This manuscript is part of a sequence of experiments during porcine respiratory ECMO support, some of which were previously published elsewhere [11]. The study was approved by the Institutional Animal Research Ethics Committee from the Hospital Sírio-Libanês in São Paulo, Brazil, and was done according to the National Institutes of Health guidelines for the use of experimental animals. Instrumentation, surgical preparation, and lung injury were performed as previously described [11].
Instrumentation and surgical preparation
Five domestic female Agroceres pigs (80 [79,81] kg) were anesthetized with thionembutal (10 mg.kg−1, thiopental, Abbott, Brazil) and pancuronium bromide (0.1 mg.kg−1, Pavulon, AKZO Nobel, Brazil) and connected to a mechanical ventilator (Evita XL Dräger, Dräger, Lübeck, Germany) with the following parameters: tidal volume 8 mL/kg, end-expiratory pressure 5 cmH2O, FiO2 initially set at 100 % and subsequently adjusted to maintain arterial saturation between 94 and 96 %, and respiratory rate titrated to maintain PaCO2 between 35 and 45 mmHg or an end-tidal CO2 (NICO, Dixtal Biomedica Ind. Com, São Paulo, Brazil) between 30 and 40 mmHg. The electrocardiogram, heart rate, oxygen saturation, and systemic pressures of the animals were monitored with a multiparametric monitor (Infinity Delta XL, Dräger, Lübeck, Germany). Central venous pressure (CVP), mean pulmonary artery pressure (PAPm), pulmonary artery occluded pressure (PAOP), and cardiac output (CO) were measured with the use of a pulmonary artery catheter. Anesthesia was maintained with midazolam (1–5 mg.kg−1.h−1) and fentanyl (5–10 mcg.kg−1.h−1), and muscular relaxation was maintained with pancuronium bromide (0.2 mg.kg−1.h−1).
A 25-cm ECMO arterial cannula (Edwards Lifesciences, Irvine, CA, USA) was introduced into the right external jugular vein. A 55-cm ECMO drainage cannula (Edwards Lifesciences, Irvine, CA, USA) was positioned close to the right atrium via the right femoral vein with the aid of transhepatic ultrasonographic visualization. Only the guidewires were inserted until the first baseline measurements, after the stabilization period (Fig. 1). Heparin infusion was then started at 1000 IU/h. After the first baseline, guidewires were replaced by the ECMO cannulas. Cannula diameter was chosen in accordance with vein diameter as measured with the aid of ultrasonography. Four animals had a 20-Fr and one had a 21-Fr drainage cannula. Three animals had a 21-Fr and two had a 20-Fr return cannula. A central venous catheter and an arterial line were placed in the left femoral vein and artery, respectively.
Stabilization and support of the animals
After surgical preparation, we allowed the animals to stabilize for 1 h. A continuous infusion of 3 mL.kg−1.h−1 of lactated Ringer’s solution was infused throughout the experiment. Fluid challenges and vasopressors were used to maintain the mean arterial pressure between 65 and 80 mmHg.
ECMO priming, starting, and maintenance
The ECMO system (Permanent Life Support (PLS) System, Jostra–Quadrox D, Maquet Cardiopulmonary, Hirrlingen, Germany) was primed with a 37 °C normal saline solution and connected to a centrifugal pump (Rotaflow, Jostra, Maquet Cardiopulmonary, Hirrlingen, Germany). Heparin infusion was titrated to keep the activated coagulation time 1.5–2.5 times its baseline value. The absence of significant re-circulation was confirmed by an oxygen saturation of less than 70 % collected from the pre-membrane port [10, 12] 10 min after the beginning of each new sweep gas flow.
Varying ECMO blood flow and sweep gas flow
The animal was kept in apnoea with 10 cmH2O of PEEP and a FiO2 = 1.0 using a concentric coil-resistor PEEP valve (Vital Signs Inc., Totowa, NJ, USA) with a humidified oxygen continuous flow at 10.0 L/min. ECMO blood flow was initially set at 5.0 L/min, and the sweep flow was set at 5.0 L/min. After 10 min, clinical and laboratorial data were collected, and blood flow was reduced to 1500 mL/min, with an initial sweep gas flow = 3.0 L/min. An arterial blood sample was collected every 10 min for up to 30 min; afterwards, an arterial blood sample was collected every 5 min until the new ECMO adjustment was in place for 50 min. Carbon dioxide equilibrium was defined as two blood gas analysis with a PaCO2 variation less than 3 %. Hemodynamic data were also collected along with blood samples. After this step, the blood flow was kept at 1500 mL/min, and the sweep gas flow was set at 1.5 L/min and subsequently at 10.0 L/min in 50-min intervals. Blood flow was then elevated to 3500 mL/min, and a sequence of sweep gas flows of 2.0, 3.5, 7.0, and 12.0 L/min for 50 min each was applied (see Fig. 1). Two different blood flows were chosen in order to evaluate the impact of blood flow in PaCO2 equilibrium. For each ECMO blood flow, sweep gas flow sequence was chosen arbitrarily. However, for a blood flow of 1.5 L/min, we chose an alternating sequence of 3.0, 1.5, and 10 L/min in order to minimize a possible carbon dioxide carry-over phenomenon.
Time to equilibrium–time constant (tau) determination
To calculate the time constant for each sweep gas and blood flow combination, data corresponding to the PaCO2 value along the time were fitted into the following equation [13]:
$$ \mathrm{Partial}\ \mathrm{pressure}\ \mathrm{of}\ {\mathrm{CO}}_2 = a + b \times \left(1-{e}^{-t/\mathrm{tau}}\right) $$
where t is the time in minutes, e corresponds to Euler’s number, tau is the time constant obtained for each flow combination, and a and b correspond to constants used in the model.
Calculations
Calculations were done using standard formulas:
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Blood oxygen content CbO2 [mL O2/100 mL of blood] = 1.36 × Hb × SatbO2 + 0.0031 × PbO2
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O2 transfer [mL/min] = (1.36 × Hb × (After - pre-membrane SatO2) + 0.0031 × (After - pre-membrane PO2)) × ECMO blood flow
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CO2 transfer [mL/min] = (CO2 partial pressure of the exhalation port of the membrane/barometric pressure) × sweep flow in mL/min
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Blood CO2 content [mL/min] [14] = (1 − ((0.0289 × Hb)/(3.352 − 0.456 × (SatbO2/100) × (8.142 − pHb)))) × 2.226 × 0.0307 + (0.00057 × (37 − temperature)) + (0.00002 × (37 − temperature)2) × PbCO2 × (1 + 10 (pHb − 6.086) + (0.042 × (7.4 − pHb)) + ((38 − temperature) × 0.00472 + (0.00139 × (7.4 − pHb))))
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Oxygen delivery DO2 [mL O2/min] = cardiac output × CaO2 × 10
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Oxygen consumption VO2 [mL O2/min] = C(a − v)O2 × cardiac output × 10
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Standard base excess [SBE - mEq/L] = 0.9287 × (HCO3− − 24.4 + 14.83 × (pH − 7.4))
Statistical analysis
Normality was assessed by the Shapiro-Wilk test. Variables are shown as mean ± standard deviation if normally distributed and median and the 25th and 75th percentiles if otherwise. Percent variations of PaCO2 at the end of the 50-min observations in each sweep gas flow were normally distributed and compared with the paired t test. The R free source statistical package and comprehensive-R archive network (CRAN)-specific libraries were used to build the graphics and analyze the data [15].