D) Net water secretion occurred in anti-CD3–treated CFTRΔF508 (ΔF508) mice or in wild-type mice perfused with 100 μM niflumic acid, indicating that Cl^– secretion via CFTR or Ca²⁺-activated chloride channels is not necessary for water secretion after T cell activation. Inhibition of Na⁺ absorption in control animals using either Na⁺-free perfusate or the NHE inhibitors HOE694 (200 μM) and S3226 (10 μM) resulted in decreased absorption but did not cause net secretion of water (n = 3). (E) Increased paracellular flux also occurred in anti-CD3–treated CFTRΔF508 mice or mice treated with niflumic acid but was not induced by perfusion with Na⁺-free perfusate or the NHE inhibitors HOE694 and S3226 without anti-CD3 treatment (n = 3).