The Danish Animal Experiments Inspectorate approved the experiment (no. 2016-15-0201-00866), which was performed in accordance with the Utstein-style guidelines for reporting laboratory cardiopulmonary resuscitation [16].
Study design: Randomized controlled experimental animal study. The statistician performing the analysis was blinded to the randomization.
Animal care
Fourteen female Danish Landrace pigs were used for this study. The animals were allowed 1 week of acclimation after arrival to the research facility. They were housed in pairs, with free access to water and feed.
The pigs were pre-anaesthetised with an intramuscular injection of a Zoletil (Virbac, Kolding, Denmark) mixture containing two dissociative medications (ketamine 8.3 mg/ml and tiletamine 8.3 mg/ml), a benzodiazepine (zolazepam 8.3 mg/ml), an opioid (butorphanol 1.7 mg/ml), and xylazine (8.3 mg/ml). Anaesthesia was maintained with a continuous infusion of propofol and fentanyl until ventricular fibrillation (VF) was induced, when all medications were withdrawn. A single dose of 2.500 IU heparin was given to inhibit blood clots in the vascular catheters. No vasopressors or other medications were used during the 1-h study period.
The animals were paralyzed with 50 mg of rocuronium prior to VF induction to inhibit a confounding effect of gasping [17].
The trachea was intubated with a 6.5-mm endotracheal tube, and the lungs were mechanically ventilated with a ventilator (Dameca Dream, Roedovre, Denmark). The tidal volume (TV) was 10 ml/kg, and the positive end-expiratory pressure (PEEP) was 5 cm H2O. The fraction of inspired oxygen (FiO2) was 0.6 during the surgical procedures. The TV was higher than recommended in guidelines for humans, but it is the normal TV range for pigs [18, 19]. The respiratory rate (RR) was adjusted to keep the partial pressure of carbon dioxide in arterial blood (PaCO2) at 4.5–5.5 kPa with an unaltered TV because a change in TV could influence the effect of an ITD.
A central 7 Fr venous catheter was inserted via the right jugular vein to the level of the right atrium for drug and fluid administration and VF induction. A 6 Fr femoral catheter was inserted into the right femoral artery for continuous blood pressure monitoring and blood tests.
Electrocardiography (ECG), bladder temperature, end tidal CO2 (ETCO2), and blood pressure (BP) were monitored continuously using a Propac MD monitor (Zoll, Medidyne, Copenhagen, Denmark).
One litre of saline was infused during instrumentation to rehydrate the animals after fasting prior to the experiments.
A bladder catheter with a temperature gauge (Degania Silicone Ltd. Degania Bet 15130, Israel) was inserted for urine drainage and temperature monitoring.
The animal’s temperature was kept at a level of normothermia using a warming blanket, if needed.
Both internal carotid arteries were exposed, and a perivascular ultrasound transit time flow probe of 4 mm (Medi-Stim ASA, Fernanda Nissensgate 3, Oslo, Norway) was fitted on the arteries to measure blood flow. The combined flow in the two arteries was considered a reflection of the blood flow to the brain.
The animals were euthanized with an overdose of pentobarbital at the end of the study period.
Experimental procedures
Before initiating VF, FiO2 was reduced to 0.23, which is the lowest possible set point for the ventilator, and induction of VF was not performed if PaO2 was above 15 kPa.
VF was induced with a pacemaker wire inserted via the central venous catheter into the right ventricle using a 9-V DC shock. CA was defined as VF and systolic BP below 25 mmHg according to the Utstein guidelines [16]. Just after induction of VF, the ventilator was adjusted to a FiO2 of 1.0, and RR was set to 10/min with unaltered tidal volume but with no PEEP. Mechanical cardiac compressions were started immediately using LUCAS1™ at 100 compressions/min, a compression:decompression relationship of 1:1 and a compression depth of 5 cm [1]. The animals were placed in a homemade pig holder for the LUCAS device (LUCAS 1, Jolife AB, Ideon Science Park, SE-223 70 Lund, Sweden) to keep the pigs securely positioned and slightly turned to the right side, which in our experience results in fewer injuries to the pig during cardiac compressions (Fig. 1).
On each of the seven study days, 2 pigs were randomized to one of the 2 groups immediately after VF. Thus, seven pigs were randomized to each group.
ITD group
Ventilation was continued using the ventilator with the same settings as described for the control group, but an ITD (ResQPod, Advanced Circulatory Systems, Roseville, MN, USA) was connected to the tube as an interface between the animal and the ventilator to inhibit air flow into the trachea during the decompression phase.
Control group
Ventilation was continued using the ventilator with an unchanged TV of 10 ml/kg and an RR of 10/min; PEEP was reduced to zero, and FiO2 was increased to 1.0.
Cardiac compressions for both groups were started immediately following VF induction using the LUCAS1 device at a frequency of 100/min and were continued for 60 min.
Exclusion criteria
The Utstein recommendations for animal experiments of resuscitation define circulatory arrest as a systolic BP below 25 mmHg [16]. However, since our primary outcome was carotid blood flow during 60 min of mechanical CPR, data were included even if BP decreased below 25 mmHg. The experiment was terminated if an animal had ROSC.
Experimental outcomes
The primary outcome measure was the change in blood flow in both carotid arteries after 60 min compared to the flow 5 min after the induction of cardiac arrest. The secondary outcome measures were systolic BP, pH, PaO2, ETCO2, plasma potassium and plasma lactate.
Measurements
The following values were measured just prior to VF induction (i.e., precardiac arrest): total blood flow in both carotid arteries (carotid BF), systolic femoral blood pressure (BP), and ETCO2. Blood gas analysis was performed using an ABL 800 Radiometer (Copenhagen, Denmark). pH, PaO2, potassium and lactate concentration values are presented in the results.
Bladder temperature was continuously measured, and the temperature was kept in the normal range using warming blankets.
All measurements were performed every 5 min after VF induction during the period of investigation up to 1 h. Cardiac compressions were paused for 5 s during each measurement to evaluate ROSC.
Statistics
Distributions are described as the means and standard deviations (SDs). Comparisons of baseline distributions between treatment groups were performed using an unpaired t-test.
To describe the time course in the two groups, spline curves were fitted to the time series data of outcome variables by means of repeated measures one-way analysis of variance (ANOVA). The levels of outcome variables at time t = 5 and t = 60 were estimated from these splines. In all analyses, bootstrapping with 10.000 replications was used for calculations of standard errors, and p-values were used to account for potential violations of assumed normality and correlation structure in the data.
A value of p < 0.05 was regarded as statistically significant.
Stata Version 15 (Stata Corporation, College Station, TX, USA) was used for all calculations and graphs.
