Ethics statement
This study was carried out in strict accordance with the guideline for animal care and use established by the Capital Medical University Animal Care and Use Committee. The study’s experimental protocol was approved by the Committee on the Ethics of Animal Experiments of Capital Medical University (Permit Number: 2019-D-014). Animals used in this study were handled in compliance with the Guiding Principles for the Care and Use of Animals expressed in the Declaration of Helsinki [14]. All animals were maintained in a specific pathogen-free environment in our facility, and were fed with standard chow and had free access to water. All surgery was performed under anesthesia and analgesia, and all efforts were made to minimize suffering.
Animal preparation
Thirty-two male domestic pigs aged 11 to 13 months with an average weight of 30 ± 2 kg were used in each part of this study [15]. The strain of those pigs is mixed breed. Those animals were supplied by a single source breeder (Experimental Animal Center of Capital Medical University, Beijing, China).The piglets were randomly assigned into 4 groups, sildenafil group (n= 8), sildenafil +NG-nitro-l-arginine methyl ester (L-NAME) (20mg/kg L) group (n=8), saline (SA group, n=8); and sham operation group (sham group, n= 8). Sildenafil was obtained from a 25-mg Viagra (Pfizer Australia) tablet that was dissolved in 50 ml saline, filtered and stored at 4℃. In sildenafil group, this solution was given once intraperitoneally in the dose of 0.5 mg/kg 30 min prior to VF [16]. In sildenafil+ L-NAME group, sildenafil (0.5 mg/kg) and L-NAME (20mg/kg), pretreatment was administered once intravenously at 30 min before VF [17]. The drugs were delivered in a randomized manner by the sealed envelope method, as we previously described [12]. The vehicle (0.9 % NaCl) was administered in the same manner and volume. After premedication with 0.5 mg/kg intramuscular midazolam, the animal was anesthetized by ear vein injection of propofol (1.0 mg/kg) and maintained in a surgical plane of anesthesia with intravenous infusion of sodium pentobarbital (8 mg/kg/h). All animals were intubated by a cuffed 6.5-mm endotracheal tube and ventilated by a volume-controlled ventilator (Servo 900C; Siemens, Munich, Germany) using a tidal volume of 8 mL/kg and a respiratory frequency of 12 breaths/min with room air. End-tidal CO2 was measured by an inline infrared cacographic (CO2SMO plus monitor; Respirometric Inc, Murrysville). Respiratory frequency was adjusted to maintain end-tidal CO2 between 35 and 40 mm Hg before CA was induced. Room temperature was adjusted to 26℃, and body temperature was maintained at 37℃ under an infrared lamp, and all efforts were made to minimize suffering. Fluid losses were compensated by an infusion of 30 mL/kg acetated Ringer’s solution during the first hour of preparation, followed by a continuous infusion of 2.5% glucose-electrolytes solution 8mL/kg/h and acetated Ringer’s solution 20 mL/kg/h. All investigators performing CPR and interpreting the outcome assessments were blinded to the medication.
An angiographic catheter was inserted from the femoral artery into the aortic arch for collecting blood samples and for measuring aortic pressure. A Swan-Ganz catheter (7 Fr; Edwards Life Sciences, Irvine, CA) was advanced from the right femoral vein and flow-directed into the pulmonary artery for measurement of right atrial pressure, mean pulmonary arterial pressure (MPAP) and cardiac output (CO). The electrocardiogram and all hemodynamic parameters were monitored with a patient monitoring system (M1165; Hewlett-Packard, Palo Alto, CA). Animals with self-adhesive defibrillation electrodes located on the chest wall. Pigs in the sham group that were not subjected to CA were used as controls. Arterial blood gas values were measured regularly using an ABL 520 Blood Gas Analyzer (Radiometer, Bronshoj, Denmark) at six time points: at baseline, 30 min, and 1, 2, 4, 6 h after ROSC. Mean aortic pressure (MAP) was monitored via the right femoral arterial catheter. The amounts of infused fluid and urine output were also monitored during the experiment. Coronary perfusion pressure (CPP) was calculated as the difference between decompression diastolic aortic and time-coincident right atrial pressure measured at the end of each minute of precordial compression. During CPR, CPP was calculated as the difference between the mean aortic and mean right atrial pressures during diastole (spontaneously beating) or decompression (CPR). CPR compression force, rate, and depth were controlled and continuously recorded during all experiments to assure that all groups received identical CPR quality.
Experimental Protocol
After establishment of vascular catheters, the animals were allowed to equilibrate for 30 min to achieve a stable resting level. Baseline measurements and arterial blood gases were obtained. Mechanical ventilation was established as described above. The temporary pacemaker conductor was inserted into the right ventricle through the right sheathing canal and connected to an electrical stimulator (GY-600A; Kaifeng Huanan Equipment Co, Ltd, Kaifeng, China) programmed in the S1S2 mode (300/200 ms), 40 V, 8:1 proportion, and 10 ms step length to provide a continuous electrical stimulus until VF [18]. VF was defined as a waveform of VF emerging on the monitor and a rapid decline in MAP toward zero. After successful induction of VF, mechanical ventilation was discontinued. Mechanical ventilation was discontinued after the onset of VF. After 8 minutes of untreated VF, CPR was performed. Manual chest compressions were immediately initiated at a rate of 100 compressions per minute for 2 minutes and ventilation conducted using a bag respirator attached to an endotracheal tube with room air. CPR was performed by the same CPR technician from our laboratory, who compressed the porcine chest to approximately one-third of the anteroposterior diameter. The quality of chest compressions was controlled by a Heart Start MRx Monitor/Defibrillator with Q-CPR (Philips Medical Systems, Best, Holland) [19]. The compression-to-ventilation ratio was 30:2. After 2 minutes of CPR, a single 120 J biphasic electrical shock was attempted with a Smart Biphasic defibrillator (Philips Medical Systems,Andover, MA). If the first defibrillation was unsuccessful, epinephrine (20 μg/kg) was given intravenously followed by 2 mins of CPR, and repeated every 2 min if ROSC was not achieved. The 150 J shocks were used for the second and all subsequent attempts. The study was blinded as to the medication used, and only the principal investigator, who did not take part in any resuscitation effort, knew the assignment of each animal. Furthermore, the investigators involved in data recording, data entry, and data analysis were also blinded to the allocation. If spontaneous circulation was still not achieved, CPR was continued for a further 2 min, and defibrillation was attempted once more.
ROSC was defined as 10 consecutive minutes of maintenance of systolic blood pressure at 50 mm Hg. If spontaneous circulation was not restored within 30 min, we regarded the animal as dead [20]. All the animals received normal saline (10 mL/kg/h) intraoperatively to replenish fluid losses. After successful resuscitation, the animals were mechanically ventilated with 100% inspired oxygen for the first 30 min, 50% for the second 30 min and 21% thereafter. With the exception of one jugular vein sheath that was used for fluid administration, all other vascular sheaths and endotracheal tube were removed after a 6 h intensive care period. The animals were allowed to recover from anesthesia, and were then placed in observation cages and monitored for a further 18 h. After a period of 24 h, post-resuscitation measurements were completed. All catheters were removed and wounds were surgically sutured. The animals were then euthanatized with 10 mL of 10 mol/L potassium chloride intravenously following a bolus of 100 mg of propofol intravenously. Myocardial specimens were harvested and snap frozen in liquid nitrogen and stored at -80℃.
Measurements
Hemodynamic and oxygen metabolism parameters collection
ECG was continuously monitored. The hemodynamic parameters including heart rate (HR), CO, MAP, and MPAP were measured continuously, and we recorded the values at baseline, and at 30 min, 1, 2, 4, 6 h after ROSC. At the end of each time point, 4°C saline was injected into the right atrium through the Swan-Ganz catheter to determine CO by the transpulmonary thermo dilution method as described previously [12]. MAP was determined by the electronic integration of the aortic blood pressure waveform. The amounts of infused fluid and urine output were also monitored during the experiment. Serum lactate level, and arterial blood gas values of which temperatures were corrected to 37°C were measured regularly using an ABL 520 Blood Gas Analyzer (Radiometer, Bronshoj, Denmark). CPP was calculated as the difference between decompression diastolic aortic and time-coincident right atrial pressure measured at the end of each minute of precordial compression. CPP during VF was defined as the difference between the mean aortic and mean right atrial pressures. Oxygen metabolism parameters, including oxygen delivery (DO2) and oxygen consumption (VO2), were calculated.
Survival
The survival rate was determined based on the animals that survived the experimental protocol starting at ROSC until 24 hours after ROSC. Animals that died during surgical recovery were excluded.
Micro-RNA isolation and expression
Total RNA samples were extracted using Trizol (Invitrogen, USA) from cultural myocytes. MiR-155-5p, miR-145-5p and eNOS level were quantified by the mirVana qRT-PCR (quantitative real-time PCR) miRNA Detection Kit (Ambion, USA) in conjunction with real-time PCR with SYBR Green I (Applied Biosystems, USA) [21]. The following primers were used for PCR detection: miR-155-5p [5’-GCGCGTTAATGCTAATTGTGA-3’(forward); 5’-AGTGCAGGGTCCGAGGTATT-3’ (reverse)]; miR-145-5p [5’- CGGTCCAGTTTTCCCAGGAA -3’ (forward); 5’ AGTGCAGGGTCCGAGGTATT -3’ (reverse)], U6 was used as an internal control. eNOS [5′-TCC CAG ACC CCA TAA CAA CAG-3′ (sense) and 5′-TGA GGG TGC AGCGAA CTT TA-3′ (antisense)]. The relative expression of miR-155-5p, miR-145-5p and eNOS mRNA was calculated using the 2−ΔΔCt method. All samples were run in triplicate from three independent experiments.
Ultra structural analysis
The remaining tissue was preserved in 10% formaldehyde and 4% paraformaldehyde to observe pathologic and ultra structural changes of the myocardium under transmission electron microscope (TEM) (H-7650; Hitachi, Tokyo, Japan). The pathologic data were assessed by reviewers blinded to the experimental groups.
Statistical analyses
Continuous variables were presented as mean ± standard deviation (SD) when data were normally distributed or as a median (25th, 75th percentiles) when data were not normally distributed. Student t test was used for comparisons between every two groups. Differences at different time points were compared with repeated-measures analysis of variance (ANOVA) with Bonferroni correction for post hoc comparison. The Kruskal-Wallis test was used to compare continuous variables in multiple groups. For these comparisons, the Bonferroni correction was applied to control for the multiple testing. Survival analysis was performed using the method of Kaplan and Meier, and comparisons between groups were made using the log-rank test. A value of p < 0.05 was considered as statistically significant. All analyses were conducted using the SPSS 17.0 software (SPSS Inc, Chicago III) and GraphPad PRISM version 6 (GraphPad Software Inc.,San Diego,CA).