All protocols were approved by the Ethical Review Board of Imperial College London, and carried out under the authority of the UK Home Office in accordance with the Animals (Scientific Procedures) Act 1986, UK. We used male C57BL6 mice (Charles River, Margate, UK) aged 10–12 weeks and weighing 25-30g. In total 68 animals were used – 23 for measurements of respiratory mechanics and alveolar inflammation, 25 for assessing alveolar fluid clearance, and 20 for lung wet/dry weight and histology scoring.
Mice were anaesthetised by intraperitoneal injection of xylazine (6mg/kg) and ketamine (60mg/kg), and given an intraperitoneal fluid bolus of 10μl/g 0.9% normal saline as preemptive fluid resuscitation. Mice were suspended vertically from their incisors on a custom-made mount for orotracheal instillation, as described previously (10 (link)) (additional details are provided in the online supplement). A fine catheter was guided 1cm below the vocal cords, and 75μl of an isoosmolar (to mouse plasma - 322mosmol/L) solution of 0.1M hydrochloric acid (pH 1.0) was instilled. For the next 4 hours, during which animals exhibited significant respiratory depression/distress as an acute result of acid aspiration-induced ALI, mice were kept in a custom-made transparent recovery box under humidified supplemental oxygen (FiO2 reduced gradually from 1.0 to 0.21). During this period animals were carefully monitored and body temperature was maintained using external heat sources, after which they were transferred to individually ventilated cages with air and free access to food and water.