Experimental sheep, allergen sensitization and airway challenges
Female Merino-cross ewe lambs (4-5 months of age) free of significant pulmonary disease  were used for these studies. All experimental animal procedures and the collection of tissues and cells were approved by the Animal Ethics Committee of Monash University, following guidelines set by the National Health and Medical Research Council (NH&MRC) of Australia.
Sheep were sensitized by immunization with solubilized HDM whole extract (Dermatophagoides pteronyssinus), and immunized sheep were classed as allergic when they showed increased HDM-specific serum IgE levels as assessed by ELISA . Naïve, control animals were not immunized with HDM. All allergic sheep were subsequently given 3 airway allergen challenges at weekly intervals to prime the respiratory tract to HDM allergen [16, 17] and BAL samples were collected using a fibre-optic bronchoscope to assess airway inflammation . The allergen challenges were followed by a rest period of 2 weeks before all animals were euthanased (barbituate overdose) for the collection of tracheal tissues.
Preparation of tracheal explant cultures
Following resection, the trachea was cut longitudinal along the midline through the trachealis, and pinned open onto a dissecting board. The mucosa was dissected gently away from cartilage and small 5 mm discs of tracheal tissue were cut away using a biopsy punch (Kai Medical, Germany). Tracheal explants were established in culture as outlined previously , with some modifications. Briefly, sterile strips of filter paper (Whatman® Grade 1; Sigma, Australia) pre-soaked in medium (DMEM10; DMEM with 10% fetal bovine serum + penicillin/streptomycin; Invitrogen, Life Technologies, Australia) were placed across a 35 mm diameter culture dish sitting within a larger 60 mm dish, to which 3 ml DMEM10 was added. For each treatment, triplicate tracheal biopsies were placed upon the strip of filter paper set up within each plate.
To investigate possible changes in epithelial morphology and tissue architecture, tracheal biopsies were collected from control (naïve) sheep and explants prepared in triplicate as detailed above. Following the application of 5 μl of DMEM containing HDM allergen (10 μg/ml) or 5 μl of control DMEM to the epithelial surface, explants were cultured for a period of 5 h, 24 h or 48 h at 37°C in 5%CO2/air. In a separate experiment, tracheal tissues were resected from allergic and control (naïve) sheep and tracheal explants established in triplicate cultures for a 24 h period.
Tissue processing and histology
After culture, tracheal explants were transferred into a small tube containing 100 μl DMEM10, briefly agitated and (medium washout) samples stored at -20°C for later cytokine analyses. Tissues were then fixed in 4% paraformaldehyde (PFA) and embedded in paraffin for histology. Paraffin-embedded tracheal tissue explants were sectioned (7 μm) and stained with hematoxylin and eosin (H&E), alcian blue/periodic acid-schiff (AB/PAS) or immunostained to identify caspase-3 positive apoptotic cells, mucosal mast cells and eosinophils.
Immunohistochemistry for caspase-3, mast cells and eosinophils
Immunostaining for caspase-3 was used to identify apoptotic cells  in tracheal tissues before and after 5 h, 24 h and 48 h in culture. Briefly, paraffin-embedded PFA-fixed tissue sections were dewaxed, then rehydrated and immersed in 0.1 M PBS containing 0.3% triton X100 (PBS-TX) followed by 0.1 M citric acid buffer and microwave treatment for antigen retrieval. Sections were washed in PBS-TX, endogenous peroxidase activity blocked upon incubation with 0.3% H2O2, again washed and incubated with 5% normal goat serum (NGS)/2% BSA in PBS-TX. Sections were then incubated with rabbit polyclonal anti-human/mouse activated caspase-3 antibody (Ab) (R&D Systems, USA), washed and incubated with biotinylated goat anti-rabbit IgG (Cayman Chemical, USA), and again washed prior to incubation with streptavidin-horseradish peroxidase (HRP) complex (Amersham Biosciences, UK). Following washing, sections were developed with 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma) and finally dehydrated and cover-slipped with Depex™ tissue mounting fluid (Fluka Biochemika, Switzerland).
For the detection of mast cells and eosinophils, paraffin embedded tissue sections were blocked as above, then incubated with 10% normal sheep serum in PBS prior to application of primary antibodies. A polyclonal rat anti-ovine tryptase Ab reactive with all ovine respiratory mast cells  was kindly provided by Professor Hugh Miller, University of Edinburgh, UK. For eosinophil detection, the ovine eosinophil-specific mouse anti-galectin-14 mAb (clone EL1.2) was used . Following incubations, sections were washed then incubated with HRP-conjugated rabbit anti-rat Ig (Dako, Denmark) or HRP anti-mouse (Dako) for the detection of mast cells and eosinophils, respectively. This was followed by further washes, then development in DAB as detailed above. Slides were air-dried and lightly counterstained with Wright's stain (Sigma), then cover-slipped in Depex™.
In AB/PAS stained sections, intensely purple staining cells within the epithelium (goblet cells) were enumerated in triplicate samples at 200× magnification using a calibrated grid. All intact tissue was examined for each treatment (maximum 15 microscopic fields) and counts were expressed as cell number per mm length of epithelium.
Caspase-3 positive cells, within the epithelial layer only, were enumerated in triplicate samples at 200× magnification using a calibrated grid. All intact tissue was examined for each treatment (maximum 15 microscopic fields) and counts were expressed as cell number per mm length of epithelium.
For mast cell and eosinophil cell counts, immunoperoxidase positive cells in the lamina propria underlying the epithelium were enumerated in triplicate samples at 200× magnification using a calibrated grid. All intact tissue was examined for each treatment (maximum 15 microscopic fields) and counts were expressed as cell number per mm2 area of lamina propria underlying the epithelium. Cell counts for both mast cells and eosinophils were performed on cells that were densely stained. Cells partially stained were classified as degranulated and were not counted.
Detection of cytokines in tracheal washout samples
Tracheal explant washout samples were collected for determination of the ovine cytokines IL-1α, IL-6 and TNF-α by enzyme-linked immunosorbent assay (ELISA), as described previously . The minimum detectable levels of IL-6 and TNF-α were 2.63 ng/ml and 1.98 ng/ml, respectively.
Results are presented as means ± SEM. A Kruskal-Wallis non-parametric test was used to compare treatments, within experimental and control groups, and a Dunn's post-hoc test was used where significant. A Mann-Whitney non-parametric test was used for comparisons between experimental and control groups. For all statistical analyses, p < 0.05 was considered significant.