A total of twenty-two, conventionally raised, 6–8-week-old male BALB/c mice weighing 18–20g were used in the study. The animals were fed a commercial diet ad libitum and housed in hygienic macrolane cages in air-conditioned rooms on a 12-h light/dark cycle. All experimental procedures complied with the requirements of the Animal Care and Ethics Committee of the Dokuz Eylul University.

Mice were divided into four groups: (I) Control (n=5), (II) Asthma-untreated (placebo) (n=5), (III) Asthma-100mg/kg SIN-treated (n=6), (IV) Asthma-1mg/kg dexamethasone treated (n=6). Mice in study groups except for the control group were sensitised on days 0 and 14 by an intraperitoneal (i.p) injection of 10μg/0.1ml chicken egg albumin (ovalbumin, grade V, ≥98% pure, Sigma, St. Lois, MO, USA) with alum as an adjuvant as described by Temelkovski et al.14 Mice in groups II, III and IV were challenged with an aerosol of 5ml, 2.5% ovalbumin in saline for 30min/day for three days of the week for eight weeks beginning from the 21st day of the study (Fig. 1). The mice in the control group received normal saline with alum intraperitoneally on days 0 and 14 of the experiment and aerosolised saline without alum for 30min per day on three days of the week for eight weeks beginning from the 21st day of the study. Exposures were carried out in a whole-body inhalation exposure system in a plexiglass chamber with 40×60×120 diameters designed for placement of cages. Temperature and relative humidity were maintained at 20–25°C and 40–60%, respectively. A solution of 2.5% ovalbumin in normal saline was aerosolised by delivery of compressed air to a sidestream jet nebuliser with a flow rate of 6L/min (Medi-cair, UK) and injected into a chamber. The aerosol generated by this nebuliser comprised >80% particles with a diameter of <4μm. Particle concentration was maintained in the range of 10–20mg/mm3 in the chamber.15

Figure 1.

Schematic presentation for a murine model of airway inflammation and the drugs administered to mice.

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During the last five days of the challenge period, group II received saline, group III received SIN (Sigma Aldrich, St. Louis, MO, USA) at dose of 100mg/kg, group IV received dexamethasone (Dekort; Deva Holding AS, Istanbul, Turkey) at dose of 1mg/kg by orogastric tube, once a day. Animals were sacrificed by an overdose of ketamine hydrochloride (200mg/kg) 24h after the last drug administration.

Two investigators who were blinded to the treatment groups interpreted the histopathology. Tissue specimens were obtained from the mid-zone of the left lung of mice. Samples were fixed in buffered 10% formalin and embedded in paraffin wax. Five-micron-thick serial sections were obtained and the first 10 samples were stained with haematoxylin and eosin (H&E). General tissue features of these samples were examined and the thicknesses of epithelium and sub-epithelial smooth muscle layers of the medium and small airways were measured. In order to evaluate the thicknesses of epithelium and sub-epithelial smooth muscle layers, measurements were performed from four points of each airway. Considering that each section contained approximately two to three airways, around 20 or more airways were evaluated for each mouse. Photomicrographs were taken by Olympus DP71 camera (Japan), which adapted on Olympus DP70 model microscope (Olympus Optical, Tokyo, Japan). Measurements were carried out with UTHSCSA Image Tool for Windows Version 3.00 software.

The consecutive 10 sections were stained with toluidine blue and the other 10 sections were stained with periodic acid-Schiff (PAS). Photomicrographs were randomly taken from five fields of each section which were stained with toluidine blue. For mast cell enumeration, a standard transparent counting frame representing an area of 16,400μm2 was manually used and eight fields in each photograph were examined for each mouse. Goblet cells stained with PAS were enumerated in 10 sections of each mouse. In each section, three to five randomly selected airways were photographed. Circumferences of all airways were measured and goblet cell numbers in these areas were recorded. For standardisation, goblet cell numbers in 100μm were analysed by dividing the total goblet cell number to the total length of airway circumferences and multiplying the result by one hundred.

Lungs were removed and washed with cold PBS three times in order to remove the blood. Lung tissues were taken into 2ml microcentrifuge tubes and stored at −80°C until analysis. On the study day, frozen lung tissues were thawed, weighed (60–80mg), transferred into different tubes on ice containing 5ml of stainless beads, 0.1% SDS protease inhibitor cocktail (Sigma–Aldrich, St. Louis, MO, USA) and 0.1mg/ml phenylmethanesulfonyl fluoride (PMSF) in PBS. Microcentrifuge tubes were transferred to pre-cooled Tissuelyser LT racks and placed into Tissuelyser (Qiagen, Germany) homogenisator. Time and frequency were set to 5 and 50min, respectively. The homogenates were then centrifuged at 15,000×g for 1h at 4°C and supernatants were obtained. Levels of IL-4, IL-5, IL-13 cytokines were quantified in the supernatants of the lung tissue by standard ELISA protocols by using commercial Mouse IL-4, IL-5, IL-13 (eBioscience Mouse ELISA kit, USA). Detection levels were 4pg/ml for IL-4 and IL-5, 2.8pg/ml for IL-13.

Nitric oxide levels were measured by ELISA in accordance with the manufacturer's recommendations in supernatants obtained from the lung tissue homogenates (Invitrogen Griess Reagent). Griess Reagent (A: 1-naphthyethylene-diamine dihydrochloride, 0.1%; B: sulfanilic acid 1%) and samples were mixed with an equal volume in microtiter plates (Greiner) and they were stored at room temperature for 30min. Results were determined at 548nm (BioTek Synergy HT, USA) using spectrophotometric methods.16

Serum levels of OVA specific IgE were measured using the ELISA commercial kit (Sun Red Biological Technology, Shanghai, China).

Caspase-3 (AB3623, Millipore, Temecula, CA, Polyclonal antibody), TUNEL (DeadEnd Colorimetric TUNEL system kit, Roche, Germany), MMP9 (anti-MMP-9 mouse monoclonal antibody, Santa Cruz Biotech, Inc. Germany), VEGF (anti-VEGF mouse monoclonal antibody, Santa Cruz Biotech, Inc. Germany), TGF-β (anti-TGF-β mouse monoclonal antibody, Santa Cruz Biotech, Inc. Germany) antibodies were used in immunohistochemistry experiments. After deparaffinisation and rehydration, sections were treated with trypsin (Cat No: 00-3008 Digest All 2A, Zymed, San Francisco, CA, USA) at 37°C for 15min (for TUNEL, MMP9, VEGF, TGF-β antibodies). For Caspase-3, samples were treated with 10mM citrate buffer (Cat No. AP-9003-125 Labvision) for five minutes to unmask antigens by using the heat treatment. Slides were cooled in buffer for 20min. For all sections, sections were incubated in a solution of 3% H2O2 for 15min and then with normal serum blocking solution in order to inhibit endogenous peroxidase activity. Sections were again incubated in a humid chamber for 18h at +4°C with primer antibodies, thereafter with biotinylated IgG, and then with streptavidin conjugated to horseradish peroxidase for 15min according to kit instructions (85-9043, Invitrogen Corporation, Camarillo, UK). Sections were finally stained with DAB (diaminobenzidine) (1718096, Roche, Mannheim, Germany) and counter-stained with Mayer's haematoxylin. Samples were analysed using a light microscope.17

Semi-quantitative grading system was used to score the quantity of primer antibody positive staining in the sections.18 The score was defined as follows: 0: no immunoreactivity; 1: remarkably little positive staining; 2: moderate positive staining; 3: strong positive staining evenly distributed across the whole image. Each section was graded by two individuals blind to the treatments, and the average score was taken.

All values were expressed as the mean±standard deviation (SD). The Kruskal Wallis (between all groups) and Mann–Whitney U-test (for two groups) were used to compare staining intensity values between groups. All statistical analyses were performed using the SPSS software for Windows, Version 15.0 SPSS, Chicago, IL, USA. The significance was accepted when the p value was lower than 0.05 (p<0.05).

The light microscopic examinations revealed normal findings in the control group (Table 1). We compared the histopathological features of the control group with the placebo group (group II) to show that the asthma model was successfully established. In the placebo group (group II), thicknesses of epithelium and smooth muscle layers as well as numbers of mast cells and goblet cells were significantly higher compared to the control group (group I). These results showed that the model of chronic asthma was established.

In 100mg/kg SIN treated group (group III), all histological parameters including sub-epithelial smooth muscle and epithelial thicknesses, number of mast cells except the number of goblet cells, were significantly better compared to the placebo (group II) and improvement was similar to the dexamethasone-treated group (group IV) in all parameters except sub-epithelial smooth muscle thickness and numbers of mast cells. All histological parameters were significantly better in the dexamethasone-treated group (group IV) compared to the placebo group (group II) (Table 1, Fig. 2A and B).

Figure 2.

(A) Histopathological findings of study groups. I; control (n:5), II; placebo (n:5), III; 100mg/kg SIN (n:6), IV; dexamethasone (n:6) A; H&E, B; PAS, C; Toluidine Blue staining. (A) Arrow with two heads; epithelial thickness (black arrow), sub-epithelial smooth muscle thickening (blue arrow), (B) arrows; goblet cells, (C) arrows; mast cells. (B) Comparison graphics of the histopathological findings of the study groups.

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Immunohistochemical scoring of MMP-9, VEGF, TGF-β, TUNEL and caspase-3 were found lower in the control group (group I) compared to the placebo group (group II). In 100mg/kg SIN treated group, all the immunohistochemical scores of MMP-9, VEGF, TGF-β, TUNEL and caspase-3 were lower than the placebo group (group II). Dexamethasone treatment led to lower immunohistochemical scoring of MMP-9, TGF-β, TUNEL and caspase-3 except the VEGF, compared to the placebo group (group II). We also observed that immunohistochemical scoring of 100mg/kg SIN (group II) and dexamethasone treated groups (group IV) were similar in all parameters (Table 2, Fig. 3A and B).

Figure 3.

(A) Immunohistochemical staining results of study groups. I; control (n:5), II; placebo (n:5), III; 100mg/kg SIN (n:6), IV; dexamethasone (n:6). (A); Caspase-3, (B); TUNEL, (C); MMP-9 (D); VEGF (E); TGF-β. Black arrows show positive staining with caspase 3, TUNEL, MMP-9, VEGF and TGF- β respectively. VEGF, vascular endothelial growth factor; MMP-9, matrix metallo proteinase-9, TGB-β, transforming growth factor-β, TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling and Caspase-3, cysteine-dependent aspartate-specific proteases. (B) Comparison graphics of the immunohistochemical staining findings of the study groups. *p<0.05 vs. Group I, Group III, Group IV, # and ¥p<0.05 vs. placebo. (C) Comparison graphics of the cytokine levels of the study groups. *p<0.05 vs. Group I, Group III, Group IV, # and ¥p<0.05 vs. placebo.

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IL-4, IL-5, IL-13, NO levels in lung homogenates and OVA specific IgE levels in serum samples were significantly lower in control group (group I) compared to the placebo group (group II).

All cytokine levels (IL-4, IL-5, IL-13, NO) in lung homogenates and OVA-specific IgE level in serum samples were significantly lower in 100mg/kg SIN treated group (group III) compared to the placebo group (group II). All cytokine levels in lung homogenates and serum except the IL-5 were significantly lower in the dexamethasone-treated group compared to the placebo group (group II).

All cytokine levels except the IL-4 and IL-5 were similar in 100mg/kg SIN (group III) and dexamethasone (group IV) treated groups. In the dexamethasone-treated group (group IV) IL-4 and IL-5 levels were significantly lower than the 100mg/kg SIN treated group (group II) (Table 3, Fig. 3C).

The number of eosinophils in BAL fluid was significantly reduced in the 100mg/kg SIN (group III) treated group when compared to the Placebo group (Table 4).

The authors declare that no patient data appears in this article.

The authors declare that no patient data appears in this article.

The authors declare that the procedures followed were in accordance with the regulations of the responsible Clinical Research Ethics Committee and in accordance with those of the World Medical Association and the Helsinki Declaration.