Acetyl β methylcholine chloride

Mice were anesthetized, steel cannulae were inserted into their tracheas, and then they were individually placed in a chamber to measure, using the Buxco FinePointe system [Data Sciences International (DSI), St. Paul, MN, USA], their lung resistance (R_L) while they were exposed to increasing doses of acetyl-β-methylcholine chloride (methacholine; Sigma-Aldrich, St. Louis, MO, USA). Dynamic airway resistance (Penh value) was noninvasively measured using unrestrained whole body plethysmography (Buxco Electronics, Wilmington, NC, USA) while they were exposed to increasing aerosol concentrations of methacholine.

Mice were intraperitoneally (i.p.) anesthetized with a mixture of ketamine (Vetoquinol S.A., France; 125 mg/kg) and medetomidine (Pfizer, The Netherlands; 0.4 mg/kg). EMKA invasive measurement of dynamic resistance (EMKA Technologies, France) in response to increasing doses of methacholine (acetyl-β-methyl-choline chloride, Sigma-Aldrich, The Netherlands; 0–25 mg/mL, 10% puff for 10 s.) was used on day 14 to assess lung function. Average lung resistance (R_L) is presented in cm H₂O/(mL/sec) (18 (link)).

Airway responsiveness was measured 24 h after the last aerosol exposure by recording respiratory pressure curves using barometric unrestrained whole-body plethysmography (Buxco; EMKA Technologies, Paris, France) in response to inhaled methacholine (acetyl-β-methyl-choline chloride, Sigma, The Netherlands) in conscious unrestrained mice. Airway responsiveness was expressed as enhanced pause (Penh) as described in detail previously (Hamelmann et al., 1997 (link)). Briefly, mice were placed in a whole-body chamber and basal readings were obtained and averaged for a 3 min period. Subsequently, increasing doses of methacholine (0–50 mg/mL), were aerosolized for 3 min, and readings were taken and averaged for 3 min after each nebulization (Vos et al., 2007 (link)).

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).

Airway hyperresponsiveness (AHR) was measured 24 h after the last intranasal challenge by whole body plethysmography (DSI, Buxco Electronics Inc., USA) in conscious unrestrained animals in response to increasing doses (0–25 mg/ml) of aerosolized methacholine (acetyl-β-methyl-choline chloride, Sigma-Aldrich). The baseline was established in response to PBS inhalation, and airway resistance was determined by calculating dimensionless parameter enhanced pause (PenH) as previously described^{57 (link)}.

The EMKA invasive measurement of dynamic resistance (EMKA Technologies, Paris, France) in response to increasing doses of methacholine (acetyl-β-methyl-choline chloride, Sigma-Aldrich) (0–25 mg/ mL, 10% puff for 10 s) was used to measure lung function in anesthetized mice. Data are presented as average lung resistance (RL) in cm H2O/mL^*sec-1 (26 (link)).

AHR was measured with Buxco whole-body plethysmography (WBP) system (Buxco Research Company, United States) in response to inhaled methacholine (acetyl-β-methyl-choline chloride, Sigma, The Netherlands). After the last challenge, mice were monitored for about 10 minutes in the chamber until their breathing went stable. After 5 minutes baseline was recorded, the responses were assessed for 5 minutes after inhaling different concentration of atomized methacholine solutions (0, 6.25, 12.5, 25, 50, and 100 mg/ml) respectively. About 5 minute intervals were given between tests to allow the respiratory intensity to return to the baseline. AHR was expressed as enhanced pause (Penh) as described in detail previously [39 (link)].