Preparation of Guinea Pig Trachea

The animals were euthanized as described in Section “Evaluation of Chronic Allergic Lung Inflammation Effects on Lung and Bronchial Morphology of Guinea Pigs.” The trachea was immediately removed and cleaned of fat and connective tissue; the tracheal rings (2–3 cm) were immersed in Krebs solution and bubbled with carbogen mixture (95% O₂ and 5% CO₂). The Krebs solution composition was (mM): NaCl (118.0), KCl (4.55), MgSO₄ (5.7), KH₂PO₄ (1.1), CaCl₂ (2.52), NaHCO₃ (25.0) and glucose (11.0), with pH adjusted to 7.4. To record isometric contractions, tracheal segments were suspended in steel rods in organ baths (6 mL), connected to a force transducer (TIM 05), attached to an amplifier (AECAD04F) and connected to an A/D converter in a computer running AQCAD^® software (São Paulo, Brazil). The system contained a thermostatic pump model BT 60 that controlled the organ bath temperature. The trachea resting time was 60 min in a preload tension of 1 g (baseline). During the organ resting phase, the solution was changed every 15 min to avoid metabolite accumulation (Tschirhart et al., 1987).

After the stabilization period, the tracheal rings were contracted with 1 μM CCh and isometric tension was recorded. When a stable contraction was attained, 0.1 mM arachidonic acid was added to the organ bath to confirm the presence of epithelium by the presence of arachidonic acid-induced relaxation equal to or greater than 50% of maximal tension. In some tracheal rings, the luminal surface was gently rubbed with Krebs-moistened cotton tip to remove the epithelial layer. The absence of epithelium was confirmed when arachidonic acid-induced relaxation was absent or lower than 10% of maximal tension (Tschirhart et al., 1987).