Assessment of tracheal ring contraction

Mice were euthanized by intraperitoneal injection of euthanasia solution (VEDCO, 0.1 ml/25 g). A segment of tracheas (4–5 mm in length) was immediately removed and placed in physiological saline solution (PSS) containing 110 mM NaCl, 3.4 mM KCl, 2.4 mM CaCl2, 0.8 mM MgSO4, 25.8 mM NaHCO3, 1.2 mM KH2PO4, and 5.6 mM glucose. The solution was aerated with 95%O2–5%CO2 to maintain a pH of 7.4. Two stainless steel wires were passed through the lumen of tracheal rings. One of the wires was connected to the bottom of organ baths and the other was attached to a Grass force transducer that had been connected to a computer with A/D converter (Grass). Tracheal segments were then placed in PSS at 37 °C. At the beginning of each experiment, 0.5 g passive tension was applied to tracheal rings. After 60 min equilibrium they were stimulated with 80 mM KCl repeatedly until contractile responses and passive tension reached a steady state. Contractile force in response to acetylcholine was then measured.