The study was designed as a single-blinded, two-group, preclinical study using an established animal model of OLV.12
Juvenile pigs were chosen because of their comparable size to pediatric patients and because of their previous application in pediatric pulmonary research.13,14
The methodology was designed to closely mimic the clinical scenario typical for OLV procedures such that the study would have more clinical relevance. This study was approved by the Institutional Animal Care and Use Committee, Department of Biomedical Research, Nemours, in accordance with the National Institutes of Health guidelines.
We studied 20 juvenile Landrace-Yorkshire pigs that were approximately 4–5 weeks old. We chose this sample size to achieve sufficient statistical power while minimizing the number of animals sacrificed and to remain consistent with our previous studies on this model. The pigs were randomly assigned to two groups: the hyperoxic ventilation group (n = 10) and the normoxic ventilation group (n = 10). An anesthetic mixture (ketamine: 23 mg/ml, azepromazine: 0.1 mg/ml, and xylazine: 0.05 mg/ml) was administered via two injections (1 ml/kg) at a 10-min interval. The animals were subsequently placed on a radiant warmer bed (Resuscitaire, Hill-Rom Air-Shields, Hatboro, PA) to maintain a rectal temperature of 36–39°C. Following initial anesthesia, access was secured to the internal jugular vein and carotid artery using 8-Fr umbilical catheters, and the trachea was intubated with a 6.0-mm, uncuffed endotracheal tube. The leak around the endotracheal tube was maintained <20–25 cm by inserting tonsil packs made of gauze around the tube. Both groups were mechanically ventilated with an 8–10 ml/kg tidal volume with no positive-end expiratory pressure applied (Ohmeda, Modulus II Plus™; GE Healthcare, Waukesha, WI). The normoxia group was ventilated with FiO2 at or below 50% for the entire experiment, and the hyperoxia group was ventilated with 100% FiO2 during the entire experiment. In both groups, the oxygen saturation (SaO2) was maintained ≥96% and the end-tidal carbon dioxide (ETCO2) was kept between 45 and 55 mmHg by adjusting, in some cases, the ventilatory rate, and inspiratory/expiratory ratio.
In both groups, following intubation and mechanical ventilation initiation, a 30-min stabilization period commenced during which the ventilatory parameters and anesthetic mixture were titrated to maintain the parameters mentioned above. Following the stabilization period, the left primary bronchus was blocked using a fiberoptic bronchoscope and a 5-Fr Arndt endobronchial blocker (Cook Medical, Bloomington, IN). The piglets were turned to the right-lateral position to simulate surgical positioning during OLV. Following local anesthesia with 0.5 ml of 1% lidocaine, a 5-mm trocar was placed through the left thoracic wall between the seventh and eighth ribs to simulate thoracoscopic instrumentation. The investigator observed the collapsed lung through the trocar using a Stryker endoscopy system (San Jose, CA). Breath sounds and the position of the bronchial blocker were checked half-hourly throughout the entire OLV period.
Throughout the duration of the experiment, vital signs were monitored using a standard pediatric anesthesia monitoring system (Model M1175A; Hewlett Packard, Palo Alto, CA), and arterial blood gas was measured half-hourly (Stat Profile pHOx arterial blood gas/critical care analyzer; Nova Biomedical, Waltham, MA). Pulmonary mechanics were measured with a noninvasive cardiac output monitor (NICO; Respironics Novametrix LLC, Wallingford, CT). Based on lung function evaluations in humans and animals, lung compliance, and lung volume are dependent upon weight,15,16
so total respiratory compliance was normalized to weight for analysis.
Anesthesia was maintained with intravenous sufentanil infusion at 0.2–0.3 μg/kg/hr and 1% inhaled isoflurane. Intravenous pancuronium (0.2 mg/kg) was administered half-hourly to maintain muscle relaxation. The depth of anesthesia was monitored using changes in vital signs as the primary criteria.
Blood samples were drawn half-hourly in sodium citrate (10% by blood volume), and after centrifugation, plasma was obtained for subsequent analysis of inflammatory mediators. These samples were drawn after a 30-min stabilization period (baseline), half-hourly during the OLV period (OLV), and again 30 min after re-expansion of the collapsed lung (endpoint).
Immediately before the piglets were killed, their anesthesia was deepened with boluses of sufentanil and ketamine, and isoflurane was increased to 4%. A modified Millonig’s buffer solution (0.11 M NaOH, 0.12 M NaH2PO4·H2O, 0.01 M glucose, 100 U/L heparin, pH 7.45) was introduced to the pulmonary artery to flush blood from the vasculature of the lungs before they were harvested. Base nondependent lung-tissue samples (approximately 5 cm3) were immediately collected from both groups and snap-frozen in liquid nitrogen. The samples were stored at −70°C until assaying for cytokines and oxidative injury markers. Lung-tissue homogenates were prepared as described below, and total protein concentrations of lung-tissue homogenates were determined using a bicinchoninic acid protein assay (BCA Protein Assay Kit; Thermo Scientific, Rockford, IL).
Myeloperoxidase (MPO) in lung tissue was assayed, as modified from the procedure developed by Goldblum et al.17
Snap-frozen lung tissue was homogenized on ice in 300 μl of 50 mM potassium phosphate, pH 6.0 (phosphate buffer). The total volume was adjusted to 1 ml. Following centrifugation at 10,000 rpm for 15 min, the pellet was resuspended in 300 μl hexadecyltrimethylammonium bromide buffer (50 mM potassium phosphate, pH 6.0; 50 mM hexadecyltrimethylammonium bromide) and re-homogenized for 30 sec. Next, 700 μl of phosphate buffer was added to the homogenate, and the mixture was homogenized for 20 sec using an ultrasonic homogenizer (BioLogics, Manassas, VA) before being snap-frozen in liquid nitrogen. The frozen samples were thawed at room temperature and homogenizing, snap freezing, and thawing were repeated twice more. After the third thaw, the samples were centrifuged at 10,000 rpm for 10 min to obtain a supernatant for measurement of MPO activity.
Myeloperoxidase in supernatants was measured using a standard spectrophotometric assay.17
One hundred microliter of the lung-tissue homogenate supernatant was mixed with 2.9 ml of phosphate buffer containing 0.167 mg/ml O
-dianisidine dihydrochloride (Sigma–Aldrich, St. Louis, MO) and 0.0005% hydrogen peroxide (Sigma–Aldrich). The optical density at 460 nm was read at 15-sec intervals for 3 min using a kinetic program on a commercial spectrophotometer (SmartSpec Plus; BioRad Laboratories, Hercules, CA). The change in A460
× g total protein−1
Superoxide Dismutase Assay
Frozen base nondependent lung tissue was homogenized on ice in 5 ml of cold lysis buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 0.5% Triton-100) per gram of tissue. The homogenate was centrifuged at 12,000 rpm for 10 min and the supernatant was collected for measurement of superoxide dismutase (SOD). The SOD activity was measured using a commercially available assay kit (Oxiselect™ Superoxide Dismutase Activity Assay; Cell BioLabs, San Diego, CA) according to manufacturer’s instructions.
Protein Carbonyl Assay
Protein carbonyls (PC) were detected and quantitated in lung homogenates using an enzyme linked immunoassay (ELISA) kit (Oxiselect™ Protein Carbonyl ELISA Kit; Cell BioLabs). Linear standard curves were optimized by diluting reduced or oxidized bovine serum albumin with sensitivities ranging from 0.00 to 7.5 nmol/mg; inter-assay and intra-assay coefficients of variance were <10% and <6%, respectively. The bovine serum albumin standards and protein samples (10 μg/ml) were derivatized with dinitrophenyl hydrazone and probed with an anti-dinitrophenyl antibody followed by a horseradish peroxidase-conjugated secondary antibody. The plate was read at 450 nm in an automated plate reader. All standards and samples were run in duplicate, and data are expressed as nmol/mg.
Snap-frozen lung-tissue samples were homogenized on ice in 300 μl phosphate-buffered saline (PBS), pH 7.4, then centrifuged for 1 min at 13,000 rpm. The supernatant was then collected in a clean tube. The pellet was rinsed in another 300 μl PBS and centrifuged as before. The supernatant was combined with the first, and the volume was adjusted to 1 ml with PBS.
The levels of tumor necrosis factor-alpha (TNF-α), IL-1β, IL-6, and IL-8 in lung homogenates and in plasma samples collected during the experiment were measured with quantitative ELISA using porcine-specific Quantikine ELISA kits (R&D Systems, Minneapolis, MN). Lung homogenates and plasma samples were appropriately diluted to fall within the detection range of each assay, and all standards and samples were assayed in duplicate. The test sensitivities for respective immunoassays were as follows: TNF-α, 3.7 pg/ml; IL-1β, ≤10 pg/ml; IL-6, 10 pg/ml; and IL-8, 0.039 pg/ml. Inter-assay and intra-assay coefficients of variance were <10%.
Preparation and Analysis of Histological Samples
Formalin-fixed lung tissue was processed and paraffin-embedded, and 5-μM sections were cut for slide preparation. Tissue was stained with hematoxylin and eosin according to CLIA-approved protocols and visualized at 10× magnification using a Nikon Eclipse 80i light microscope (Nikon, Tokyo, Japan) equipped with a digital camera (Digital Sight DS-SM; Nikon). Random fields from each slide were digitally imaged for qualitative analysis (ACT-2U; Nikon).
Confounding variables were analyzed for equality of groups by t test for age and weight. Plasma cytokines and physiological and hemodynamic parameters were analyzed by repeated measures ANOVA. Two-factor ANOVA was used to analyze biomarkers in the lung-tissue homogenates following log-transformation of the data. The software used for statistical analysis was SPSS 17.0 for Windows (SPSS, Chicago, IL). Probability values <0.05 were considered significant.