L methodological approaches are needed to delineate molecular signatures in such complex biological systems. Mass spectrometry based proteomics allows extensive and sensitive profiling of your protein expression pattern in biological samples [5]. We hypothesised that the pathogenic mechanisms underlying these asthma models could be reflected in the protein pattern in BAL. To this finish, we therefore employed an integrated approach combining mass spectrometry-based protein evaluation collectively with screening of a multiplex array of inflammatory biomarkers, in BAL in experimental asthma.Figure 1 Schematic outline on the animal experiments. Two groups, resembling eosinophilic (A) and neutrophilic asthma (B), were subjected to sensitization by means of i.p. injection and challenge by means of inhalation of ovalbumin (OVA). For the neutrophilic asthma model, animals were additionally challenged with lipopolysaccharide (LPS). A third group of animals within the neutrophilic asthma group, received steroid (GC) therapy 1 h prior challenge and lung mechanic assessment. As controls a final fourth group, received only vehicle (PBS) remedy for the duration of inhalation. Lung function testing was performed for all groups at day 17 followed by BAL fluid collection, differential cell count and proteomic evaluation.MethodsAnimalsFemale BALB/c mice (Taconic M B, Denmark) were applied in this study. They have been housed in plastic cages with absorbent bedding material and have been maintained on a 12 h daylight cycle. Meals and water have been provided ad libitum. Their care and the experimental protocols had been authorized by the Regional Ethics Committee on Animal Experiments in Uppsala (C86/5 and C64/8). Mice had been 6 weeks of age when the airway inflammation protocol started and 90 weeks when BAL was collected (n = 5-6 mice per group).Asthma modelssodium succinate, 0.375 g/kg) straight away just before OVA + LPS challenge (days 146). Lastly, a group of mice (n = 5) served as control (C) with no exposure to any identified airway irritant and was treated with car (PBS).Lung mechanics and airway responsivenessFor the eosinophilic asthma group (OVA, n = 5), airway inflammation was induced as described previously [3] by intraperitoneal (i.p.) injections of ten g ovalbumin (OVA, Sigma-Aldrich, St. Louis, MO, USA) emulsified in Al(OH) 3 (Sigma) on day 0 and day 7. The animals were then inhaled aerosolised 1 OVA diluted in phosphate-buffered saline (PBS, Sigma) for 30 min on days 146 (Figure 1). The aerosol exposure was performed inside a chamber coupled to a nebuliser (DeVilbiss UltraNeb Sunrise Health-related Ltd, U.K.). The chamber was divided into pieshaped compartments with individual boxes for each and every animal, offering equal and simultaneous exposure to the allergen.Ingenol Mebutate A second group of mice (n = six), resembling neutrophilic asthma, received i.Tolcapone p.PMID:23557924 OVA and was exposed to inhaled aerosolised LPS (Escherichia coli serotype 0111: B4; Sigma) dissolved in ddH2O) diluted in PBS simultaneously with OVA as described above on days 146 (Figure 1). The concentration of LPS inside the nebuliser was 0.005 w/v (the OVA + LPS group). A third group (n = five) received glucocorticoid (GC) treatment (hydrocortisoneDynamic lung mechanics have been evaluated as described in detail elsewhere [3]. Briefly, airway reactivity was characterised by murine ventilator and forced oscillation method (FOT) where Newtonian resistance (RN), tissue damping (G) and elastance (H) were determined. Airway responsiveness was determined by investigating the maximal response o.
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