The protocol for this study was approved by the Science and Technological Committee and the Animal Use and Care Committee of the University School of Medicine, Nanjing, China. Domestic pigs (Sus scrofa domesticus) were purchased from a local farmer (Qinglongshan animal breeding farm, JiangShu, China). Animal experiments were performed in accordance with the Guidance for the Care and Use of Laboratory Animals11.
Pigs were housed on straw in a cage and fed with a standard diet12. Prior to the study, the animals were fasted overnight. Ten healthy male pigs (body weight 50.3±1.5 kg) were anesthetized with an intramuscular injection of ketamine hydrochloride (3 mg/kg), atropine (2 mg/kg) and fentanyl citrate (2 mg/kg) and an intravenous infusion of propofol (1-2 mg/kg·h), fentanyl citrate (0.5-1 μg/kg·h), midazolam (0.1 mg/kg·h), and atracurium (0.4 mg/kg·h) and placed in the supine position on a thermo-regulated operating table. During surgery, pigs received balanced electrolyte solution (5 ml/kg/h), pigs’ body temperature was maintained at 37.5°C, and pigs’ mean arterial pressure (MAP) was maintained > 60 mmHg with rapid infusions of 0.9% saline (20 ml/kg), as needed.
Following anesthesia, tracheotomy was performed, and pigs were mechanically ventilated (Servo-i ventilator, Solna, Sweden) using volume-control mode at a tidal volume (VT) of 6 mL/kg, a respiratory rate of 30 breaths/min, FiO2 of 1.0, a inspiration-to-expiration time ratio (I:E) of 1:2, and PEEP of 5 cmH2O. Arterial blood samples were collected using a thermistor-tipped Pulse Contour Cardiac Output (PiCCO) catheter (Pulsion Medical System, Munich, Germany) inserted in the right femoral artery. Central venous pressure (CVP) and pulmonary arterial wedge pressure (PAWP) were measured using a Swan–Ganz catheter (Arrow International, Reading, PA, USA) inserted in the internal jugular vein. Cardiac output (CO) was measured with the Swan–Ganz catheter, and MAP was monitored with the PiCCO catheter.
Baseline measurements (TBaseline) were made after pigs had stabilized for 30 minutes. Subsequently, a pig model of ARDS was established using bilateral lung lavage with isotonic saline (30 ml/kg; 38°C) infused through a funnel. Negative pressure was applied to the proximal portion of an endotracheal tube to remove excessive fluid. Alveolar lavage was repeated every 10 min until the P/F ratio decreased to less than 100 mmHg and remained stable for 30 min (TARDS); then, FiO2 was set at 0.4.
ARDS pigs underwent three sequential recruitment maneuvers, including SI, IP and PCV applied in random order according to a random number table, with 30 mins at a PEEP of 5 cmH2O between maneuvers (Figure 1). Circulatory and lung mechanics recovered in 30 min after recruitment maneuvers13 and a PEEP of 5 cmH2O represented physiologic PEEP. SI was performed using continuous positive airway pressure (CPAP) held at 40 cmH2O for 40 secs14. For IP, PEEP was increased from 5 cmH2O to a maximum of 40 cmH2O in 5 cmH2O increments, with each increment lasting 30 secs, and retuned to 5 cmH2O in the reverse process. For PCV, peak pressure was 40 cmH2O, inspiratory to expiratory ratio was 1:2, and PEEP was 20 cmH2O for 2 min. For IP and PCV, respiratory rate was set to 30 breaths/min. Respiratory mechanics, hemodynamic parameters, arterial blood gas, and EIT were recorded at TBaseline, TARDS, and before and after each recruitment maneuver. MAP, CVP, and PAWP were monitored using calibrated pressure transducers. Blood gases were evaluated with an automated blood gas analyser (Nova M; Nova Biomedical, Waltham, MA, USA).
EIT Measurements and Analysis
EIT measurements (PulmoVista 500; Dräger Medical GmbH, Lübeck, Germany) were performed for 3 minutes each at TBaseline, TARDS, and before and after each recruitment maneuver as previously described15. EIT data were generated by applying small alternate electrical currents through 16 electrodes located equidistant apart on a belt positioned around the pigs’ thorax, 5cm above the xyphoid process. A reference electrocardiogram (ECG) electrode was positioned on the abdomen. Current applications and voltage measurements were automatically selected to be compatible with the image reconstruction algorithm. The images were continuously recorded and reconstructed at 40 Hz (Draeger EIT Data Analysis Tool 61).
Four regions of interests (ROI) of the same size and shape consisting of contiguous pixels were identified within EIT images obtained during tidal breathing. A cross section of the lung (ventral to dorsal) was divided into four equal parts, namely ROI1, ROI2, ROI3 and ROI4.16 Tidal volume distribution within the lung was quantified using the GI, as previously described.17 For each breathing cycle, the median value of a tidal image, in which each pixel represented the difference in impedance between end-inspiration and end-expiration, was calculated. The absolute difference between the median value and every pixel value was summed to indicate the variation in the tidal volume distribution. EIT- estimated regional compliance for the gravity-dependent and nondependent lung was calculated as VtROI/driving pressure18. The GI index was adjusted by normalization to the sum of the impedance values. A smaller GI index represented a more homogeneous distribution, and a larger GI index indicated a more inhomogeneous ventilation. The decrease in GI (ΔGI) with each recruitment maneuver was calculated as the difference in GI before and after recruitment.
General anesthesia was maintained throughout the study. After completion of the experiments, the animals were euthanized while in deep anesthesia by an intravenous injection of thiopental.
Statistical analyses were performed using SPSS v20 (Chicago, IL, USA). Differences in global inhomogeneity and changes in global and regional end-expiratory lung impedance among different recruitment maneuvers were investigated. Comparisons were made between values obtained before and after each recruitment maneuver. For non-normally distributed data, results are expressed as median and interquartile range, and comparisons were made with the Wilcoxon rank test. For data that was normally distributed, results are expressed as mean and standard deviation, and comparisons were made with paired samples t tests and Bonferroni correction. p < 0.05 was considered statistically significant.