Extra particulars on the strategies are supplied on-line (see supplementary data file, SIF).Animal protocolFive-week-old male C57/Bl6 mice have been bought from the Janvier laboratory (Le Genest-Saint-Isle, France) and have been housed in our “small animal” facility. They acquired one week of adaptation with their normal meals. On the age of 6 weeks, they have been divided into two teams. One group (management group) acquired a food plan through which 10% of the energy have been derived from fats within the type of lard (D12450B, SSNIFF, Soest, Germany), whereas the opposite group (overweight group) was fed a food plan through which 60% of the energy have been derived from fats within the from of lard (D12492, SSNIFF). Each teams got meals and water advert libitum and have been uncovered to a 12:12 h light-dark cycle. On the age of 18 weeks and after 12 weeks of the food plan, animals have been weighed after which fasted for 12 hours with free entry to water earlier than the experimentation. The physique mass index (BMI) was calculated as beforehand described components: BMI = weight (g)/(naso-anal size (cm))2 47. Mice have been then anesthetized with intraperitoneal pentobarbital (30 µg/g of physique weight, Hospira, Meudon La Forêt, France) adopted by steady 1.5% isoflurane (Abbott, Rungis, France). The larynx was surgically uncovered and the trachea was intubated orally beneath direct imaginative and prescient with a steel cannula (inside diameter of 1 mm, Harvard Equipment, Les Ulis, France). The tracheal cannula was correctly secured with surgical thread (Ethicon three–zero, Ethicon, Auneau, France) earlier than connection to mechanical ventilator so as to keep away from leaks.Mechanical ventilationAfter intubation, animals have been left for five min in spontaneous respiration and half of the mice (overweight and management) have been sacrificed. The opposite half of have been ventilated by way of a small-animal ventilator (flexiVent, Scireq, Montreal, Canada) as follows: VT = eight mL/kg of physique weight, respiratory charge = 180/min, end-expiratory stress = 1.5 cmH2O, and impressed fraction of oxygen = zero.four–zero.6. This mixture of VT (eight ml/kg) and respiratory charge (180/min) has been proven to supply satisfactory minute air flow and PaCO2 in mice48. Mechanical air flow lasted 2 hours with steady anesthesia maintained by 1.5% isoflurane and muscle paralysis utilizing intraperitoneal pancuronium on the onset of the experiment (zero.eight mg/kg of physique weight, Organon, Puteaux, France) to make sure passive mechanical situations. Physique temperature was monitored utilizing a rectal probe and was maintained at 36.5 °C with a blanket related to a homeothermic regulator (Homeo-blanket system 50–7221 F, Harvard Equipment, Les Ulis, France). Mice acquired one intraperitoneal heat fluid bolus (physiological serum) on the onset of the experiment (20 mL/kg of physique weight). The dosage of all medicines administered was calculated on precise weight. In ventilated overweight mice, tidal quantity was calculated both on precise weight or excellent weight. As no components is accessible to calculate predicted physique weight in mice, excellent weight was primarily based on the imply weight of management mice (particularly 31.2 g) (see SIF for particulars).Experimental designThe experimental design included 5 teams: NVC (non-ventilated management mice), VC (ventilated management mice), NVO (non-ventilated overweight mice), OVTaw (ventilated overweight mice with VT calculated on the precise weight) and OVTiw (ventilated overweight mice with VT calculated on a great weight) (Fig. 7). The variety of animals utilized in every experiment is detailed within the legend of all outcome desk or determine.Determine 7Design of experimental teams. (a) Mice have been fed both a food plan containing 10% fats (management mice) or 60% fats (overweight mice) for 12 weeks. Non-ventilated management or overweight mice (NVC and NVO) underwent common anesthesia, have been intubated and have been sacrificed after 5 min of spontaneous air flow. (b) Mice have been fed the identical food plan (10% or 60% fats). Ventilated management and overweight mice underwent common anesthesia, have been intubated and have been sacrificed after 2 hours of mechanical air flow, with a tidal quantity calculated on precise weight for management mice (VC) or on both precise weight (OVTaw) or excellent weight (OVTiw) for overweight mice.Respiratory mechanicsMean and peak airway pressures have been recorded at initiation of mechanical air flow after which hourly all through the experimental interval. Quasi-static compliance of the respiratory system was decided from a steady pressure-volume curve carried out at initiation of mechanical air flow and on the finish of MV.Specimen collectionAfter 2 hours of MV, mice have been sacrificed by exsanguination. Two totally different specimen assortment procedures have been used: For one half of the mice, instantly following thoracotomy, the pulmonary circulation was flushed by injection of two mL of saline into the proper ventricle, and the lungs have been then inflated by tracheal injection of four% formaldehyde at a stress of 20 cmH2O, mounted in four% formaldehyde and at last paraffin-embedded. For the opposite half of the mice, bronchoalveolar lavage was carried out with two tracheal injections of 1 mL of saline through the tracheal tube. A pulmonary circulation flush was then carried out as beforehand described and each lungs have been mechanically disrupted for circulate cytometry evaluation.Histologic examinationAfter formalin fixation and paraffin embedding, 5 µm thick sections of the left lung have been minimize and stained with hematoxylin, eosin and saffron (HES). An optical microscope (BX51 Olympus) with digicam (Camedia 5060, Olympus) was used for histologic examination and images. An inflammatory rating, derived from the VILI rating49,50, assessing the diploma of neutrophil infiltration in alveolar areas, was decided on every lung (see SIF for particulars). In apply, HES-stained lung sections have been examined at excessive energy discipline (x40) and solely alveolar areas, situated at a distance of a couple of alveolus from bronchial areas, have been analyzed. Ten random fields have been examined and the neutrophil rely was scored from zero to three: zero for <5 neutrophils per discipline, 1 for five to 10 neutrophils per discipline, 2 for 10 to 20 neutrophils per discipline, and three for >20 neutrophils per discipline. All lung sections have been examined beneath blinded situations, with out data of the mouse standing (overweight or not, ventilated or not).Bronchoalveolar lavageThe complete cell rely was decided for a recent fluid specimen utilizing slides with counting grids (Hycor biomedical, Indianapolis, IN, USA). The cell pellet was diluted in saline, and differential cell counts have been carried out on cytospin preparations (Cytospin three; Shandon Scientific, Cheshire, UK) stained with Diff-Fast stain (Baxter Diagnostics, McGaw Park, IL, USA). Bronchoalveolar lavage fluid was centrifuged (1500 rpm, 13 min at four °C), and cell-free supernatants have been saved at −80 °C for subsequent evaluation of protein content material (utilizing a colorimetric BCA assay, Thermo Scientific, Rockford, IL, USA).Movement cytometric analysisAfter performing BAL, a pulmonary circulation flush was carried out as beforehand described and each lungs have been eliminated and positioned in 2 mL of cell tradition medium (RPMI 16/40 supplemented with zero.1% L-glutamine and zero.1% of nonessential amino acids) after which in ice. On the finish of the experiment, lungs have been positioned in the identical medium with 5% fetal bovine serum (FBS), zero.35 mg/mL collagenase XI (Sigma-Aldrich, Saint Louis, MO, USA) and 5 mg/mL kind IV bovine pancreatic DNAse (Sigma-Aldrich), mechanically disrupted with a scalpel, and positioned in an incubator at 37 °C with 5% CO2 for 35 min. The motion of the enzymes was stopped by including 9 mL of RPMI medium containing 10% FBS, and digested lungs have been additional disrupted by gently pushing the tissue by a nylon display (70 µm). The only-cell suspension was then washed and centrifuged at 900 rpm. To lyze contaminating crimson blood cells, the cell pellet was incubated for five min at room temperature with 5 mL of Gey’s resolution (NH4Cl and KHCO3). Cells have been then washed with PBS, recentrifuged and the pellet was resuspended in four mL of PBS. Cell rely was then carried out utilizing slides with counting grids (Hycor biomedical) after Trypan blue staining.In cytometry tubes, 500,000 cells in 50 µL have been incubated with 50 µL of Fc Block (Anti-CD16/CD32 antibody, BD Biosciences, San Jose, CA, USA) at four °C for 15 min after which stained with fluorophore-conjugated anti-mouse antibodies for CD11b, CD11c, Gr-1 (Ly6C/G), F4/80 and CD62L (L-selectin) or applicable isotype-matched controls.Samples have been analyzed utilizing a CyAn cytometer and Summit software program (Beckman Coulter, Brea, USA). Macrophages have been recognized as excessive CD11c and low CD11b cells. As a way to differentiate and quantify monocytes, low side-scatter, excessive CD11b and low CD11c occasions have been gated. Neutrophils have been differentiated from monocytes as excessive side-scatter, excessive CD11b and low CD11c cells. F4/80 was used to facilitate differentiation between monocytes and neutrophils. Cell activation was assessed primarily based on L-selectin and CD11b adhesion molecule expression for neutrophils and L-selectin, CD11b and Gr-1 expression for monocytes. Particulars of the leukocyte identification process are represented in Supplementary Fig. S1.Statistical analysisData have been analyzed utilizing Prism software program model 5.zero (San Diego, CA, USA). Steady knowledge have been expressed as median [IQ25-IQ75]. Statistical comparisons between two teams used the Mann-Whitney or the Wilcoxon take a look at. Statistical comparisons between greater than two teams used the Kruskal-Wallis take a look at adopted by a pairwise Mann-Whitney take a look at with Benjamini-Hochberg correction for a number of testing. Two-tailed p values lower than zero.05 have been thought of vital.Moral approvalAll the experiments have been carried out in accordance with the official laws of the French Ministry of Agriculture and the US Nationwide Institute of Well being pointers for the experimental use of animals, have been accepted by the animal institutional evaluation board of the Paris-Est Creteil Val de Marne College, and have been performed in a selected “little animal” plateform (Plateforme Exploration Fonctionnelle du Petit Animal, INSERM U955, Créteil, France).