This study was a randomized, controlled laboratory experiment based on a pig model of cardiac arrest. All the experimental procedures were performed based on the methods of our one previous study, in which the animal model has been well established[29]. Ethics committee approval was obtained from the Second Affiliated Hospital, Zhejiang University School of Medicine (No. 2019004). The study included 32 healthy male domestic pigs purchased from same vendor (Shanghai Jiagan Biotechnology Inc., Shanghai, China), weighting 36 ± 2 kg. The pigs were housed under controlled pressure, temperature, humidity and lighting conditions. They were given water and food regularly, washed regularly and disinfected in closed cages. All pigs received care based on the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals.
Animal preparation
The night before the experiment food was withdrawn from all animals, but they were available to water intake. Induction of anesthesia in pigs was achieved by a combination of intramuscular ketamine injection at 20 mg/kg and sodium pentobarbital injection at 30 mg/kg in an ear vein. Then, to maintain the anesthesia, sodium pentobarbital at 8 mg/kg/h as well as fentanyl at 2mg/kg/h were given intravenously. Ventilation was maintained using a volume-controlled ventilator (SynoVent E5, Mindray, Shenzhen, China) with the following setting: tidal volume, 12 mL/kg; peak flow, 40 L/min and FiO2, 21%. End-tidal carbon dioxide (ETCO2) was measured using an ETCO2/SPO2 monitor (PMSH-300, SunLife Science Inc., Shanghai, China), maintaining at 35mmHg ~ 40mmHg by respiratory frequency. The standard lead II electrocardiogram surface electrode was secured.
A double-lumen catheter (11 F, Gambro Kathetertechnik Hechingen, Hechingen, Germany) was placed into the left femoral vein to establish vascular access of CRRT. A fluid-filled catheter (8 Fr, C.R. Bard Inc., Salt Lake, UT) was placed via the right femoral artery to the thoracic artery to measure aortic pressure. A thermodilution catheter (7 Fr, Abbott Critical Care # 41216, Chicago, IL) was placed via right femoral vein to the right atrium to monitor right atrial pressure and blood temperature. Intermittent heparinised saline flushes were made to avoid clogging of the catheters. A pacing catheter (5 F, EP Technologies Inc., Mountain View, CA) was placed via the right external jugular vein to the right ventricle to induce ventricular fibrillation (VF). Animals were placed supine on a heating blanket to maintain the normal temperature at 38.0 ± 0.5℃.
Experimental protocol
Baseline characteristics were recorded after a 10-min stabilization. Pigs were randomly allocated to 1 of 4 groups: normothermia (NT, n = 9), surface cooling (SC, n = 9), CRRT cooling (CRRT, n = 9), or sham control (Control, n = 5). Pigs in the NT and Control groups had the body temperature maintained at 38.0 ± 0.5℃ using the Blanketrol III (Cincinnati Sub-Zero, Cincinnati, OH). For pigs in the other 2 groups, TH was started at 5 min after CPR, and then maintained at the temperature of 33 ± 0.5℃. The hypothermia induced in the CRRT group was achieved by 8-hr CRRT, using an AN69ST hemofilter (Gambro Industries Inc., Meyzieu, France), followed by 16-hr SC; while the cooling in the SC group was achieved by 24-hr SC with the Blanketrol III. Then, a 1℃/h rate of rewarming were followed.
For the CRRT group, a 180 ml/min rate of the blood flow was determined initially, immersing the circuit in 4℃ ice water until the target temperature of 33℃ arrived. Then, the temperature was maintained and the blood flow reduced by 60 mL/min. The rates of liquid replacement and ultrafiltration were 30 mL/kg/h and 20 mL/kg/h, respectively. Immediately when CRRT started, a load dose of 1000-IU heparin was given for anticoagulation, followed by a dose of 150 IU, 300 IU, 450 IU for the first three hours, respectively, and 600 IU/h for the rest 5 hours.
For all pigs, we induced VF by delivering alternating current of 1 mA. Anesthesia and ventilation were disconnected, and the animals underwent an 8-min period of untreated VF. Then, cardiac pulmonary resuscitation (CPR) was started, with a ratio of 30: 2 of compression to ventilation. The chest compressions were achieved at a rate of 100 ~ 120/min (reaching 50 ~ 60mm deep) by a monitor defibrillator (ZOLL Medical Inc., Chelmsford, MA). A dose of 20mg/kg epinephrine was administered at 2.5 min during resuscitation. Biphasic defibrillation at 150 J was performed at 5 min of CPR. ROSC was determined as an organized rhythm and mean arterial pressure of > 50 mmHg sustaining for > 5 min. If not achieved, CPR was held on for another 2 min prior to the next defibrillation. This cycle was duplicated every 2 min, and administration of epinephrine was carried out every 3 min until successful ROSC or 15 min had elapsed. If achieved, 30-hr mechanical ventilation and infusion of normal saline were subsequently continued to keep fluid balance. After completion of the study, euthanasia and a following necropsy were executed to confirm potential injuries of thoracic or abdominal viscera due to experimental intervention. The experimental flow diagram was shown in Fig. 1A.
Measurements
Blood samples of veins and arteries were collected at 1, 3, 6, 12, 24, and 30h post resuscitation. Then the researchers separated serums from venous blood samples and stored them at -80℃ prior to further analyzing the levels of creatinine (Cr), blood urea nitrogen (BUN), intestinal fatty acid binding protein (IFABP) and diamine oxidase (DAO). To evaluate the inflammatory response and oxidative stress, kidney and intestine tissues from renal parenchyma and middle part of small intestine were harvested immediately after euthanasia, and subsequently frozen in liquid nitrogen prior to analysis. Levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) were analyzed using enzyme-linked immunosorbent assay kits (ELISA, Meixuan Biotechnology Inc., Shanghai, China). The contents of malondialdehyde (MDA) were measured by thiobarbituric acid reactive substances assay, and activities of superoxide dismutase (SOD) were measured by xanthine oxide assay[30](Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
The extent of apoptosis of the kidney and intestine were measured using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. The proportion of apoptotic cells was determined as the percentage of TUNEL-positive cells/total cells, and the cleaved caspase-3 protein was detected using immunohistochemistry. The staining intensity of cleaved caspase-3-positive undergone semiquantitative analysis through integrated optical density based on a previous study[31] with Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD).
Statistical analysis
Continuous variables were described as mean ± standard deviation (SD) or median (25th ~ 75th percentiles) for data normally distributed or not. For comparisons among multiple groups, one-way analysis of variance was used for data normally distributed, Kruskal–Wallis test for data not normally distributed. Bonferroni test was used to account for any two group comparisons when the overall comparison was significant. Categorical data were analyzed using Fisher exact test. A two-sided P value of < 0.05 was considered as statistically significant.