Animal experiments were performed at the animal facilities of the Center of Biomedical Research of the Medical University of Vienna. The study was conducted after approval from the Ethics Committee for Animal Research and the permission of the Federal Ministry of Education, Science and Research according to §26 TVG 2012 (Ethics Protocol Number: 162/115/97/98). To ensure optimal animal handling prior to the experiments, pigs were housed in groups, kept on dry straw beds and fed in a manner appropriate for their species.
The study was structured in two parts: (a) development of unilateral ALI (n=9) and (b) systemic evaluation of key parameters defining ALI (n=6), which was regarded as the main study. Fig. 1 depicts the main study design and timeline of the experimental interventions.
Preparation of a modified double lumen tube.
In pigs, a tracheal bronchus branch, named the bronchus trachealis, originates from the trachea proximal to the carina to aerate the upper parts of the right lung (Fig. 2)(15). To fit the anatomy of the bronchial tree of pigs, we modified a commercial left-sided double–lumen tube for left bronchial intubation in humans (37Fr, Hudson RCI®, Sheridan® Sher-i-bronch®, Teleflex Medical, Morrisville, NC, USA). This adaption was mandatory to avoid blockage of the bronchus trachealis so that ventilation of the right upper lobe was guaranteed. Use of a commercially available left double–lumen tube would have resulted in atelectasis of the right upper lobe. For this, the tracheal cuff was detached from the double lumen tube at the distal end and everted 4 cm proximally. The cuff was fixed and glued (Silastic®, Medical Adhesive Silicon Type A, Dow Corning Corporation, Midland, MI, USA) to the tube in the proximal position (Fig. 3). The cuff was then partially inflated with 2 to 3 mL of air to avoid undesirable adherence of the cuff material to the tube in the adjunct areas of the fixation zone. After a drying period of at least 72 hours the cuff was tested for air leakage with a cuff pressure of 30 cmH2O. If no cuff pressure drop was observed within 5 minutes the cuff was deemed to be airtight, and the double lumen tube could be used in the pig experiments.
Animal preparation
All pigs were premedicated with ketamine 5-6 mg•kg-1 i.m., midazolam 0.2-0.3 mg•kg-1 i.m. and medetomidine 0.05-0.1 mg•kg-1 i.m. and placed in the supine position on a heating mat. After cannulation of an ear vein, propofol 2.5-5 mg•kg-1 i.v. and 100 µg fentanyl i.v. was administered after which the pig was orotracheally intubated with a commercial singlelumen endotracheal tube (Rüschelit® Super Safety Clear, Teleflex Medical, Co. Westmeath, Ireland) with an ID of 8.0 mm. Pigs were ventilated using a human ventilator (Dräger Primus®, Dräger Medical, Luebeck, Germany) using a volume-controlled mode with tidal volumes of 6-8 mL•kg-1 and positive end–expiratory pressure (PEEP) of 5 cmH2O, resulting in maximum inspiratory pressures (Pmax) of ~ 20 cmH2O, at a respiratory rate of 12-20 breaths per minute. Anesthesia was maintained with continuous infusion of 2% propofol (20 mg•kg-1•h-1), fentanyl (5 µg•kg-1•h-1) and rocuronium (1 mg•kg-1•h-1). A central venous catheter was placed in the external jugular vein under ultrasound guidance, and an arterial line was placed in the left or right common carotid artery. A suprapubic urinary catheter (Zystofix®, B. Braun SE, Melsungen, Germany) was placed for urinary output measurements under ultrasound guidance. After instrumentation and preoxygenation of the pigs, the commercial single lumen tube was replaced by the modified double–lumen tube (DLTm) over a tube exchange catheter (Cook Medical, Limerick, Ireland). The position of the double–lumen tube was bronchoscopically verified (Pentax® FI-13RBS, Pentax Europe GmbH, Hamburg, Germany) in all animals. The position of the tube was adjusted until the tracheal bronchus, the carina and the left and right main stem bronchus were confirmed. After correct positioning of the double–lumen tube, the left and right lung were ventilated separately using two ventilators, one ventilator for ventilation of the right lung and another ventilator for ventilation of the left lung (Fig. 4). For both lungs, we used volume-controlled mode with tidal volumes of 3 mL•kg-1 for the left lung and 5 mL•kg-1 for the right lung. A Positive end–expiratory pressure (PEEP) of 5 cmH2O, resulted in maximum inspiratory pressures (Pmax) of ~ 20-25 cmH2O in the right lung and ~ 15-20 cmH2O in the left lung, at a respiratory rate of 12-20 breaths per minute. Then, animals underwent a median sternotomy, and bilateral pleural opening was performed to have free access to the right and the left lung. Lung separation was followed by temporary closure of the thorax with clamps and hypoxic preconditioning of the left lung. Hypoxic preconditioning was performed by transient termination of ventilation of the left lung while ventilating the right lung with 100% oxygen for 10 minutes. Hypoxic preconditioning was performed to account for the Euler-Liljestrand mechanism(16) and, thus, support hemodynamic stability and to minimize shunt fraction during induction of lung injury. After 10 minutes of hypoxic preconditioning, pigs underwent three cycles of rinsing of the left lung with 0.9% NaCl and Triton® X-100. Animals in the pilot study underwent rinsing in the supine position whereas animals in the main study were positioned in the left lateral decubitus position before cyclic rinsing. The correct tube position was reconfirmed via fiberoptic bronchoscopy in all animals of the pilot study and the main study.
Pilot study
A pilot study with nine pigs was conducted as a first step to achieve strictly unilateral ALI in pigs weighing 49-64 kg. This included the optimal production of the DLTm. We evaluated the optimal position of the tracheal cuff to avoid atelectasis of the upper parts of the right lung. In three pigs, the lung block was harvested with the DLTm tube in place, and the distance between the everted tracheal cuff and the tracheal bronchus was measured. We found that a proximal shift of the tracheal cuff of 4 cm was sufficient to avoid blockage of the tracheal bronchus.
Despite optimal positioning of the tracheal cuff and optimal blockage of the left main bronchus with the DLTm spillover of the rinsing fluid occurred in 6 out of 9 pigs, thus injuring parts of the right lung. After unsuccessful production of unilateral conditions of lung injury in 6 out of 9 pigs we decided to perform the rinsing procedure in the left lateral decubitus position. This step proved to be essential in producing strictly unilateral ALI.
Main study
Six male Austrian Landrace pigs (Sus scrofa domesticus) with an average body weight of 62 kg (range 56 to 76 kg) were used for the experiments. Cyclic rinsing of the left lung was performed in all pigs of the main study in the left lateral decubitus position. Otherwise, the experiments were conducted in a manner comparable to the pilot study.
Induction of unilateral lung injury
Lung injury was induced in all animals of the pilot study and the main study by rinsing the left lung with a mixture of 150 mL 0.9% saline and 0.3% Triton® X-100 (Sigma Aldrich, Saint Louis, MO, USA). Rinsing was performed in the supine position in all animals of the pilot study and in the left lateral decubitus position in all animals of the main study. During the purging of the left lung, continuous bronchoscopic control via the tracheal lumen was used to detect any spill-over of the rinsing solution into the right lung in all animals of the pilot study and the main study. These measures were undertaken to guarantee strict unilateral rinsing. To ensure optimal distribution of the rinsing fluid in the peripheral lung tissue, the left lung was ventilated with increasing Pmax of up to 40 cmH2O for 5 minutes. Thereafter, the remaining rinsing liquid in the left bronchial system was removed via suction. This rinsing procedure was repeated twice; thus, 3 cycles of rinsing were performed in all pigs. A mean arterial pressure > 70 mmHg was set as the target to guarantee hemodynamic stability during the vulnerable rinsing period. Boluses of phenylephrine (Biorphen®, Sintetica, Muenster, Germany) 0.2 mg were administered, if required.
Ventilation
After induction of lung injury, ventilation of the right lung was adjusted to reach the predefined target values for respiratory and metabolic stability, wherein we targeted a PaO2 > 60 mmHg, a PaCO2 < 65 mmHg, and SpO2 > 90%. The left lung was ventilated using lung–protective ventilation with VT of 3 mL•kg-1, PEEP of 8 cmH2O and Pmax of < 30 cmH2O. In cases of desaturations or hypercapnia, ventilatory settings were adjusted. Eight out of nine pigs were ventilated for 7 hours in the pilot study (one animal died prematurely). Two pigs were ventilated for one hour, and four pigs were ventilated for seven hours in the main study.
Lung lavage and tissue sampling
BAL was performed in all pigs (n = 9) of the pilot study and three pigs of the main study for measurement of interleukins (IL-6 and IL-8). All of these animals underwent mechanical ventilation for at least 7 hours. No BAL was performed in animals with ventilation time ≤ 1 h. One animal died prematurely and was therefore excluded from the analysis of IL-6 and IL-8 in BAL. Bronchoalveolar lavage of the left and right lung was carried out at fixed time intervals (at 1, 3, 5, and 6 hours) to measure typical inflammatory parameters, such as interleukin-6 (IL-6) and interleukin-8 (IL–8). BAL in the left and right lung was performed with a saline bronchial flush of 40 mL saline 0.9%, which was sampled again via suction. Samples were stored immediately on ice and were centrifuged at 2000 g at 4°C for 10 minutes, and shock frozen at –80°C within 3–5 minutes thereafter. For analysis, samples were thawed and centrifuged at 1000 g for 20 minutes. Cytokine levels were determined using commercial ELISA kits for IL–6 (MyBioSource, San Diego, CA) and IL–8 (Thermo Fisher Scientific, Waltham, MA).
At the end of the experiment, pigs were sacrificed with an overdose of fentanyl 500 µg i.v. and 30 mL potassium chloride 7.45% i.v.. Thereafter, the lungs were removed en-bloc. Six lung tissue samples were obtained from standardized areas in the left and right lung (Fig. 5), thus, 12 tissue samples were collected from each pig. Lung samples were processed and analyzed by an independent and blinded pathologist to test for histological signs of inflammation and destruction. Then, 7.5% buffered paraformaldehyde was used to fix the lung tissue for at least 12 hours. Fixed lung tissue was then cut to size, placed in tissue capsules, drained overnight, and embedded in paraffin the next morning. Sections of two micrometers thickness were cut and stained with hematoxylin-eosin or with trichrome staining for better visualization of fibrin (pink) and erythrocytes (orange) using acid fuchsin orange G aniline blue (according to Mallory & Cason). The tissue samples were stored in paraformaldehyde at 4°C until evaluated and graded according to the Lung Injury Scoring System (LISS) adapted from Matute-Bello et al., and used previously by other authors for numerical evaluation of lung injury(17).
Statistical Analysis.
For the main study we defined LISS as main outcome parameter, because histopathological changes are rated crucial in defining ALI in the consensus paper of the American Thoracic Society.(17) A LISS of zero is associated with healthy lung structure and a LISS of one is associated with completely destroyed lung structure. The precondition of strictly unilateral ALI is a substantial difference in LISS between the right and the left lung. Therefore, we demanded a mean difference in LISS between the right and left lung of 0.5 and assumed a rather large standard deviation in differences of 0.2. With alpha of 0.05 a power of 0.9 can be achieved with six pigs in paired samples. Thus, a sample size of six pigs was chosen for the main study.
Because of the small sample size Wilcoxon-signed-rank-test was used for comparison of outcome parameters throughout. Descriptive statistics were expressed as mean ± SD, the significance level was set at p ≤ 0.05. IBM SPSS Statistics software (Version 27.0.1.0) was used for data processing.