Compound Muscle Action Potential (CMAP) recordings

Rats were anesthetized with isoflurane (Piramal Healthcare, Bethlehem, Pennsylvania) at a concentration of 3.0-3.5% diluted in oxygen. Animals were placed supine and the region just below the rib cage was shaved and cleaned with 70% ethanol. Phrenic nerve conduction studies were performed with stimulation at the neck via near nerve needle electrodes placed along the phrenic nerve. A reference electrode was placed on the shaved surface of the right costal region. The phrenic nerve was stimulated with a single burst at 6mV (amplitude) for a 0.5 millisecond duration. Each animal was stimulated between 10-20 times to ensure reproducibility, and recordings were averaged for analysis. ADI Powerlab8/30stimulator and BioAMPamplifier (ADInstruments, Colorado Springs, CO) were used for both stimulation and recording, and Scope 3.5.6 software (ADInstruments, Colorado Springs, CO) was used for subsequent data analysis. Following recordings, animals were immediately euthanized with a triple-dose of ketamine/xylazine/acepromazine and tissue was collected (as described below).