Buxco small animal whole body plethysmography

Respiratory function was monitored in unrestrained mice using Buxco small animal whole-body plethysmography (Data Sciences International, New Brighton, MN) and FinePointe software (Data Sciences International, New Brighton, MN). The system was calibrated each day prior to data collection. On the day of data collection, animals were placed in individual chambers and given 30 min to acclimate; followed by 60 min of data collection. The software averaged the data over each minute and recorded a value every minute for 60 min. To ensure data was representative, breath frequency was used to ensure the mouse had not held its breath, buried its head under its body or was breathing too rapidly. Mean breath frequency was calculated and data which fell outside 1SD of the mean was excluded from the data analysis (Roberts et al., 2015 (link)).

Airway inflammation was induced in female 6–8 weeks old BALB/c mice with ovalbumin (OVA; grade V; Sigma-Aldrich, St. Louis, MO) as previously described [17 (link)]. Briefly, mice were intraperitoneally (i.p) sensitized with 10 µg OVA and 65 µg aluminiumhydroxide (alum; SERVA Electrophoresis, Heidelberg, Germany) or PBS/alum in 150 µl on days 0 and 14. On days 21–24, mice were anesthetized with 5% isoflurane (CP-pharma, Burgdorf, Germany) and treated with 100 µg OVA in 30 µl PBS intranasally (i.n.) or 30 µl PBS alone. Thirty minutes prior to each OVA application, mice received i.n. 0.1 µg (group OMVs 0.1 µg/OVA), 1 µg of EcO83-OMVs (group OMVs 1 µg/OVA), or 0.9% NaCl (groups Sham/PBS and Sham/OVA). On day 25, airway hyperresponsiveness was tested using unrestrained whole-body plethysmography (Buxco® Small Animal Whole-body Plethysmography, Data Sciences International; St Paul, MN). Mice were subjected to increasing doses (0–50 mg/mL) of nebulized methacholine (Sigma-Aldrich, St. Louis, MO) and the enhanced pause was measured as an index for airway obstruction. Mice were terminally anesthetized on day 26, spleens and lungs were harvested and bronchoalveolar lavage (BAL) and blood were taken.