Animals
Male Wistar–Kyoto rats (450–550 g) were randomly divided into a group of normal rats that did not receive ECLS (sham; n = 11), a group of normal rats treated with ECLS (NC + ECLS; n = 11) and a group of asphyxial cardiac arrest rats rescued by ECLS (CA + ECLS; n = 11). Two rats were housed per cage, and they had free access to Purina chow and water under a 12-h light–dark cycle. The experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, and the study protocol was approved by the Animal Care and Use Committee of National Taiwan University (IACUC number: 20140024).
Anesthesia and surgical preparation
Sodium pentobarbital (Sigma Chemical Co., St. Louis, MO, USA; 50 mg kg−1) was administered intraperitoneally to anesthetize the rats, and intravenous reinjections (35 mg kg−1) were performed every hour. The surgical sites (neck and right and left groin) were shaved, and the lungs were ventilated using a ventilator (Model 131, New England Medical Instruments, Medway, MA, USA). Ventilation was performed with room air through a 14G plastic catheter (B. Braun Medical, Bethlehem, PA, USA) at a tidal volume of 8 mL kg−1 and respiratory rate of 70 breaths per min. The rats were placed in the supine position and a rectal temperature probe (TP-K01 and TES-1300, TES Electrical Electronic Corp., Neihu Dist., Taipei, Taiwan) was inserted for the continuous monitoring of rectal temperature. Rectal temperature was maintained at 36 °C by using a circulating warm water blanket (B401H, Firstek Scientific Co. Ltd., Xinzhuang Dist, New Taipei City, Taiwan; TP22G, Gaymar Industries, Inc., Orchard Park, NY, 14,127 USA) and a heating lamp. The electrocardiogram (ECG) of lead II was recorded using a Gould ECG/Biotech amplifier (Gould Electronics, Cleveland, OH, USA).
The surgical sites were shaved and disinfected with betadine. The left femoral artery was cannulated using a Millar catheter (model SPC 320, size 2F; Millar Instrument, Houston, TX, USA) for the continuous monitoring of arterial pressure, and the left femoral vein was cannulated using PE-50 tubing for drug administration throughout the experiment. A 20G IV catheter (B. Braun Medical, Bethlehem, PA, USA) was inserted into the right femoral artery for ECLS inflow and arterial blood sampling. Cannulation of the right external jugular vein for ECLS venous outflow was then performed using a customized template-modified 5-hole 14G catheter, which was advanced to the junction of the right atrium and superior vena cava. This catheter was heparin-locked with 500 UI of heparin to prolong the activated clotting time to longer than 300 s. The arterial blood gas, arterial lactate, and hematocrit (i-STAT CG-4+ and 6+ cartridge, Abbott Point of Care, Princeton, NJ, USA) were determined at the baseline. The same procedure was used for all groups.
Cardiac arrest and ECLS
After instrumentation and the collection of the baseline parameters, a neuromuscular blockade was performed through the intravenous administration of pencuronium bromide (Sigma Chemical Co., St. Louis, MO, USA; 1 mg kg−1) for 5 min. Subsequently, the ventilator was switched off for 5 min to induce asphyxial cardiac arrest. Circulatory arrest was defined as a mean arterial blood pressure (MAP) of <20 mmHg because this pressure is commensurate with the cessation of all tissue blood flow.
The ECLS circuit (Fig. 1) designed for the rats consisted of an open venous reservoir (TERUMO®, Tokyo, Japan; 5-mL syringe), a membrane oxygenator (Micro-1 Rat Oxygenator, Dongguan Kewei Medical Instrument Co., Ltd., Guangdong, China), a heat exchanger (Radnoti Glass Technology Inc., Monrovia, CA, USA), silicone tubing (ID 1.6 mm), and a roller pump (Masterflex, Barrington, IL, USA) that was primed using 19–20 mL of Plasma-Lyte A (Baxter, Deerfield, IL, USA). After 5 min of asphyxia, reperfusion was initiated by starting the ECLS system, which was continued for 30 min.
Discontinuation of ECLS and intensive care unit phase
After 30 min of ECLS, the rats were weaned off the system by removing the right jugular catheter within 5 min, and the incision was closed in two layers by using an absorbable suture for the subcutaneous layer and skin. Arterial samples for blood gas analysis, hematocrit, and lactate were collected at 30, 60, 90, and 120 min after the commencement of reperfusion, defined as the start of ECLS. We did not perform a blood transfusion or administer drugs such as bicarbonate, inotropes, and vasopressors. The pump was primed only with crystalloid throughout the experiment. All rats were humanely euthanized at the end of the experiment.
Estimation of inflammatory response
To estimate the inflammatory response, plasma levels of CRP (Immunology Consultants Laboratory Inc., Portland, OR, USA), IL-6 (BioLegend Inc., San Diego, CA, USA), TNF-α (BioLegend Inc., San Diego, CA, USA), and IL-10 (Abcam, Cambridge, UK) were measured using enzyme-linked immunosorbent assay kits.
Statistical analysis
All data are reported as the mean ± standard error of the mean (SEM). The Mann–Whitney U test was used to compare differences between the groups at the same time points. Kaplan–Meier curves were plotted to depict the survival trend. The log-rank test was used to compare the risk between the two groups. In the mortality cases, the survival time was defined as the duration from the baseline to death; among the survivors, the duration was from the baseline to 120 min after the start of reperfusion. Statistical significance was defined as P < 0.05.
