Flexivent ventilator

The procedures of low and high tidal volume ventilation were performed as previously reported [12 (link)]. Briefly, after anesthesia, mice were mechanically ventilated with room air and end-expiratory pressure of 3 cmH₂O on a FlexiVent ventilator (SCIREQ, Montreal, QC, Canada) continuously for 3 h. A tidal volume of 30 ml/kg body weight at a rate of 60/min was used for injurious ventilation (IV), and 6 ml/kg body weight at a rate of 150/min was used for normal ventilation (NV). Lung tissues were harvested after 3 h of ventilation for RNA isolation or Western blot.

Mice were anaesthetised by intraperitoneally administration of sodium pentobarbital (90 mg/kg), ensuring the mouse was at surgical levels of anaesthesia throughout the procedure. The mouse was placed under a heat lamp, the trachea exposed and cannulated. Mechanical ventilation was initiated immediately using a computer-controlled piston Flexivent ventilator (Flexivent, Scireq Scientific Respiratory Equipment Inc) system. Prior to airway function measurements, a deep inflation of the lung was performed to recruit closed lung areas and standardize lung volume history. The absence of spontaneous inspiratory efforts was also confirmed using a test pressure volume curve measurement (PVs-V). AHR to inhaled aerosolised methacholine (Acetyl-β-Methylcholine Chloride (cat#A2251-25G, Sigma Aldrich, UK)) (0-100 mg/ml) was then carried out using the forced oscillation technique [63 ]. All operational scripts for ventilation and force oscillation technique measurements, including data acquisition were handled using Flexiware software (Version 7.6.3).

To determine whether expression of hORMDL3 in ASM influenced spontaneous AHR to MCh, AHR was assessed in naive hORMDL3^{Myh11eGFP-cre} and naive WT mice at 12 weeks of age (n = 12 mice/group). Mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) i.p., intubated, and ventilated (FlexiVent ventilator; Scireq) as previously described (8 (link)). The dynamic airway resistance and elastance were determined using Scireq software in mice exposed to nebulized PBS and methacholine doses at 0, 3, 24, and 48 mg/mL. Ventilator settings include tidal volume set at 10 mL/kg, frequency at 150/min, and positive end-expository pressure at 3 cmH₂0.

To assess the pressures and rates at which alveoli in the injured lungs closed, de-recruitability tests at different PEEP (positive end-expiratory pressures) as well as quasi-static pressure volume-loops using a Flexivent ventilator (SCIREQ, Canada) were performed (27 (link)). The de-recruitability tests consist of two recruitment maneuvers (up to 30 cmH₂O followed) followed by 5 min of low-tidal volume ventilation (10 ml/kg body weight) interspersed with 8 s multi-frequency forced oscillation perturbations at 30 s intervals. Tissue elastance (H), tissue damping (G) and tissue hysteresivity (G/H) were calculated by fitting the constant phase model to impedance spectra. After each de-recruitability test, 3 quasi-static PV loops were recorded, and quasi-static compliance was calculated according to the Salazar-Knowles equation.

Lung function was assessed with FlexiVent ventilator (SCIREQ, Canada) for small animals when mice were stable. At the 24th hour after the final exposure, mice were anesthetized intraperitoneally and tracheostomized. After intubation, mice were mechanically ventilated to assess lung function. Using pressure–volume (PV) loops, lung function was measured including total lung capacity (TLC) and lung compliance (chord compliance, Cchord).

On day 0, 14 and 21 CREM^−/−, CD2CREMαtg and wt C57Bl/6*129/SV, and FVB mice were sensitized with ovalbumin (OVA) and aluminumhydroxide (Alu). On day 28 and 29, sensitized animals were exposed to nebulized OVA (1%) for 30 min each day, whereas controls were exposed to NaCl (0.9%). On day 35, mice were mechanically ventilated with the flexiVent ventilator (SCIREQ, Canada) and lung functions were measured by the forced oscillation technique. AHR was provocated with rising acetylcholine (Ach) aerosol concentrations.