The GSP samplers were mounted with 0.8 μm polycarbonate filters with airflow of 3.5 L/min. All filters were extracted in 5 mL sterile 0.05% Tween-80 in 0.9% NaCl solution by shaking for 15 min at room temperature (500 rpm) in orbital shaking glass flasks and serial dilutions were made for determination of CFU (see above).

Determination of respiratory parameters for assessment of irritation in upper respiratory tract, conducting airways and alveolar region, respectively was performed as thoroughly described [18]. Briefly, three types of effects CP-690550 nmr from the respiratory system can be studied simultaneously: a) Sensory irritation. In humans, chemicals stimulating the trigeminal nerve endings of the upper respiratory tract cause irritation that may increase to burning and painful sensations, termed ‘sensory irritation’. Formaldehyde, ammonia and methacrolein are examples of compounds being sensory irritants [18–20]. Sensory irritants decrease the respiratory rate in mice due to a reflex causing a break at the end of the inspiratory phase [21].   b) Bronchial constriction. Airflow limitation due to bronchial constriction or inflammation of the

conducting airways causes a lengthening of the duration of expiration and thus causes an associated decrease in respiratory rate. To quantify this effect, the airflow rate during expiration is measured.   c) Pulmonary irritation is caused by AZD0156 ic50 stimulation of vagal nerve endings at the alveolar level [22]. Stimulation of this reflex is characterized by a pause at the end of expiration, which is a specific marker of pulmonary irritation. Ozone is an example Selleck LY2835219 of a substance inducing pulmonary

irritation [18].   about The assessments and quantifications of the respiratory frequency, time of inspiration, time of expiration, time from end of inspiration until the beginning of expiration termed “”time of brake”", time from end of expiration until beginning of the next inspiration termed “”time of pause”", tidal volume and mid-expiratory flow rate were performed using the Notocord Hem software (Notocord Systems SA, Croissy-sur-seine, France) as described in details previously [23]. For the comparison of CFU recovered from total lung homogenate to that of CFU recovered from BAL fluid, a pilot inhalation experiment with 8 mice was performed. BAL procedure The BAL procedure was performed as previously described with minor modifications (Larsen et al., 2007). Briefly, the lungs were flushed four times with 0.8 mL saline (0.9%) and the recovered fluids were pooled for each mouse. From the BAL fluid of mice that have received bacterial inocula, a 250 μL of total fluid was removed before centrifugation for CFU determination. Cells were counted and differentiated by morphology into neutrophils, lymphocytes, macrophages, epithelial cells and eosinophils. For each sample, 200 cells were differentiated.