Animals.

The Institutional Animal Care and Use Committee at the University of Florida, Gainesville, reviewed and approved all procedures. Twenty-four adult male Sprague-Dawley rats (300-450 g) were studied (Harlan Laboratories, Indianapolis, IN). The animals were housed in a 12-h light/12-h dark cycle with free access to standard rat pellets and water.

Animals were anesthetized with isoflurane anesthesia (3–5% in O₂). Subcutaneous injections of carprofen (5 mg/kg body wt) and buprenorphine (0.03 mg/kg body wt) were administered preoperatively for management of pain and discomfort. After an absence of corneal and paw-withdrawal reflexes was confirmed, a 1-in. midline incision was made on the ventral surface of the neck. After isolating the extrathoracic trachea, a saline-filled inflatable cuff (Vivo Metric, Fine Science Tools) was placed around the trachea and the ends of the cuff sutured together. The actuator tube of the cuff was externalized by routing the tube subcutaneously to an incision between the scapulae. Small cutaneous incisions were made bilaterally on the chest wall at level T5-T7. The EI muscles were visualized by blunt dissection. An area between the parasternal and anterior axillary lines was exposed, and bipolar wire electrodes were sutured through the exposed EI muscles (Omnetics Connector, Minneapolis, MN). The EMG wires were also routed subcutaneously to the rats' dorsal incision between the scapulae. The incision in the dorsal scapular surface was closed with sutures, with the externalized actuator tube and connector head accessible for use during experiments. The incisions on the ventral surfaces were sutured. Rats were administered warm normal saline (0.01–0.02 ml/g body wt), penicillin (0.1 ml/kg PenG 30,000 units/ml), and gradually weaned off the isoflurane anesthesia. Postoperative analgesia was administered once every 24 h for 3 days using carprofen (5 mg/kg body wt) and buprenorphine (0.03 mg/kg body wt). Animals recovered for 1 wk before the experimental protocols were initiated.

Instrumented animals were randomly divided into two groups: ITTO group (n = 16) and control group (n = 8). Conscious animals were placed in a whole body restrainer for the entire experimental trial duration. The EMG signals were amplified (P511 series, Grass Instruments, Quincy, MA) and band-pass filtered (30-1,000 Hz). Analog outputs were digitized at 5 kHz (Model 1401, Cambridge Electronics Design), computer processed (Spike2, Cambridge Electronics Design), and stored for subsequent analyses. Trial and data recording began after allowing the animals sufficient time (2–4 min) to acclimate to the restrainer. For the ITTO conditioning group, rats were placed in the restrainer and left undisturbed for 2 min of baseline recording. After 2 min, the ITTO group animals received 3–5 s of tracheal occlusion via cuff inflation by using saline pressure, followed by 10–15 s of unoccluded breathing. This was repeated for a total of 20 min. Cuff pressure was recorded with a differential pressure transducer connected to the actuator tube and syringe.

A total of ∼35–40 tracheal occlusions were presented over the 20 min. After ITTO trials were completed, the animals were allowed to recover for 2 min post-ITTO while recording EI EMG activity. Control animals were placed in the restrainer, EMG activity recorded, and no ITTOs presented throughout the 24-min trial duration. At the end of the trial, the animals were removed from the restrainer and returned to their cage. This procedure was repeated every day for a total of 10 days. The timing of ITTO trials was maintained throughout the 10 days of trials for every animal. Animals were euthanized after 10 days of experimental trials by overdosing with isoflurane gas anesthesia (5% in O₂). After the breathing had stopped the diaphragm was cut to create a pneumothorax.