Animal preparation and experimental protocol
This study was approved by the Research Ethics Committee of the Federal University of Rio de Janeiro Health Sciences Center (CEUA 019). All animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the U.S. National Academy of Sciences.
Guide for the care and use of laboratory animals
Thirty-six male Wistar rats (weighing 256 ± 15 g) were kept in controlled-temperature conditions (23 °C) and maintained on a 12:12 h light–dark cycle with free access to water and food. Animals were randomly divided into two groups. In the emphysema group, rats were administered porcine pancreatic elastase (2 IU suspended in saline solution to a total volume of 100 μl, Sigma Chemical Co., St. Louis, MO, USA) intratracheally (it), once weekly for 4 weeks, whereas control animals received saline solution alone (100 μl) under the same protocol. Before each intratracheal instillation, rats were premedicated with intraperitoneal (ip) diazepam (10 mg/kg, Compaz®, Cristália, Itapira, SP, Brazil) and anesthetized with 1.5–2.0 % isoflurane (Cristália, Itapira, SP, Brazil) by mask.
Eight weeks after the first instillation (Fig. 1), rats (weighing 398 ± 23 g) were sedated with diazepam (10 mg/kg ip, Compaz®, Cristália, Itapira, SP, Brazil) and anesthetized with ketamine (100 mg/kg ip, Ketamin-S+®, Cristália, Itapira, SP, Brazil) and midazolam (2 mg/kg ip, Dormicum, União Química, São Paulo, SP, Brazil). The tail vein was cannulated (Jelco 24G, BD, New Jersey, USA) for continuous infusion of 50 mg/kg/h ketamine, 2 mg/kg/h midazolam, and 7 ml/kg/h Ringer’s lactate (B. Braun, Rio de Janeiro, Brazil) during mechanical ventilation. The adequacy of anesthesia was assessed by the response to a nociceptive stimulus before surgery.
Anesthetized animals were placed in the dorsal recumbent position and tracheotomized via a midline neck incision after subcutaneous injection of lidocaine (Xylestesin® 2 %, Cristália, Itapira, SP, Brazil). The right internal carotid artery was cannulated (18G, Arrow International, USA) for blood sampling and mean arterial pressure (MAP) measurement.
Heart rate (HR), MAP, and rectal temperature were continuously recorded (Networked Multiparameter Veterinary Monitor LifeWindow 6000V, Digicare Animal Health, Florida, USA). Body temperature was maintained at 37.5 ± 1 °C using a heating bed. Gelafundin® (B. Braun, São Gonçalo, RJ, Brazil) was administered intravenously in 0.5-ml increments to keep MAP ≥ 70 mmHg.
Once animals were hemodynamically stable, they were mechanically ventilated (Servo-i, MAQUET, Solna, Sweden) in PSV mode for 5 min, with a tidal volume (V
T) of 6 ml/kg, zero end-expiratory pressure (ZEEP), and fraction of inspired oxygen (FiO2) set to 0.4, to evaluate whether the degree of lung damage was similar in emphysema groups. Arterial blood (300 μl) was drawn into a heparinized syringe for the measurement of arterial oxygen partial pressure (PaO2), arterial carbon dioxide partial pressure (PaCO2), arterial pH (pHa), and bicarbonate (Radiometer ABL80 FLEX, Copenhagen NV, Denmark). At this time (baseline), data on MAP, rectal temperature, and respiratory parameters were collected for functional data analysis (FDA). Following this step, control and emphysema animals were randomly assigned by the sealed-envelope method to receive mechanical ventilation in PCV or PSV mode (n = 6/each). During PCV, animals were paralyzed by intravenous administration of pancuronium bromide (2 mg/kg, Cristália, Itapira, SP, Brazil). In PCV and PSV, driving pressure was adjusted to achieve a V
T of 6 ml/kg, and positive end-expiratory pressure (PEEP) was set at 3 cmH2O and FiO2 = 0.4. In addition, in PCV, the respiratory rate (RR) was controlled to keep minute ventilation constant at 160 ml/min. Six animals from each group (control and emphysema) were not ventilated (NV) and used as controls for experimental emphysema characterization and molecular biology analysis. Blood gases and respiratory parameters were analyzed immediately after randomization (time point 0, T0) and after 2 h (T2) and 4 h (T4) of mechanical ventilation, whereas echocardiography was performed at T0 and T4. At the end of the experiment, heparin (1000 IU) was injected into the tail vein, a laparotomy was performed, and animals were killed by intravenous injection of sodium thiopental (50 mg/kg, Cristália, Itapira, SP, Brazil). The left and right lungs were extracted at an airway pressure equivalent to PEEP for histological and molecular biology analysis, respectively. The lungs and diaphragm of NV animals were extracted for lung histology and molecular biology analysis. Schematic flowcharts of study design and a timeline representation of the protocol are shown in Fig. 1.
Echocardiography
Animals were placed in the dorsal recumbent position and the precordial region was shaved. Transthoracic echocardiography was performed by an expert (IPR) blinded to group allocation, using a 7.5-MHz probe (Esaote model, CarisPlus, Firenze, Italy). Images were obtained from the parasternal views. The left ventricular ejection fraction and fractional shortening were calculated in one-dimensional mode analysis of the left ventricle guided by the parasternal short-axis view. Pulsed-wave Doppler was used to measure pulmonary artery acceleration time (PAT) and pulmonary artery ejection time (PET), and the PAT/PET ratio was used as an indirect index of pulmonary arterial hypertension. Measurements were obtained in accordance with American Society of Echocardiography Guidelines [12, 13].
Respiratory data acquisition and processing
A pneumotachograph (internal diameter = 1.5 mm, length = 4.2 cm, distance between side ports = 2.1 cm) was connected to the tracheal cannula for airflow (V’) measurements. The pressure gradient across the pneumotachograph was determined using a SCIREQ differential pressure transducer (UT-PDP-300, SCIREQ, Montreal, Canada). V
T was calculated by digital integration of the flow signal. Tracheal pressure (Paw) was measured with a SCIREQ differential pressure transducer (UT-PDP-75, SCIREQ, Montreal, QC, Canada). Changes in esophageal pressure (Pes), which reflect chest wall pressure, were measured with a 30-cm-long water-filled catheter (PE205) with side holes at the tip connected to a differential pressure transducer (UT-PL-400, SCIREQ, Montreal, Canada). The catheter was passed into the stomach and then slowly returned into the esophagus; its proper positioning was assessed using the “occlusion test” [14]. Transpulmonary pressure (P,L) was calculated during inspiration and expiration as the difference between tracheal and esophageal pressures. Mean (Pmean,L) and peak transpulmonary pressures (Ppeak,L) were calculated. The respiratory rate (RR) was calculated from Pes swings as the frequency per minute of each type of breathing cycle. The ratio between inspiratory and total time (Ti/Ttot) was calculated, as well as the coefficients of variation of V
T, RR, and Ti/Ttot. Moreover, the esophageal pressure generated 100 ms after onset of inspiratory effort (P
0.1) and the pressure–time product (PTP) per minute (PTP/min) (integral of ΔPes over time) were calculated.
Airflow and tracheal and esophageal pressures were continuously recorded throughout the experiments with a computer running software written in LabVIEW® (National Instruments; Austin, Texas, USA) (Additional file 1: Figure S1). All signals were filtered (200 Hz), amplified by a 4-channel conditioner (SC-24, SCIREQ, Montreal, Quebec, Canada), and sampled at 200 Hz with a 12-bit analogue-to-digital converter (National Instruments; Austin, Texas, USA). All mechanical data were computed offline by a routine written in MATLAB (Version R2007a; The Mathworks Inc, Natik, Massachusetts, USA).
Lung histology
Morphometric analysis was performed in excised lungs at end-expiration with a PEEP of 3 cmH2O. Immediately after excision, the left lung was flash-frozen by immersion in liquid nitrogen, fixed with Carnoy’s solution, and embedded in paraffin. Slices (4 μm thick) were mounted and stained with hematoxylin–eosin. Morphometric analysis was done by viewing through an integrating eyepiece with a coherent system made of a 100-point grid consisting of 50 lines of known length, coupled to a conventional light microscope (Axioplan, Zeiss, Oberkochen, Germany). The volume fraction of collapsed pulmonary areas and the fraction of the lung occupied by large-volume gas-exchanging air spaces (hyperinflated structures with a morphology distinct from that of alveoli and wider than 120 μm) were determined by the point-counting technique, at a magnification of ×200, across ten random, non-coincident microscopic fields [15]. Briefly, points falling on collapsed pulmonary or hyperinflated areas were counted and divided by the total number of points in each microscopic field. Lung tissue distortion was assessed by measuring the mean linear intercept between alveolar walls (Lm) at a magnification of ×400 [16]. Lm is an estimate of the average difference between gas exchange surfaces. The investigators (LFHB, CLB) were unaware of the origin of the examined material.
Transmission electron microscopy of diaphragm tissue
Three slices (each 2 × 2 × 2 mm) were cut from three different segments of the diaphragm and fixed in 2.5 % glutaraldehyde and phosphate buffer 0.1 M (pH = 7.4) for electron microscopy analysis (JEOL 1010 Transmission Electron Microscope; Japan Electron Optics Laboratory Co, Tokyo, Japan). The following parameters were observed qualitatively: (1) fibrillar disarrangement, (2) thickened Z-line, (3) smooth cell proliferation, (4) abnormal mitochondria, and (5) enlarged endoplasmic reticulum [17]. To assess pathological findings, a five-point, semi-quantitative, severity-based scoring system was used as follows: 0 = normal diaphragm, 1 = changes in 1 to 25 % of examined tissue, 2 = changes in 26 to 50 % of examined tissue, 3 = changes in 51 to 75 % of examined tissue, and 4 = changes in 76 to 100 % of examined tissue. Scores were calculated as the product of severity and extent of each feature, ranging from 0 to 16. This analysis was performed by a pathologist (VLC) blinded to group allocation.
Molecular biology analysis of lung and diaphragm tissue
Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to measure biological markers associated with cell mechanical stress (amphiregulin), inflammation (cytokine-induced neutrophil chemoattractant (CINC-1)), epithelial cell mechanotransduction (surfactant protein (SP)-D), endothelial cell damage (vascular endothelial growth factor (VEGF), vascular cell adhesion molecule (VCAM)-1, and angiopoietin (ANG)-2), extracellular matrix organization (lysyl oxidase-like (LOXL)1), and fibrogenesis (type III procollagen (PCIII)) in the lung, as well as markers of muscle proteolysis (muscle atrophy F-box (MAFbx) and muscle RING finger (MuRF)-1). Central slices were cut from the right lung and diaphragm, collected in cryotubes, flash-frozen in liquid nitrogen, and stored at −80 °C. Total RNA was extracted from frozen tissues using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) for the lungs and RNeasy Fibrous tissue Mini Kit (Qiagen, Hilden, Germany) for the diaphragm, following the manufacturer’s recommendations. The RNA concentration was measured by spectrophotometry in a Nanodrop ND-1000 system. First-strand cDNA was synthesized from total RNA using a Quantitec reverse transcription kit (Qiagen, Hilden, Germany). The primers used are described in the Supplementary Material (Additional file 2: Table S1). Relative messenger RNA (mRNA) levels were measured with a SYBR green detection system using real-time PCR (ABI 7500; Applied Biosystems, Foster City, CA, USA). For each sample measured in triplicate, the gene expression was normalized to that of a housekeeping gene (acidic ribosomal phosphoprotein P0, 36B4) [18] and expressed as fold change relative to non-ventilated control and emphysema animals, using the 2−ΔΔCt method, where ΔCt = Ct (reference gene) minus Ct (target gene). This is a suitable method to analyze relative changes in gene expression from real-time quantitative PCR experiments [19].
Statistical analysis
The number of animals per group was based on a previous study [17]. A sample size of six animals per group (providing for one animal as dropout) would provide the appropriate power (1-β = 0.8) to identify significant (α = 0.05) differences in mean transpulmonary pressure obtained after PSV and PCV, taking into account an effect size d = 1.6, a two-sided test, and a sample size ratio = 1 (G*Power 3.1.9.2, University of Düsseldorf, Germany).
Data were tested for normality using the Kolmogorov-Smirnov test with Lilliefors’ correction, while the Levene median test was used to evaluate the homogeneity of variances. If both conditions were satisfied, two-way ANOVA followed by Tukey’s test was used. To compare the time course of respiratory parameters and arterial blood gases, one-way repeated-measures ANOVA followed by Bonferroni’s test was used. A t test with Bonferroni correction was used to compare PCV and PSV in control and emphysema groups. For nonparametric data, the Kruskal-Wallis test followed by Dunn’s post hoc test was used. Parametric data were expressed as mean ± standard deviation (SD), and nonparametric data, as median (interquartile range). Spearman’s correlations were used. All tests were performed using the GraphPad Prism v6.01 statistical software package (GraphPad Software, La Jolla, California, USA). Significance was accepted at p < 0.05.
