Adult ferrets (1.7 to 2.3 kg, random sex) obtained from Marshall Farms (North Rose, NY) were killed by intraperitoneal injection of pentobarbital sodium (150 mg/kg body weight), and the trachea from the larynx to the carina was immediately removed. Each trachea was divided into four or eight roughly equal segments from the upper trachea to the carina. The segments were weighed and then immersed in 10 mL of Krebs-Henseleit solution (KHS) [containing 6.9 g/L of NaCl, 0.35 g/L of KCl, 0.16 g/L of KH2PO4, 0.141 g/L of MgSO4, 0.373 g/L of CaCl2, 2 g/L of D-glucose: pH 7.4, 255 mOsm/L]. This was agitated gently in a shaking water bath at ferret body temperature, 38°C, with 0.5 L/min of 100% oxygen provided into the incubator bath. Measured fraction of inspired oxygen varied from 0.40 to 0.45. Isolated tracheae were rested in KHS for 2 h to stabilize airway secretion. review
The incubation solution was changed, and the segments were incubated for 20 min with only KHS to measure the constitutive secretion (period 1). These were then incubated for another 20 min with test agent: KHS with added 1 g/dL of saline solution (597 mOsm), 3 g/dL of saline solution (1,192 mOsm), 5 g/dL of saline solution (1,823 mOsm), 10 g/dL of saline solution (3,612 mOsm), or 15 g/dL of mannitol solution (1,040 mOsm), or with only KHS (0.69 g/dL of saline solution [288 mOsm]) as a negative control (period 2). After the experiment, the bathing solution was collected and stored at – 70°C until analyzed.
The relative contribution of mucous and serous cells to the secretion was evaluated by measuring the amount of mucin, a marker for mucous cell secretion, and lysozyme as a marker of serous cell secretion. A secretory index (SI) expressing the relative increase in mucin or lysozyme under experimental conditions was calculated for each tracheal segment as concentration after a 20-min exposure to the test agent (period 2) divided by the concentration measured after a 20-min exposure to KHS alone (period 1).