Murine Model of VIDD.

The experimental design has been described in a recent study (20) and has been reviewed and approved by the Animal Care and Use Committee Languedoc-Roussillon and recorded under reference CEEA-LR-12078.

In brief, 35 adult male (10–12 wk old, 25–30 g) C57/BL6 mice were separated into three groups.

Mice in the first group were intubated with a 22-gauge angio-catheter and mechanically ventilated for 6 or 12 consecutive hours using a volume-driven small-animal ventilator (Minivent; Harvard Apparatus). Tidal volume was established at 10 µL/mg body weight with a respiratory rate of 150 breaths per min, a PEEP level from 2 to 4 cmH₂O, and a fraction of inspired oxygen of 0.21. Nonspontaneous ventilation was defined as a lack of diaphragm contractile activity attested by repetitive stereotypical deflections observed in the airway pressure curve. The mice mechanically ventilated from this first group were subdivided into 5 subgroups. The first subgroup contained only mice ventilated for 6 (MV) or 12 h (MV12h). The second subgroup was ventilated for 6 h (MV-Trolox) and received a priming dose of Trolox (0.125 mL of saline containing 5 g/L Trolox, corresponding to ∼20 mg/kg) i.v. (IV) infused over a 5-min period, 20 min before the start of MV (MV-Trolox). During MV, a constant IV infusion of Trolox at a rate of 4 mg⋅⁻¹kg⋅h⁻¹ (∼0.025 mL/h) was maintained. The third subgroup of mice ventilated for 6 h (MV-S107) or 12 h (MV12h+S107) was treated for 7 d before the start of MV, with S107 in their drinking water (final concentration, 0.25 mg⋅ml⁻¹) as reported (34, 48, 49) and received the same volume of IV saline 20 min before starting and during MV. The mice drank approximately 3 mL/d (water consumption was variable, and we recorded water bottle and body weight to monitor consumption) for a daily dose of ∼0.75 mg (∼37.5 mg⋅kg⁻¹⋅d⁻¹). The fourth subgroup of mice ventilated for 6 h or 12 h (MV+propanolol, MV12h+propranolol) was treated 30 min before the start of MV with propranolol (3 mg/kg) and one injection every hour. The fifth subgroup of mice ventilated for 6 or 12 h (MV+ICI118551, MV12h+ICI118551) was treated 30 min before the start of MV with ICI118551 (10 mg/kg), respectively, and one injection every hour.

The second group of mice was intubated and treated in an identical manner to the mechanically ventilated mice but breathed spontaneously with a CPAP of 3–4 cmH₂O for 6 h (CPAP). For this purpose, the angio-catheter was connected to an air compressor to deliver a high inspiratory flow rate (1 L/min room air) while the expiratory port was placed under a water seal to obtain PEEP.

All groups mechanically ventilated or under CPAP (first and second group) received the same general care. Mice were anesthetized with i.p. injection of pentobarbital sodium (50 mg/kg body weight) and orally intubated with a 22-gauge angiocatheter. General care applied during the experiments also included continuous reheating by using a homeothermic blanket (Homeothermic Blanket Control unit; Harvard Apparatus, set at 35 °C), and hourly i.p. injection of 0.05 mL of Ringers Lactate solution to maintain hemodynamic stability and compensate insensible losses, as well as bladder expression, eye lubrication, and passive limb movements.

The third group was considered a control group and was divided into three subgroups. The first subgroup was only treated by serum saline (Control, n = 8), the second subgroup (Control+ICI118551) was treated with ICI118551 according to the same protocol previously described for the mechanically ventilated mice. The third subgroup (Control+S107) was treated with S107 according to the same protocol previously described for the mechanically ventilated mice. All control mice received the same amount of volume compared with the first group.