This study was conducted in conformance to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health [20] and the ARRIVE Guidelines [21] and was approved by the Danish Animal Experiments Inspectorate (License number: 2015-15-0201-00744).
Danish landrace piglets aged 2–3 weeks (≈ 8 kg) underwent surgical pulmonary artery banding (n=5). The pulmonary artery was exposed through a left lateral thoracotomy. Growth-induced stenosis was created by fitting two silicone bands around the pulmonary artery, as described in a previous study [18]. After a growth period of 16 weeks, the animals developed right ventricular dysfunction and were re-examined while under anaesthesia and compared with an age- and weight-matched control group (n=6).
Pulmonary artery banding
The animals were pre-medicated immediately upon arrival at the surgical facilities with midazolam (0.5 mg/kg) and azaperone (0.5 mg/kg), to allow for the establishment of intravenous access. Propofol (3 mg/kg) and pancuronium (0.1 mg/kg) were administered to allow endotracheal intubation. The animals were ventilated (S/5 Avance Datex Ohmeda, GE Healthcare, WI, USA) with a tidal volume of approximately 10 mL/kg, a fraction of inspiratory oxygen of 35% and a positive end-expiratory pressure of 4 cm H2O. Ventilation was adjusted to maintain partial pressure of carbon dioxide in the arterial blood at approximately 5 kPa. Arterial blood gases were analysed, and lactate concentration measured (ABL800; Radiometer) every 30 minutes to ensure normal ventilation and oxygenation. Lactate levels were also analysed. Body temperature was maintained within the normal range (38°C–39°C) using a warming blanket. Continuous analgesia and anaesthesia were maintained with Fentanyl (25 μg/kg/hour) and Sevoflurane inhalation at 2.7%. Prior to surgery, the animals were administered prophylactic antibiotics (penicillin 20,000 IU/kg (Ceva Animal Health, Vejle, Denmark) and analgesics (Flunixine 25 mg), both as intramuscular injections. Following the surgery, the animals were aroused and extubated when they started moving and breathing steadily on their own.
Over the following five days, veterinary technicians administered the animals with Flunixine 25 mg and Penicillin (100.000 IU) once daily and paracetamol 250 mg (Actavis, Gentofte, Denmark) orally twice daily.
Re-examination
On the day of re-examination, the animals were anaesthetised and ventilated using the same protocol but omitting pancuronium. Four sheaths were inserted bilaterally into the jugular veins and carotid arteries of the animals. They were then administered 150 IU/kg of heparin prior to catheter insertion, to avoid clotting within the sheaths. Following the administration of 500 μg fentanyl euthanasia was performed by excision of the heart.
Haemodynamic measurements
A Swan-Ganz catheter (7.5F CCOmbo, Edwards Lifescience, Irvine, USA) was inserted into the pulmonary artery to continuously determine cardiac output (CO) and mixed venous oxygen-saturation (SvO2, %). Mean arterial pressure (MAP, mmHg), mean pulmonary artery pressure (mPAP) and central venous pressure (CVP, mmHg) were measured at the level of the phlebostatic axis and recorded continuously using pressure transducers (TruWave, Edwards Lifescience, Germany) throughout the experiment. Heart rate (HR, beats/min) was determined from the arterial pressure curve.
Ventricular measurements
Volumes were calibrated using cine MRI images obtained prior to catherization [18]. Guided by fluoroscopy, pressure-volume catheters (Ventric-Cath 510, Millar Instruments, USA) were inserted antegrade into the right ventricle and retrograde into the left ventricle. A Fogarty occlusion catheter (Boston Scientific, Denmark) was positioned in the inferior caval vein to induce preload reduction. Calibration and preload occlusion measurements were performed for each ventricle, with the other catheter disconnected to avoid the possibility of crosstalk.
The maximum rate of pressure change (dP/dtmax, mmHg∙s-1), preload recruitable stroke work (PRSW, mmHg∙ml∙ml-1), end-systolic pressure-volume relationship (ESPVR, mmHg∙ml), end-systolic pressure–volume relationship x-axis intercept (ESPVR Vo, ml) and maximum ventricular pressure (Pmax, mmHg) were used as measures of systolic function. The end-diastolic pressure–volume relationship (EDPVR, mmHg∙ml-1) was used as a parameter for ventricular stiffness, and the isovolumic relaxation constant (Weiss method) (tau, ms) was employed as a parameter for active diastolic relaxation. Arterial elastance (Ea mmHg∙ml-1), a measure of the arterial load, was calculated as the ventricular end-systolic pressure (mmHg) divided by the stroke volume (ml) and calculated for both ventricles. The arterio-ventricular coupling was defined as ESPVR divided by Ea. Signals were sampled using an MPVS Ultra (Millar Instruments, USA), processed in PowerLab 16/35 (ADinstruments, UK), recorded at 2 kHz and analysed in LabChart 7 Pro (Adinstruments, UK).
Calculations
The systemic vascular resistance index (SVRI, dyn·s/cm5/kg) and the pulmonary vascular resistance index (PVRI, dyn·s/cm5/kg) were calculated as 80∙(MAP – CVP)/CO/kg and 80∙ (right ventricular mean pressure – left ventricular minimum pressure (Pmin))/CO/kg, respectively. CO, stroke volume (SV) and ventricular volumes were indexed to body surface area (BSA, m2) [21].
NT-proBNP
Venous blood was sampled at 16 weeks, following pulmonary artery banding. Lithium-Heparin coated vacutainers were used for sampling and centrifuged for 15 minutes at 1000 x G at 2-8°C. The plasma was stored at -80°C. All samples were thawed and analysed at the same time. NT-proBNP was assessed using an ELISA KIT (Porcine N-terminal pro-brain natriuretic peptide ELISA Kit (Porcine NT-proBNP, Mybiosource, USA).
Medication protocol
Following instrumentation, the animals were subjected to a 30-minute stabilization period, after which baseline measurements were taken. The animals were then randomised to start incremental intravenous infusions with either norepinephrine (0.05, 0.1, 0.25, 0.5 µg∙kg∙min-1) or dobutamine (1.0, 2.5, 5.0, 10 µg∙kg∙min-1). Each step was performed for 10 minutes and concluded with new measurements. After a washout period of 30 minutes, new baseline measurements were taken and the second treatment was started in incremental steps (Figure 1). Concentrations were selected after pilot experiments (n=3, not included in data) were conducted to achieve approximately 100% increase in contractility, estimated by PRSW for both norepinephrine and dobutamine.
Statistics
Differences between the baseline measurements of pulmonary artery banded animals and controls were measured at the first baseline and presented as mean (±SD). Significant differences were assessed using a two-tailed Student’s t-test for normally distributed data and Wilcoxon rank-sum test for non-parametric data. Normal distribution was verified using quantile–quantile plots and histograms.
The hypothesis of differences between norepinephrine and dobutamine with incremental doses was tested using repeated two-way ANOVA (between-group differences) and Bonferroni multiple comparisons post-test, using the GraphPad Prism 5.04 software (GraphPad Software Inc., La Jolla, California, USA). If significant difference was detected between baseline and post-washout, the data were analysed as relative changes from baseline. A p-value of 0.05 was considered to be statistically significant.