Subjects
All the experiments involving animals were approved by University of Nebraska Lincoln (UNL) Institutional Animal Care and Use Committee (IACUC) (protocol # 1674).
Female pigs (~ 50 kg, n = 21) were housed in Animal Science Complex, UNL in experimental pens and cared for according to USDA (United States Department of Agriculture) guidelines. Animals were acclimated to the facility for 4–7 days and received food reward training to ease handling and blood draws. Six animals died during the experiments due to either anesthesia/surgical complications or smoke inhalation related complication. We continued our next phase of study (involving efficacy of a novel therapeutic for hypoxia) in some of these animals to reduce cost and number of animals used.
Smoke Delivery System
Upon completion of all surgical procedures, animals were exposed to oak wood smoke from a custom-made smoke generator connected in parallel to the endotracheal tube. Schematic is shown in Fig. 1.
Surgical Procedures
Sedation for peripheral intravenous catheter (PIV) placement and endotracheal intubation was achieved with a mixture of telazol (4.4 mg/kg), ketamine (2.2 mg/kg) and xylazine (2.2 mg/kg) delivered via intramuscular injection. To assist with intubation, an intravenous bolus dose of fentanyl (0.05 mg/kg) and/or propofol (2-4.4 mg/kg) was given as needed. Baseline chest x-rays were obtained (portable x-ray unit EPX-F2800, Ecotron Co. Ltc; wireless digital flat panel detector Mars1417V-TSI, iRay Technology, Shanghai, China) prior to smoke inhalation, and at 24 and 48 hours after smoke inhalation. An endotracheal tube (#7–8 cuffed; MWI Animal Health, Boise, ID, USA) was inserted into the trachea and animals were ventilated at a tidal volume (TV) of 6 mL/kg and peak end expiratory pressure (PEEP) of 5 cmH2O (Newport HT70, Medtronic, Minneapolis, MN). Respiratory rate (RR) was adjusted to maintain eucapnia as monitored by end-tidal CO2 (ETCO2). The fraction of inspired oxygen (FiO2) was set at 50% during surgical procedures (central venous & arterial catheter placement), then titrated down to 21% and maintained throughout the experiment. Non-invasive monitoring included blood pressure taken by cuff placed around the animal's hind leg, peripheral oxygen saturation (SpO2), heart rate (HR) and ETCO2 recorded via the Surgivet monitor (Smiths Medical, Dublin, OH). Continuous IV sedation containing midazolam (0.4–0.7 mg/kg/h), fentanyl (0.03–0.1 mg/kg/h) and propofol (0.2–0.4 mg/kg/min) and maintenance IV fluids (10 mL/kg/h normal saline) were given throughout the procedure via a quadruple-lumen central venous catheter (8.5Fr x 16 cm, Arrow International) placed in the internal jugular vein. Core temperature was monitored by rectal probe and a circulating warming blanket was used to prevent body cooling. A urinary catheter was placed to monitor output.
Using sterile technique and ultrasound guidance (Butterfly iQ, Butterfly Network, New York City, NY), carotid artery (CA) and femoral artery (FA) access catheters were placed for serial lab draws and invasive blood pressure monitoring (18 GA 16 cm; Femoral Arterial Line Catherization Kit; Teleflex, Morrisville, NC). Pulmonary artery (PA) catheter (8F x 110 cm Swan-Ganz CCOmbo Thermodilution Catheter; Edwards Lifesciences, Irvine, CA) was placed in the internal jugular vein under ultrasound guidance. The CA and PA access ports were connected to Surgivet monitor and Vigilance II monitor, respectively (Edwards Lifesciences, Irvine, CA) with transducers (Meritans DTXPlus, Disposable Pressure Transducer with EasyVent; Merit Medical, South Jordan, UT, USA). Invasive arterial blood pressure, central venous pressure (CVP), pulmonary artery pressure (PAP), cardiac output (CO), mixed venous oxygen saturation (SmvO2), and central (core) temperature were monitored throughout the study. Blood samples were drawn from the CA, FA and PA catheters for the measurement of baseline blood gas prior to smoke inhalation and at pre-determined time intervals throughout the study period (ABL80 FLEX CO-OX, Radiometer, Brea, CA). To maintain patency, catheters were flushed throughout the experiment with 3–5 mL of sterile saline, and a heparin solution (1:500 dilution in 50% dextrose solution) was infused to fill the volume of the port chosen as a "lock" solution. Sedated/anesthetized animals from survival surgeries were continuously monitored until sternal recumbence was regained. All catheters were removed after smoke inhalation was completed. The surgical procedures were repeated at 48 h after smoke inhalation.
Smoke Inhalation
Upon completion of surgical procedures, animals in the SI group were exposed to oak wood smoke from the custom-made smoke chamber (Fig. 2) through the endotracheal tube. The duration of the smoke exposure was 1 to 2 hours starting from 0h time point. The volume of smoke inhaled was approximately 500 liters per hour. Invasive and noninvasive parameters were monitored continuously during the experiment. Following smoke exposure, blood samples were collected from arterial ports and PAC. Animal was continuously monitored until recovery from general anesthesia. Smoke exposure was stopped immediately if the animal developed hemodynamic instability, which was determined by hypotension (systolic blood pressure less than 60) and irreversible desaturation (SpO2 less than 70% despite rescue maneuvers such as increase in inspired percentage of oxygen).
Ventilator Parameters
On the day of the smoke inhalation, the ventilator parameters were maintained at values: tidal volume, 6 mL/kg = 270-360L/min; respiratory rate, 18–30/min; PEEP 5mmHg; and FiO2, 21–34% (Table 2). During the 48h after smoke inhalation surgery, animals were extubated and maintained on room air.
Table 2
Parameters | Baseline | 2h | 24h | 48h |
---|
TV (mL/kg) | 270–360 | 270–360 | -- | 220–280 |
RR (bpm) | 18–30 | 18–30 | -- | 16–24 |
FiO2(%) | 21–34 | 21–34 | 21 | 21 |
PEEP (cmH2O) | 5 | 5 | --- | 0 |
TV, tidal volume; RR, respiratory rate; bpm, beats per minute; FiO2, fraction of inspired oxygen; PEEP, positive end expiratory pressure; n = 13. |
Post-Surgical Animal Monitoring and Care
After recovery from surgical anesthesia, all animals were transferred to the post-surgery recovery pen and were monitored 24h/day by trained personnel. Chest x-rays were taken and blood samples drawn for ABG analysis at 24h and 48h following final smoke exposure. Animals (n = 7) were humanely euthanized at 48h post SI and a full necropsy was performed; the remainder of animals were utilized to continue our lab’s therapeutic investigations.
Plasma Sample Extraction
Blood samples were collected from the CA catheter at baseline, 2h, and 48h time points in lithium heparin BD Microtainer tubes (Becton, Dickinson and Company, Franklin Lakes, NJ). Tubes were immediately inverted 8–10 times to assure anticoagulation and centrifuged at 8000 g for 4 minutes. Supernatants were collected as plasma samples and stored at -20 ºC until analysis. IL-6 immune assay was performed in samples of 10 animals using IL-6 Quantikine® ELISA kit, catalog No#P6000B (R&D Systems, Inc., Minneapolis, MN) on plasma samples following manufacturer’s protocol.
Bronchoalveolar Lavage (BAL)
BAL of pig lungs was performed at baseline, 2h, and 48h time points using a bronchoscope in a set of 6 intubated animals. 10 ml of sterile normal saline was instilled to the secondary and tertiary bronchi through the bronchoscope and ~ 5ml of the fluid was collected for analysis. BAL fluid samples were centrifuged immediately at 4000 g at 4°C for 10 minutes and supernatants were at stored at -20 ºC until analysis. Total protein quantification was performed in samples using Pierce™ BCA (Bicinchoninic Acid) Protein Assay Kit (Thermo Fisher Scientific Inc. Waltham, MA) following manufacturer’s protocol. IL-6 immune assay was performed using IL-6 Quantikine® ELISA (Enzyme-linked immunosorbent assay) kit, catalog No#P6000B (R&D Systems, Inc., Minneapolis, MN) on BAL fluid samples following manufacturer’s protocol.
Tissue Collection
Necropsy was performed in 7 animals. At necropsy, lung tissues were collected from all five lobes; upper, middle and lower lobes of right lung and upper and lower lobes of left lung for histological examination and pulmonary edema assessment. Tissues for histology were immediately placed in 10% neutral buffer formalin fixative for approximately 24 hours. Formalin fixed tissues were placed into 70% ethanol and transferred to the University of Nebraska Medical Center (UNMC) Tissue Science Facility (TSF) for further tissue processing and embedment in paraffin blocks.
Lung Injury Score
The lung tissue in 10% neutral formalin was dehydrated in graded concentrations of ethanol solution and cleared in xylene. The tissue samples were then paraffin-embedded, sectioned with 4-µm thickness, and stained with hematoxylin and eosin at the UNMC Tissue Sciences Facility using automated Ventana Discovery Ultra (Roche Diagnostics, Indianapolis, IN) as per manufacturer’s protocol. An independent pathologist performed a blinded examination of the tissues under light microscope. Ten fields of each lung tissue section were examined at magnification x400. The severity of the lung injury was scored by the criteria of alveolar edema, intra-alveolar hemorrhage, and leukocyte infiltration. Alveolar edema and intra-alveolar hemorrhage were scored on a scale from 0 to 3; where 0 = < 5% of maximum pathology, 1 = mild (< 10%), 2 = moderate (10–20%), and 3 = severe (20–30%). Leukocyte infiltration was also scored on a scale from 0 to 3; where 0 = absence of extravascular leukocytes, 1 = < 10, 2 = 10–45, and 3 = > 45 leukocytes.
Wet-to-dry weight (W/D) ratio
Lung tissues (n = 6) were dried in an incubator at 60°C for 5 days and weighed again (dry weight). The W/D ratio was calculated as the ratio of the wet weight to the final dry weight as described elsewhere [23].
Ki67 Immunohistochemistry
Immunostaining for Ki67 was performed on pig SI and control formalin-fixed, paraffin‐embedded lung tissues sections at UNMC Tissue Sciences Facility using automated Ventana Discovery Ultra (Roche Diagnostics, Indianapolis, IN) in lung tissue samples of 4 animals as per manufacturer’s protocol. Specimens were processed on the same day to eliminate any variability in conditions. An independent pathologist performed a double-blinded examination of the tissue slides under a light microscope. A total of 2000 cells were counted at magnification of X400 and the percentage of Ki67 positive cells were calculated.
Lung Tissue Lysate Preparation
Fresh frozen lung lobe tissues with highest injury score (n = 5) were homogenized using VWR® Mini Bead Mill Homogenizer (VWR International LLC., Radnor, PA) following manufacturer’s protocol. Briefly, frozen tissues of two control and three SI animals were washed in cold X1 PBS, and 30 mg of each tissue was placed separately in a 2 mL tube containing 2.8 mm ceramic beads and 750 µl of lysis buffer containing RIPA buffer (Thermo Fisher Scientific Inc. Waltham, MA) and protease inhibitor cocktail (Sigma Aldrich Inc., St. Louis, MO) at room temperature. The samples were homogenized at speed 4 for 60 seconds. This was followed by incubation in ice for 30 minutes and centrifugation at 13,000 rpm for 20 minutes at 4 ºC. Protein concentration was determined using Pierce™ BCA (Bicinchoninic Acid) Protein Assay Kit (Thermo Fisher Scientific Inc. Waltham, MA) following manufacturer’s protocol.
Immunoblot Analysis
Protein (50 µg) was separated by SDS- polyacrylamide gel electrophoresis and transferred onto PVDF (polyvinylidene fluoride) membrane (Bio-Rad Lab Inc., Hercules, CA) by electro blotting. The membrane was blocked with 5% nonfat dry milk in X1 TBST (50mM Tris, pH 7.5, 150mM NaCl, 0.01% Tween 20) for 1 hour at room temperature. The membrane was then incubated in primary antibody, IL-6 antibody (#ab6672, Abcam Inc, Cambridge, MA) or β-actin (#4970, Cell Signaling Technology Inc., Danvers, MA) at 1:1000 dilution in X1 TBST with 5% bovine serum albumin (Sigma Aldrich Inc., St. Louis, MO) overnight at 4°C. The membrane was washed three times with X1 TBST for 10 minutes each and incubated with HRP‐conjugated secondary antibody (#7074, Cell Signaling Technology Inc., Danvers, MA) at 1:5000 dilution in X1 TBST with 5% nonfat dry milk for 1 hour at room temperature. Following washes in X1 TBST, proteins were detected using the enhanced chemiluminescence system (Bio-Rad Lab Inc, Hercules, CA).
Statistical Analysis
Statistical analysis was performed using GraphPad prism 8. One-way ANOVA with Tukey’s post-hoc analysis and paired t test were utilized to generate adjusted “p” values. P-values < 0.05 were considered statistically significant.