Mechanical ventilation.

Rats were tracheostomized and connected to a volume-cycled rodent ventilator (Kent Scientific PhysioSuite, Torrington, CT). Arterial O₂ saturation (SaO2, %) was continuously monitored with a rodent pulse oximeter (MouseSTAT Jr.; Kent Scientific, Torrington, CT) placed around the right hindlimb foot. Immediately after initiation of MV, a microcapnograph (Microstream Capnostream 20; Medtronics, Minneapolis, MN) sampling line was inserted into the expiratory tube to measure end-tidal CO₂ (PETCO2). To ensure diaphragm quiescence, PETCO2 was maintained at ≤30 mmHg and diaphragm activation (or inactivation) was monitored and visually verified. The ventilator was maintained at an average breathing frequency of 70 ± 10 breaths/min and a tidal volume of 6–7 mL/kg body wt. The level of PEEP (1 or 9 cmH₂O) was applied through the automatic PEEP settings on the rodent ventilator (Kent Scientific PhysioSuite).