All experiments were conducted between January and November 2020 in accordance with the guidelines and approval of the Animal Care and Use Committee (Health Sciences), University of Alberta (AUP00001764), presented according to the ARRIVE guidelines12, and registered at preclincialtrials.eu (PTCE0000148). A graphical display of the study protocol is presented in Figure 1. The authors declare that all supporting data are available within the article.
Randomization
Piglets were randomly allocated to two groups (“CC rate 180/min or “90/min”). Randomization was 1:1 with variable block sizes using a computer-generated randomization program (http://www.randomizer.org). Sequentially numbered, sealed, brown envelopes containing the group allocation were opened during the experiment (Figure 1).
Sample Size and Power Estimates
The primary outcome measure was the time of CPR to achieve ROSC. Our previous studies showed a mean (standard deviation) ROSC of 220 (25) sec with a CC rate of 90/min. We hypothesized that a CC rate of 180/min would reduce the time to achieve ROSC. A sample size of 7/group would be sufficient to detect a clinically important (20%) reduction in time to ROSC (i.e., 176 sec vs. 220 sec) with 90% power and a 2-tailed alpha error of 0.05.
Blinding
TFL opened the randomization envelope and set the rate on the automated CC machine. GMS assessed cardiac arrest and was blinded to group allocation prior to the start of CC. However, due to the varying acceleration speed of the plunger, blinding was not feasible during CC. The statistical analysis was blinded to group allocation and unblinded after completion.
Inclusion and exclusion criteria
Newborn mixed-breed piglets (0-3 days of age) obtained on the day of experimentation from the University Swine Research Technology Center were included. There were no exclusion criteria.
Animal Preparation
Piglets were instrumented as previously described with some modifications13. Following the induction of anesthesia using isoflurane, piglets were intubated via tracheostomy, and mechanical ventilation (Sechrist infant ventilator model IV-100; Sechrist Industries, Anaheim, CA) was commenced at a 20/min rate, peak inspiratory pressure of 25 cmH2O and positive end-expiratory pressure of 5 cmH2O. Oxygen saturation was kept within 90-100%, glucose level and hydration were maintained with an intravenous infusion of 5% dextrose at 10 mL/kg/h. During the experiment, anesthesia was maintained with intravenous propofol 5-10 mg/kg/hr and morphine 0.1 mg/kg/hr. Additional doses of propofol (1-2 mg/kg) and morphine (0.05-0.1 mg/kg) were also given as needed, and their body temperature was maintained at 38.5-39.5°C by using an overhead warmer and a heating pad.
Hemodynamic Parameters
A 5-French Argyle® (Klein-Baker Medical Inc. San Antonio, TX) double-lumen catheter was inserted into the femoral vein for fluid administration and medications. A 5-French Argyle® single-lumen catheter was inserted above the right renal artery via the femoral artery for continuous arterial blood pressure monitoring and arterial blood gas measurements. The right common carotid artery was exposed and encircled with a real-time ultrasonic flow probe (2 mm; Transonic Systems Inc., Ithica, NY) to measure carotid blood flow. A Millar catheter (MPVS Ultra, ADInstruments, Houston, TX) was inserted into the left ventricle (LV) via the left common carotid artery for continuous measurement of stroke volume, end-diastolic volumes, dp/dtmax (maximal rate of rise of left ventricular pressure), and dp/dtmin (minimum rate of change of ventricular pressure), which served as a surrogate for cardiac output. Because of the size difference between the Millar catheter and LV longitudinal axis, which poses a limitation for the accuracy of in vivo volume measurement, an alpha factor = 0.46, based on comparison between Millar’s recording and direct echocardiographic measurements in three piglets, was used to correct the conductance volume14.
Piglets were placed in the supine position and allowed to recover from surgical instrumentation until baseline hemodynamic measures were stable (minimum of one hour). The ventilator rate was adjusted to keep the partial arterial CO2 between 35-45 mmHg as determined by periodic arterial blood gas analysis. Arterial blood pressure, heart rate, and percutaneous oxygen saturation were continuously measured and recorded throughout the experiment with a Hewlett Packard 78833B monitor (Hewlett Packard Co., Palo Alto, CA).
Respiratory Parameters
A respiratory function monitor (NM3, Respironics, Philips, Andover, MA) continuously measured tidal volume, airway pressures, gas flow, and end-tidal CO2. The sensor was placed between the endotracheal tube and the ventilation device. Tidal volume was calculated by integrating the flow signal, and end-tidal CO2 was measured using a nondispersive infrared absorption technique15,16. The accuracy for gas flow was ±0.125 L/min, and the end-tidal CO2 was ±2 mmHg.
Automated Chest Compression Machine
The settings for the automated CC machine were anterior-poster depth 33%, acceleration of compression 500 cm/s2, speed of recoil 50 cm/s, a simulated two-thumb technique, and a CC rate of 90/min or 180/min according to group allocation8,9.
Force Measurement
A FlexiForce A201 sensor (TekScan, Boston, MA) was placed on the bottom of the plunger of the automated CC machine to measure the applied compression force. The applied compression force was recorded with Arduino Software (Somervile, MA) with a sample rate of 200 Hz8,9.
Experimental Protocol
Piglets were randomized into two groups: “CC rate 180/min” or “CC rate 90/min”. Following surgical instrumentation and stabilization, the piglets were placed onto the automated CC machine, which was placed on the surgical bed. The piglets’ chest diameter was measured from the sternum to the vertebrae touching the bed (anterior to posterior) with a measuring tape, and then the anterior-posterior depth of 33% was calculated8,9. Piglets were then exposed to 45 minutes of normocapnic hypoxia, which was followed by asphyxia. Asphyxia was achieved by disconnecting the ventilator and clamping the endotracheal tube until asystole. Asystole was defined as zero carotid blood flow and no audible heartbeat during auscultation. Fifteen seconds after asystole, positive pressure ventilation was provided for 30 sec with a Neopuff T-Piece (Fisher & Paykel, Auckland, New Zealand) with 21% oxygen, peak inspiratory pressure of 30 cmH2O, positive end-expiratory pressure of 5 cmH2O, and gas flow of 8 L/min. After 30 sec of positive pressure ventilation, mechanical CC was initiated8,9 using 21% oxygen17,18, with an antero-posterior chest diameter depth of 33%8,9, and continuous CC was initiated during sustained inflation (CC+SI) with a peak inspiratory pressure of 30 cmH2O for 30 sec19–21. Sustained inflation was interrupted for 1 sec before a further 30 sec of sustained inflation was provided, and this was continued until ROSC13. Epinephrine (0.02 mg/kg per dose) was administered intravenously 2 minutes after the start of positive pressure ventilation and every 3 minutes until ROSC with a maximum of three doses1,2, as the maximum resuscitation time was 10 minutes. The administration of epinephrine was immediately followed by a saline flush of 3 ml. ROSC was defined as an unassisted heart rate >100/min for at least 15 sec. After ROSC, piglets recovered for one hour before being euthanized with an intravenous overdose of sodium pentobarbital (120 mg/kg). If there was no ROSC, piglets were euthanized immediately with an intravenous overdose of sodium pentobarbital (120 mg/kg).
Data collection and statistical analysis
Demographics of study piglets were recorded. Transonic flow probe, heart rate and pressure transducer outputs were digitized and recorded with LabChart® programming software (AD Instruments, Houston, TX). To analyze the hemodynamic data until time to ROSC (i.e., arterial blood pressure, central venous pressure, and carotid blood flow), the duration of CC time was divided into 10 epochs. To analyze stroke volume, end-diastolic volume, dp/dtmax, and dp/dtmin, the pressure–volume loops were compared between groups. Airway pressures, gas flow, tidal volume, and end-tidal CO2 were measured and analyzed using Flow Tool Physiologic Waveform Viewer (Philips Healthcare, Wallingford, CT, USA). For all respiratory parameters, the median values for each piglet during CPR were calculated first, and then the mean of the median was calculated for comparison.
The data are presented as the mean (standard deviation - SD) for normally distributed continuous variables and median (interquartile range - IQR) when the distribution was skewed. The data were tested for normality (Shapiro–Wilk and Kolmogorov–Smirnov test) and compared using t tests or rank sum for normally or skewed distributed data. P values are 2-sided, and p<0.05 was considered statistically significant. Statistical analyses were performed with SigmaPlot (Systat Software Inc, San Jose, USA).