Studies were approved by the Institutional Animal Care and Use Committee of Case Western Reserve University, Cleveland, OH, USA.
Studies were performed on seven dogs (mean weight 16.9 ± 0.5 kg). Animals were initially anaesthetized with pentobarbital sodium (25 mg kg−1) given intravenously. Additional doses were given, as required, based upon the status of corneal reflexes and response to noxious stimuli, both of which were suppressed. After completion of the experiments, animals were killed with pentobarbital sodium (100 mg kg−1 given i.v.).
In each animal, a large bore cuffed endotracheal tube (10 mm ID) was sutured into the trachea in the mid cervical region. A femoral vein catheter was placed to administer fluid and supplemental anaesthesia. A femoral arterial catheter was placed to monitor blood pressure and heart rate (Waveline Pro multi-function monitor, DRE Inc., Louisville KY, USA). A heating blanket (Harvard Apparatus, Holliston, MA, USA) was used to maintain body temperature at 38 ± 0.5°C. End-tidal was monitored at the trachea and oxygen saturation from the earlobe (Waveline Pro multi-function monitor). Tidal volume was recorded by electrical integration of the flow signal from a pneumotachograph (Series 3700, Hans Rudolph Inc., Shawnee, KS, USA).
Following a laminectomy at the T4 level, an eight-plate lead with 4 mm contacts (Carefusion, San Diego, CA, USA) was positioned under direct vision on the ventral surface of the spinal cord and advanced to the T2 level (as previously described) (DiMarco & Kowalski, 2009, 2010). A Grass square-wave pulse stimulator (model S88, Grass Technologies, West Warwick, RI, USA) equipped with a stimulus isolation unit (SIU5, Grass Technologies) was used to provide electrical stimulation. Stimulus train duration was set at 1.3 s since this duration approximated that occurring during spontaneous breathing.
Respiratory displacements of the chest wall were monitored by inductance plethysmography (Braebon Medical Corporation, Kanata, ON, Canada). The effort belts were placed around the rib cage at the lower border of the sternum and around the abdomen at the level of the umbilicus, respectively. Gains of the two signals were adjusted during spontaneous efforts following airway occlusion.
Bipolar Teflon-coated, stainless steel fine-wire electrodes, uninsulated at their terminal ∼5 mm, were used to assess multiunit inspiratory muscle EMG recordings of the external intercostal muscles (dorsal and ventral portions of the 3rd interspace and dorsal portions of the 5th and 7th interspaces). Inspiratory muscle activation was further characterized by single motor unit (SMU) recordings. SMU recordings were made using Teflon-coated stainless steel electrodes with an uninsulated portion of ∼1 mm, to provide greater selectivity. EMG potentials were amplified (1000–10,000 times) and filtered (50 Hz to 5.0 kHz) (model BMA-830, CWE, Inc., Ardomore, PA, USA). All recordings were monitored and stored on an eight-channel data-acquisition recorder (model Dash 8X, Astro-Med, Inc., West Warwick, RI, USA) for subsequent analysis (AstroView X, Data Review Software, AstroMed).
Following measurements obtained during spontaneous breathing, the cervical spinal cord was sectioned at the C2 level in each animal using watchmaker forceps. A hook forceps was passed across the area of transection to verify complete section.
Protocol 1 (n= 7) EMG recording electrodes were positioned in each of the external intercostal muscles to assess their pattern of activation. Multiunit and SMU measurements of each muscle were initially taken during spontaneous breathing. Following C2 section, EMG measurements were also made during HF-SCS (300 Hz; 0.2 ms pulse width) at the T2 level; stimulus amplitude was adjusted to approximate the magnitude of inspired volume observed during spontaneous breathing (mean stimulus amplitude of 0.47 ± 0.05 mA). Five to ten sites were sampled from each muscle for SMU recordings. At each site, one to four SMUs were distinguished on the basis of their morphology. Several breaths were analysed at each site.
Protocol 2 (n= 4) Since preliminary trials indicated that the rib cage contribution to inspired volume was greater during HF-SCS compared to spontaneous breathing, stimulus amplitude was adjusted to approximate the magnitude of rib cage movement observed during spontaneous breathing (mean stimulus amplitude of 0.24 ± 0.04 mA). This additional manoeuvre was performed to determine the distribution of inspiratory activity to the external intercostal muscles under conditions in which activation was more comparable to spontaneous breathing. Otherwise, the protocol procedure was the same as in Protocol 1.
Data analysis During spontaneous breathing, the time of onset of multiunit EMG of the external intercostal muscles (To) was determined relative to the onset of inspiratory flow; during HF-SCS, the onset of multiunit EMG of these same muscles was determined relative to the onset of the stimulus pulse. Inspiratory time (Ti) was determined from the flow signal. As an additional index of the inspiratory timing of EMG signals, To was also expressed as a percentage of Ti. Values were averaged over five consecutive breaths.
Single motor unit analyses included determination of mean peak discharge frequencies, which were assessed over several consecutive breaths. The discharge frequency of each SMU during spontaneous breathing and HF-SCS was plotted against the number of motor units recorded.
Comparisons were made, where applicable, using one-way ANOVA and post hoc Newman–Keuls tests. A P value < 0.05 was taken as statistically significant. Data are reported as means ±s.e.m.