Design
This was a blinded block-randomized placebo-controlled intervention study in pigs to compare initial resuscitation success and post-arrest cardiac function with intravenous esmolol (Brevibloc® 10 mg/ml, Baxter, Deerfield, IL, USA) versus control (9 mg/ml NaCl) during resuscitation with veno-arterial extracorporeal membrane oxygenation (VA-ECMO) following acute myocardial infarction and cardiac arrest. Non-participating personnel had access to the randomization list and prepared the study drug. Investigators remained blinded to randomization until all data were collected and analysed. The study was performed at The Intervention Center, Oslo University Hospital. Equipment used in the experiments is presented in detail in the additional materials.
Anaesthesia and animal preparation
We included 20 healthy crossbreed Norwegian Landrace pigs (48 kg (range 46–51)) of either sex in the study. The animals were anaesthetized and surgically prepared as previously described in detail [24]. In brief, after fasting overnight aside from free water access, the animals were pre-medicated in the animal facility (Department of Comparative Medicine, University of Oslo) by an intramuscular injection with a mixture of 30 ml ketamine 50 mg/ml (30 mg/kg), 4 ml azaperone 40 mg/ml (3 mg/kg) and 1 ml atropine 1 mg/ml (20 μg/kg) and then transported to the operating theatre. Further anaesthesia was provided with a weight standardized mixture of pentobarbital 4 mg/kg/h, morphine 2 mg/kg/h and midazolam 0.15/kg/h suspended in Ringer`s acetate solution infused at 10 ml/kg/h. Rocuronium was infused at 3 mg/kg/h. The pigs were mechanically ventilated with tidal volume 10 ml/kg, respiratory rate (RR) 18/min, positive end-expiratory pressure (PEEP) 5 mmH2O, fraction of inspired oxygen 0.4 and ratio of inspiratory to expiratory time (I:E ratio) 1:2. PaO2 and PaCO2 were adjusted according to blood gas analyses throughout the experiment.
The surgical preparation included a tracheostomy and placement of a left intraventricular pressure catheter, a carotid arterial pressure catheter and a pulmonary artery (PA) catheter. In addition, ECMO cannulas were positioned through the right internal jugular vein and the left femoral artery [24].
Experimental protocol (Fig. 1)
After the surgical preparation and a following 30-min stabilization period, pre-arrest (baseline) haemodynamic measurements and cardiac MRI were obtained and blood samples were withdrawn for analyses of serum concentrations of cardiac Troponin T (cTnT) and aspartate transaminase (ASAT).
The pigs were connected to the Ringer`s acetate primed VA-ECMO circuit with the circuitry set to stand-by and the vascular connections clamped. An intravenous injection of heparin 2 mg/kg followed by 0.5 mg/kg/h infusion was provided to prevent blood clotting in the ECMO circuit. Activated clotting time (ACT) was targeted to ≥ 300 s.
A myocardial infarction was induced by fluoroscopy-guided intravascular balloon occlusion of the proximal coronary circumflex artery. After 15 min of occlusion, VF was induced using a 9 V trans-thoracic current device and confirmed by electrocardiography (ECG) and a rapid aortic arterial pressure drop. The VF was left untreated for 10 min with ventilation on hold. Resuscitation with low-flow (2.5 l/min) VA-ECMO was then initiated and mechanical ventilation restarted with RR set to 10/min. VA-ECMO flow rate was selected to mimic flow generated by optimal CPR. Identical volumes of study drug, (either esmolol 1 mg/kg or NaCl 9 mg/ml—control) were administered 1 min after the initiation of VA-ECMO, 2 min prior to 1 mg adrenaline administration and 4 min prior to the first defibrillation attempt to allow adequate circulation of drugs. The first attempt to terminate VF was thereby initiated 15 min after VF induction using biphasic 200-Joule defibrillation(s) (maximum 3). If the first defibrillation failed, adrenaline was re-administered after 3 min, and defibrillation(s) (maximum three) was repeated 2 min after adrenaline injection. The study protocol allowed a maximum of two 5-min cycles with adrenaline and up to six defibrillations. Pilot experiments suggested resuscitation beyond 10 min of ECMO and six defibrillations to be futile. The balloon in the circumflex artery was deflated after a 40-min occlusion-time.
Successful defibrillation was followed by a 1-h ECMO-support at the maximum achievable ECMO blood flow. During ECMO-support, lower limits of mean arterial pressure (MAP) and pulse pressure were 50 mmHg and 15 mmHg, respectively, sustained if necessary by fluid (max 1 l of Ringer’s acetate) and dobutamine infusions (maximum 5 μg/kg/min). The ECMO-support was reduced by 1/6 of maximum flow every 5 min during a 30-min weaning period in accordance with a standardized weaning protocol. The total VA-ECMO duration was median 100 min for the esmolol group (range 95–110 min) and median 95 min for the placebo group (range 95–103 min). After successful ECMO weaning, dobutamine was discontinued. Post-arrest haemodynamic measurements and cardiac MRI were re-assessed after an additional 30-min stabilization period. Analysis of serum concentrations of cTnT and ASAT were also repeated post-arrest.
The animals were euthanized with an intravenous injection of potassium chloride 1 mmol/kg, morphine 1 mg/kg and pentobarbital 20 mg/kg. Immediately post-mortem, a median sternotomy was performed and the heart was excised, sliced and stained in triphenyl tetrazolium chloride (TTC) to estimate myocardial infarction size.
Cardiac function assessments
Arterial, pulmonary artery and left ventricular pressures were measured continuously. Maximum and minimum left ventricular pressure pressures (LVPmax, LVPmin,) and the related first time-derivate of LVP (LV dP/dtmax and dP/dtmin) were also determined. Left ventricle (LV) function at baseline and post-arrest were assessed by MRI and included measurements of stroke volume (SV), cardiac output (CO = SV × heart rate (HR)), end-diastolic volume (EDV), end-systolic volume (ESV), ejection fraction (EF = SV/EDV), mitral annular plane excursion (MAPSE) and mid-LV radial wall thickening.
Cardiac MRI was performed as previously described in detail [24], and in accordance with the recommendations from the Society for Cardiovascular Magnetic Resonance [25]. Cardiac output was assessed by phase-contrast imaging with blood velocity encoded through-plane images of the mid-ascending aorta, a technique considered to provide robust and accurate cardiac output measurements [24, 26]. The images were analysed using the software Medviso Segment version 2.1 R6005 (http://segment.heiberg.se) [27]. Intra- and inter-observer analyses were performed.
MRI-assessed infarct size and histological staining
Before euthanasia, each pig received a dose of gadolinium contrast medium to enhance the infarcted area on the ex vivo MRI scans of the heart. A combination of manually drawing and auto-detection tool was used to estimate myocardial infarct size with the software Medviso Segment version 2.1 R6005 [27].
After euthanasia and post-mortem MRI, the left ventricle was excised and cut into approximately 0.5 cm thick slices before staining in tetrazolium chloride (TTC) at 38 °C for 20 min. Infarct size was determined as percentage of the left ventricle [28, 29] using Photoshop CC2017, version 18.01.
Statistics
Statistical analyses were performed using IBM SPSS Statistics Software version 25. Values are reported as medians with 95% confidence intervals. Groups were compared using Pearson’s chi-squared test or Wilcoxon-Mann-Whitney U test as appropriate. Based on initial pilot and previous experiments, we estimated that post-arrest cardiac output would be 3.3 ± 0.5 l/min in the control group. Assuming cardiac output improved by 15–20% with esmolol (up to 4.0 ± 0.5 l/min), we estimated needing approximately 10 animals in each group with α = 0.05 and power 1-β = 0.9.
Interobserver and intraobserver variability
Four different MRI baseline measurements and two different MRI post-arrest measurements were randomly selected to investigate inter- and intraobserver variability of ejection fraction and cardiac output. The intraclass correlation coefficient of inter- and intraobserver variability for ejection fraction were 0.96 (95% CI 0.7–1.0; p < 0.001) and 0.98 (95% CI 0.87–1.0; p < 0.001), respectively, and for cardiac output 1.0 and 1.0 (95% CI 0.99–1.0), respectively.