This study was approved by the Animal Care and Use Committee (CEUA: 122/18) of the Health Sciences Center, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil. 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. Animals were housed at a controlled temperature (23°C) and controlled light–dark cycle (12–12 h), with free access to water and food.
Animal Preparation and Experimental Protocol
Thirty-five male Wistar rats (age 8-10 weeks, body weight 291±75 g) were used. Rats were anesthetized by inhalation of sevoflurane 1.0% (Sevorane®; Cristália, Itapira, SP, Brazil) and received Escherichia coli lipopolysaccharide (LPS: 9.6×106 EU/mL in 200 μL of saline solution) intratracheally (i.t.) to induce experimental acute respiratory distress syndrome  Twenty-four hours after ARDS induction, animals were premedicated intraperitoneally (i.p.) with 10 mg/kg diazepam (Compaz®, Cristália, Itapira, SP, Brazil), followed by 100 mg/kg ketamine (Ketamin-S®, Cristália, Itapira, SP, Brazil) and 2 mg/kg midazolam (Dormicum®, União Química, São Paulo, SP, Brazil). After local anesthesia with 2% lidocaine (0.4 mL), a midline neck incision and tracheostomy were performed. An intravenous (i.v.) catheter (Jelco 24G, Becton, Dickinson and Company, New Jersey, NJ, USA) was inserted into the tail vein, and anesthesia induced and maintained with midazolam (2 mg/kg/h) and ketamine (50 mg/kg/h). A second catheter (18G, Arrow International, USA) was then placed in the right internal carotid artery for blood sampling and gas analysis (Radiometer ABL80 FLEX, Copenhagen NV, Denmark), as well as monitoring of mean arterial pressure (MAP) (Networked Multiparameter Veterinary Monitor LifeWindow 6000V; Digicare Animal Health, Boynton Beach, FL, USA). Heart rate (HR), MAP, and rectal temperature were continuously monitored (Networked Multiparameter Veterinary Monitor LifeWindow 6000V, Digicare Animal Health, Florida, USA). Body temperature was maintained at 37.5±1°C using a heating bed. Animals in dorsal recumbency were paralyzed with pancuronium bromide (2 mg/kg, i.v.) and their lungs mechanically ventilated (Servo-i, MAQUET, Solna, Sweden) in volume-controlled mode (VCV) with constant inspiratory airflow, VT = 6 mL/kg, respiratory rate (RR) to maintain V′E = 160 mL/min, zero end-expiratory pressure (ZEEP), FiO2 = 0.4, and an inspiratory-expiratory ratio of 1:2 (BASELINE). Arterial blood gases and echocardiography were evaluated. PEEP was then increased to 9 cmH2O and, at the same time, animals were randomized to receive standard [(10 ml/kg (NORMO)] or high [30 mL/kg/h (HIGH)] volume of Ringer’s lactate (B. Braun, Crissier, Switzerland), continuous intravenous administration until the end of the study. For 30 min, all animals were mechanically ventilated with tidal volume=6 mL/kg and PEEP=9 cmH2O (to keep alveoli open), then randomized to undergo abrupt or gradual (0.2 cmH2O/min for 30 min) PEEP decrease, from 9 to 3 cmH2O (Fig. 1). After this period, animals were further ventilated for 10 minutes at PEEP=3 cmH2O. At the end of the experiment, arterial blood gases, echocardiography, and respiratory system mechanics were assessed (FINAL), and heparin (1000 IU) was injected into the tail vein. All animals were killed by overdose of sodium thiopental (60 mg/kg i.v.) and the trachea was then clamped at PEEP=3 cmH2O. Lungs and kidney were then extracted for histology and molecular biology analysis. Seven animals received LPS intratracheally but were not mechanically ventilated [non-ventilated (NV) animals] and, after 24 h, were euthanized and had their lungs and a kidney removed for molecular biology analysis.
Data Acquisition and Processing
Airflow and airway pressure were continuously recorded throughout the experiments [5, 13, 14]. VT, RR, V′E were calculated. Respiratory system mechanics was assessed by occluding the airways at end-inspiration for 5 seconds until a respiratory system plateau pressure (Pplat,RS) was reached. Respiratory system driving pressure (DP,RS) was calculated as the difference between Pplat,RS (post end-inspiratory pause) and PEEP. All signals were amplified in a four-channel signal conditioner (SC-24, SCIREQ, Montreal, QC, Canada), and sampled at 200 Hz with a 12-bit analog-to-digital converter (National Instruments; Austin, Texas, USA). Mechanical data were computed offline by a routine written in MATLAB (Version R2007a; The Mathworks Inc., Natick, Massachusetts, USA).
Shaved animals were placed in the dorsal recumbent position. Transthoracic echocardiography was performed by an expert (NNR) blinded to group allocation, using an UGEO HM70A system (Samsung, São Paulo, Brazil) equipped with a linear phased-array probe (8–13 MHz). Images were obtained from the subcostal and parasternal views. Transthoracic echocardiography was performed  and the following parameters analyzed: right (RV) and left ventricular (LV) areas, as well as right ventricular cardiac output (RVCO). Pulsed-wave Doppler was used to measure the ratio between pulmonary acceleration time (PAT) and pulmonary ejection time (PET), which is an indirect index of pulmonary arterial hypertension . All parameters followed American Society of Echocardiography and European Association of Cardiovascular Imaging recommendations .
Diffuse Alveolar Damage
The lungs and heart were removed en bloc. The left lung was frozen in liquid nitrogen and immersed in formaldehyde solution (4%), embedded in paraffin, cut longitudinally in the central zone by means of a microtome into three slices, each 4 μm thick, and stained with hematoxylin–eosin for histological analysis [5, 13]. Photomicrographs at magnifications of ×100, ×200, and ×400 were obtained from eight non-overlapping fields of view per section using a light microscope (Olympus BX51, Olympus Latin America Inc., Brazil). Diffuse alveolar damage (DAD) was quantified using a weighted scoring system by two investigators (M.V. and V.L.C.) blinded to group assignment and independently, as described elsewhere . Briefly, scores of 0 to 4 were used to represent interstitial edema, overdistension, alveolar collapse, septal inflammation, and alveolar hemorrhage, with 0 standing for no effect and 4 for maximum severity. Additionally, the extent of each scored characteristic per field of view was determined on a scale of 0 to 4, with 0 standing for no visible evidence and 4 for complete involvement. Scores were calculated as the product of severity and extent of each feature, on a range of 0 to 16. The cumulative DAD score was calculated as the sum of each score characteristic and ranged from 0 to 80, as described elsewhere .
Acute Kidney Injury Score
Kidney slices were stained with hematoxylin–eosin and periodic acid–Schiff and observed under light microscopy for qualitative and quantitative analysis. Semiquantitative data were obtained from high-resolution photomicrographs. Fifteen non-overlapping images of tubular tissue (cortex and outer medulla) were randomly obtained with a ×40 objective lens, from each kidney section (n=8/group) stained with H&E and PAS (tubular profiles). Histological findings were graded from 0 to 4 (0, no change; 1, changes affecting 25% of the field of view; 2, changes affecting 25–50%; 3, changes affecting 51–75%, and 4, changes affecting >75% of the field), according to the area affected by the features of interest (edema, tubular cell vacuolization, deranged brush border in proximal tubular epithelia, tubular cell death/desquamation, and inflammation). The final kidney injury score in each rat was expressed as the sum of all values of all features obtained and ranged from 0 to 20 .
Transmission Electron Microscopy
Three slices (2 × 2 × 2 mm) were cut from three different segments of the left lung and fixed (2.5% glutaraldehyde) for electron microscopy. On each lung electron microscopy image (20 fields per animal), degree of interstitial edema, damage to basement membrane, extracellular matrix damage, type II epithelial cell damage, and endothelial cell damage were graded on a five-point, semiquantitative, severity-based scoring system as follows: 0 = normal lung parenchyma, 1 to 4 = changes in 1 to 25%, 26 to 50%, 51 to 75%, and 76 to 100% of examined tissue, respectively .
All histological analyses were performed in a blinded manner.
Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) was used. In lung tissue, gene expression of biomarkers associated with inflammation (interleukin [IL]-6), tight junction (zona occludens-1 [ZO-1], epithelial cell damage (club cell secretory protein 16 [CC16]), extracellular matrix damage (versican and syndecan-1), and endothelial cell damage (vascular endothelial growth factor [VEGF]) were measured. In kidney tissue, gene expressions of biomarkers associated with renal injury (kidney injury molecule [KIM]-1 and neutrophil gelatinase associated lipocalin [NGAL]) and inflammation [IL-6] were evaluated. The primer sequences are listed in Additional File 1, Table S1. Central slices of the right lung and kidney were cut, flash-frozen by immersion in liquid nitrogen, and stored at −80 °C. For each sample, the expression of each gene was normalized to the acidic ribosomal phosphoprotein P0 (36B4) housekeeping gene  and expressed as fold change relative to NV group, using the 2–∆∆Ct method, where ΔCt = Ct (target gene) – Ct (reference gene) .
Sample size was calculated based on pilot studies, which detected differences in IL-6 between abrupt and gradual PEEP decrease under high fluid administration. A sample size of 7 rats per group would provide the appropriate power (1-b=0.8) to identify significant differences in IL-6, taking into account the effect size d=2.0, a two-sided t test, and a sample size ration of 1 (G*Power 126.96.36.199., University of Dusseldorf, Dusseldorf, Germany). Normality and the equality of variance were evaluated by Kolmogorov-Smirnov test with Lilliefors’ correction and Levene’s median test, respectively. Two-way ANOVA followed by Tukey’s test was used to compare abrupt and gradual PEEP release under standard and high fluid volume conditions. Parametric data were expressed as mean ± SD, while non-parametric data were expressed as median (interquartile range). All tests were carried out in GraphPad Prism 8.00 (GraphPad Software, La Jolla, CA, USA). Significance was established at p value less than 0.05.