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, ?=?7) or regional anticoagulation using 4Â % trisodium citrate (group 2, ?=?7).
Each experiment was conducted over a duration of 14Â 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 (10Â mg/kg),
midazolam (0.5Â mg/kg), and atropine (0.04Â mg/kg). Induction of anaesthesia and analgesia
was achieved by an intravenous injection of thiopental sodium (5Â mg/kg), midazolam
(0.5Â mg/kg), and sufentanyl citrate (6Â ?g/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 3Â cm of H
2
O, a fraction of inspired oxygen (FiO
2
) 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 (4Â ?g/kg/h), and pancronium bromide (0.1Â 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 6Â ml/kg/min,
while maintaining other baseline parameters (RR 10–12 breaths/min, PEEP 3 cm H
2
O, FiO
2
0.5, and an I:E ratio of 1:2). The reduced Vt of 40Â % was maintained for the remainder
period of the experiment.
CO
2
dialysis unit set-up and cannulation procedure
The CO
2
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 (0.8Â m
2
, 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 (20Â IU/kg) and the right internal jugular vein was catheterized
using a regular single double lumen dialysis catheter (14 Fr.?×?10â€, 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 80Â 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, 100Â 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.
Fig. 1. In vivo extracorporeal carbon dioxide removal study unit set-up; the hemodialyzer
is replaced by diffusion membrane oxygenator (0.8Â m
2
, Quadrox-iD paediatric), blood flow set at 200Â mL/min, and sweep gas flow at 5Â L/min
(100Â % O
2
)
Intervention phase
The sweep gas flows (100Â % O
2
) through the diffusion membrane were titrated to 5Â 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 4Â % trisodium citrate at the inflow port of
the catheter, which chelated the ionized calcium of inflowing blood. A continuous
infusion of calcium gluconate 10Â % 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 4Â % 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 CO
2
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
(150Â 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.
CO
2
transfer rate was calculated using the following formula:
where Qgas
is the gas flow through the oxygenator and percentage of CO
2
is the CO
2
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.