The experimental protocol was approved by the institutional ethical committee for animal research (CIPA # 4I17025PFp) in compliance with the Canadian Council on Animal Care and the National Institute of Health’s Guide for the Care and Use of Laboratory Animals.
Donor pig management
White-hybrid pigs (55–65 kg) are used in this study. General anesthesia is induced. Under continuous electrocardiogram and blood pressure monitoring, the pigs are mechanically ventilated with an inspiration: expiration ratio of 1:2. Respiratory rate is adjusted to keep an end-tidal CO2 between 35 and 45 mmHg. Saline solution is infused at a rate of 10 mL/kg/h.
In the control group (n = 5), after baseline measurement of the respiratory parameters and collection of blood samples, a sternotomy is performed, and the first lung biopsy is collected (in-situ biopsy). After clamping the ascending aorta, the right and left atrium are opened, and blood collected with Cell Saver (C.A.T.S., Fresenius HemoCare Gmbh, Bad Homburg, Germany). The lungs are flushed with 1 liter of cold Perfadex–LPD solution (Vitrolife AB, Gothenburg, Sweden) through a cannula secured in the main pulmonary artery. The lungs are then procured in a standard fashion and preserved in cold Perfadex for 2 hours.
In the LPS group (n=6), a right carotid artery catheter is installed for systemic arterial pressure monitoring and blood sampling. A central line with a pulmonary artery catheter is installed in the right internal jugular vein for measurement of pulmonary artery pressure (PAP, mmHg), blood sampling, and drug administration. Respiratory and hemodynamic parameters are recorded continuously. An LPS infusion induces systemic inflammation. LPS (Escherichia coli; serotype O111:B4, Sigma, Saint Louis, MO, USA) is dissolved in 100 mL saline solution and infused intravenously at a concentration of 20 μg/kg over one hour, followed by a 90-minute observation period. The dose of LPS was determined based on an initial small-scale feasibility study where higher concentrations of LPS (50-100 μg/kg) over 30-60 minutes caused deleterious effects on the lungs rendering them unsuitable for EVLP perfusion. Boluses of 1 mg/ml Milrinone are administered intratracheally to treat pulmonary hypertension. Hemodynamic instability (mean arterial pressure, MAP < 65 mmHg) is treated with increasing the rate of saline infusion to 15 mL/kg/h and an infusion of phenylephrine. Blood samples are collected at baseline (before LPS administration) and at 30-minute intervals for a total of 150 minutes. The samples are centrifuged (1500g/15 min), and plasma is preserved at -80°C for later analysis of cytokines. Blood gases, chemistry, and metabolite contents are analyzed in the blood samples at baseline, at the end of LPS infusion, and before lung procurement. At the end of the observation periods, the lungs are procured and flushed with cold Perfadex. LPS-treated lungs are compared to the lungs from the control group.
Ex-vivo Lung Perfusion Procedure
In preparation for EVLP, one cannula is secured in the main pulmonary artery and the second in the trachea. The left atria cuff is left open per the Lund EVLP protocol10. A second biopsy (Pre-EVLP biopsy) is performed. Lungs are weighed (pre-EVLP lung weight). A temperature probe and a perfusate sample catheter are secured inside the left atrium. The lungs are then connected to the perfusion system.
EVLP is performed with Vivoline LS1 (Vivoline Medical AB, Lund, Sweden) using a modified Lund protocol adapted for pig lungs. The circuit is primed with 2.6 L of a perfusion solution containing human serum albumin and 500 mL of washed red blood cells, previously collected from the donor pig during the procurement with Cell Saver. Before reperfusion, baseline hematocrit is adjusted to 10-12%. Heparin and antibiotics (Imipenem 100mg) are added to the perfusion solution, and pH corrected to 7.35-7.45 using tris(hydroxyl)aminomethane (THAM).
Porcine cardiac output (CO) is estimated to be 100 ml/min/kg body weight. The final target flow used in our EVLP protocol was 50% of CO, corresponding to blood flow between 2.8 and 3.2 L/min. Based on the work from Steinmeyer et al. and Roma et al., a reduced flow rate of less than 100% of CO is more suitable for the delicate lungs of young pigs11,12. This has been consistent with our experience. We noticed significant lung edema when utilizing a flow rate of 100% of CO. Since we have modified the protocol with the reduced flow rate as per Roman et al., we have seen improved outcomes using the Lund protocol. Lung reperfusion is initiated at a flow rate of 0.5 L/min and gradually increased to 1.0-1.2 L/min over the first 10 minutes or until the temperature of the lungs reaches 25ºC. Bronchoscopy and bronchoalveolar lavage fluid (BALF) collection are performed. The flow rate is then increased over 30-40 minutes to 50% CO. When the perfusate temperature reaches 32ºC, ventilation using 50% FiO2 is started in volume-controlled mode at an initial tidal volume of 4 ml/kg of donor weight, a positive end-expiratory pressure of 5 cmH2O and a rate of 5 breaths/min. Tidal volume is increased gradually to a maximum of 6 mL/kg. When a temperature of 37ºC and a target 50% flow are reached, a recruitment maneuver is performed with an inspiratory hold on 20 cmH2O over 1 minute and repeated hourly.
Hemodynamic and ventilation parameters
The following hemodynamic parameters are monitored and collected every 15 min: PAP (mmHg), perfusate flow (L/min), pulmonary vascular resistance (PVR; Dyn.s.cm-5), and temperature (ºC). The ventilation parameters collected or calculated every 15 min were: peak airway pressure (cmH2O) and airway plateau pressure (cmH2O), airway resistance (cmH2O/l/sec), static and dynamic compliance (ml/cmH2O) and end-tidal carbon dioxide (mmHg).
At the end of the reconditioning phase, the evaluation phase starts with disconnecting the oxygen supply to the oxygenator, which serves to deoxygenate the perfusate using a gas mixture of 7% CO2 and 93% nitrogen. Lung function is evaluated after ventilation with 100% FiO2. Blood gas analyses are performed on samples collected from the left atrium. The pO2/FiO2 (P/F) ratio is calculated. Electrolytes and metabolites are also measured in these samples and the perfusates collected at the start (T0), at 2 hours and 3 hours of EVLP. The lungs are then weighed (post-EVLP lung weight) to estimate total lung water accumulated during EVLP, followed by a third biopsy (post-EVLP biopsy).
Acute Lung Injury Profiling
The parameters used to evaluate the acute lung injury following LPS infusion include the weight of the graft, wet-to-dry weight ratios; the presence of edema on bronchoscopic evaluation; BALF analysis for total protein. These parameters are measured before and after EVLP.
Inflammation Profiling during EVLP
Kinetics of the cytokine response is explored during two different phases: after LPS infusion in the donor pig and during EVLP. Cytokines are measured in the serially collected arterial plasma samples and perfusates. BALF samples that were collected using bronchoscopy before and after EVLP were also centrifuged and used for the analysis of cytokines. Porcine TNFα, IL-6, IL-10, and IL-1β are analyzed using kits from DuoSet® ELISA Development System (R&D Systems Inc., MN, USA).
Lung Histology
Lung tissue biopsies collected in-situ, at the end of 2-hour cold ischemic time and the end of 4 hour-EVLP periods are fixed in neutral buffered 10% formalin solution for 24-48 hours. Biopsies were embedded in paraffin, sectioned at 5-µm thickness, stained with hematoxylin and eosin, and examined under light microscopy for pathologic changes.
Statistical Analysis
Data in the graphs are presented as means ± standard deviation (SD). The lung weight of the donors in control and LPS groups was compared using the unpaired t-test. Within the LPS donor group, repeated measures during the observation period were compared to baseline data using the paired t-test. For the data collected during EVLP, a 2-way ANOVA test with repeated measures was used to compare the two groups over time. Statistical analyses were performed using GraphPad Prism software (GraphPad Software Inc, La Jolla, CA, USA), and a p-value <0.05 was considered significant.