This in vivo study was conducted at the Department of Cardiothoracic Surgery, Experimental Laboratory, Maastricht University, the Netherlands. The study was approved by the local animal investigation committee of the Maastricht University. Animal handling was in accordance with the guide and use of laboratory animals (published by the National Institute of Health, USA).
Overview of study
Fourteen healthy domestic landrace pigs weighing 51 ± 3 kg were studied. The animals were divided into two groups based on either systemic anticoagulation with heparin (group 1, n = 7) or regional anticoagulation using 4 % trisodium citrate (group 2, n = 7).
Each experiment was conducted over a duration of 14 h, divided into four subsequent phases: stabilization phase (0–60 min), hypercapnic phase (60–360 min), intervention phase (360–720 min), and post-intervention phase (720–840 min).
Animal preparation
The animals were sedated with a mixture of intramuscular administrated ketamine (10 mg/kg), midazolam (0.5 mg/kg), and atropine (0.04 mg/kg). Induction of anaesthesia and analgesia was achieved by an intravenous injection of thiopental sodium (5 mg/kg), midazolam (0.5 mg/kg), and sufentanyl citrate (6 μg/kg). Following endotracheal intubation, mechanical ventilation (Datex Engstrom, Instrumentarium Corporation, Helsinki, Finland) was ensued and animals were stabilized on a volume-controlled setting with a respiratory rate (RR) of 10–12 breaths/min, a tidal volume (Vt) of 10 mL/kg/min, a positive end expiratory pressure (PEEP) of 3 cm of H2O, a fraction of inspired oxygen (FiO2) of 0.5, and an inspiratory is to expiratory (I:E) ratio of 1:2.
Anaesthesia, analgesia, and muscle relaxation were maintained with intravenous administration of midazolam (1.0 mg/kg/h) additionally supported by Isoflurane (0.5–1 %), sufentanyl citrate (4 μg/kg/h), and pancronium bromide (0.1 mg/kg/h). Limb lead electrocardiogram (ECG) and oxygen saturation (ML320/E Oximeter POD, PowerLab, AD Instruments, Oxfordshire, UK) were recorded and monitored continuously. Body temperature was maintained within normal range (37.5–38.0 °C). Intravenous infusion of Ringer’s lactate was provided. The femoral vessels (artery and vein) and pulmonary artery were catheterized to measure the mean arterial pressure, central venous pressure, and cardiac output.
Hypercapnic phase
The hypercapnic phase was induced by resetting the ventilator to a lower Vt of 6 ml/kg/min, while maintaining other baseline parameters (RR 10–12 breaths/min, PEEP 3 cm H2O, FiO2 0.5, and an I:E ratio of 1:2). The reduced Vt of 40 % was maintained for the remainder period of the experiment.
CO2 dialysis unit set-up and cannulation procedure
The CO2 dialysis unit consisted of a continuous veno-venous hemofiltration device (Baxter BM11, Baxter Healthcare Corporation, Deerfield, IL, USA), a Bio-line coated diffusion membrane oxygenator (0.8 m2, Quadrox-iD paediatric, Maquet Cardiopulmonary, Rastatt, Germany) that replaced the hemoconcentrator (as shown in Fig. 1) and was primed with 200 mL of 4 % Gelofusine (B. Braun AG, Melsungen, Germany). At end of the hypercapnic phase, the animals in group 1 received an intravenous bolus injection of heparin (20 IU/kg) and the right internal jugular vein was catheterized using a regular single double lumen dialysis catheter (14 Fr. × 10”, 25 cm; Arrow international, Reading, PA, USA). A continuous infusion of heparin at the inflow port of the dialysis catheter maintained the activated partial thromboplastin time (aPTT) between 60 and 80 s, measured hourly. In group 2, the regional anticoagulation was started as soon as the catheter was placed through the inflow port of the dialysis catheter. In both groups, 100 mL of priming volume was discarded immediately after the circuit was connected to the dialysis catheter. The correct placement of the dialysis catheter was confirmed by fluoroscopy.
Intervention phase
The sweep gas flows (100 % O2) through the diffusion membrane were titrated to 5 L of oxygen, and the blood flow was maintained at 200 mL/min. In group 2, Ringer’s lactate infusion was tapered accordingly, since there was continuous infusion of 4 % trisodium citrate at the inflow port of the catheter, which chelated the ionized calcium of inflowing blood. A continuous infusion of calcium gluconate 10 % at the outflow port of the catheter maintained the systemic ionized calcium. Monitoring of anticoagulation in this group was performed by hourly measurement of the ionized-calcium levels at the post-oxygenator and in the animal. The infusions of 4 % trisodium citrate and calcium gluconate were titrated accordingly to maintain the ionized-calcium level between 0.25 and 0.35 mmol/L [15] in the dialysis circuit and a systemic ionized-calcium level of 1.12–1.20 mmol/L.
Post-intervention phase
The CO2 dialysis was terminated, and the dialysis circuit was disconnected after clamping the inflow and outflow ports. The animals continued to be on mechanical ventilation, while the disposable parts of the circuits were separated, flushed with saline, and visually inspected for clot formation. Parts of the diffusion membrane were collected for scanning electron microscopy (SEM) analysis, and following post-intervention phase, the animals were euthanized under general anaesthesia using a single dose of pentobarbital (150 mg/kg).
Measurements
Hourly samples for blood gases were drawn and analysed (Gem Premier 3000, Instrumentation Laboratory, Lexington, MA, USA). Blood samples for liver function and renal function tests were collected to monitor the acute metabolic effects of citrate. The aPTT was measured using a Hemochron Jr. Signature (Edison, USA). Cardiac output was measured by thermo-dilution.
CO2 transfer rate was calculated using the following formula:
$$ \mathrm{C}{\mathrm{O}}_2\kern0.5em \mathrm{transfer}\ \mathrm{rate} = {Q}_{\mathrm{gas}}*\ \mathrm{Percentage}\ \mathrm{of}\ \mathrm{C}{\mathrm{O}}_2*\kern0.5em 10 $$
where Q
gas is the gas flow through the oxygenator and percentage of CO2 is the CO2 at the gas outlet port of the oxygenator, measured by capnograph (Novametrix Capnogard, Philips Respironics, Best, The Netherlands).
Statistical analysis
Statistical analysis was performed with SPSS 21.0 software (SPSS, Chicago, IL, USA). Continuous data are presented as mean ± standard deviation or median with interquartile range as appropriate. The continuous variables were subjected to normality distribution. Accordingly, continuous variables of paired samples within the group were analysed by the Wilcoxon signed-rank test, and differences between two continuous variables were analysed by the Mann-Whitney U test. A p value <0.05 was considered statistically significant.