*2.1. Horses*

Eleven horses were enrolled in the study, six EA-affected horses in asymptomatic phase, and five young just slaughtered horses as control, healthy at the pre-mortem visit and without evidence of pulmonary disease on post-mortem examination.

The EA-affected group consisted of five Italian Saddle horses and one Appaloosa, four mares and two geldings, 15.7 ± 1.9-year-old, with body weight ranging from 400 to 520 kg. The horses lived outdoor and had a history of signs of acute recurrent onset of asthma when housed in a stable with shavings for bedding and hay for feed. None of the horses received medications for at least three months prior to the assessment. Throughout the experimental time, a daily clinical examination was performed.

The control group comprised lungs removed immediately after slaughter from three Italian saddle horses and two Standardbreds, four mares and one male, 1.4 ± 1.5-year-old, with body weight ranging from 420 to 540 kg.

## *2.2. Experimental Design*

EA-affected horses in asymptomatic phase (T0), were sedated with acepromazine maleate at 0.02 mg/kg bw iv (Prequillan, Fatro, Ozzano dell'Emilia BO, Italy) and detomidine hydrochloride at 0.01 mg/kg bw iv (Domosedan, Vetoquinol Italia, Bertinoro FC -Italy) and underwent a bronchoscopy (Pentax EG290P diameter 9.8 mm; length 120 cm).

For each endoscopic procedure performed in EA-affected horses, the instrument was introduced in the right and left principal bronchus. The airways were anesthetized by spraying of lidocaine, and four (two left and two right) epithelial biopsy specimens were obtained from four sites by use of endoscopic forceps. Biopsy sites were chosen between: 1) Right cranial bronchus and principal bronchus; 2) second right lateral segmen<sup>t</sup> and

principal bronchus; 3) left cranial bronchus and principal bronchus; and 4) second left lateral segmen<sup>t</sup> and principal bronchus [24] (Figure 1).

**Figure 1.** Position of bronchial biopsies: (1) Between right cranial bronchus and principal bronchus; (2) between second (II) right lateral segmen<sup>t</sup> and principal bronchus; (3) between left cranial bronchus and principal bronchus; and 4) between second (II) left lateral segmen<sup>t</sup> and principal bronchus.

Randomization of biopsy sequence was applied, and biopsy was performed by grabbing the lateral part of a bronchial branch with a 2.3 mm biopsy forceps, by avoiding the vessel from sliding flowing on the floor of the bronchus.

After the first bronchoscopy, the horses were brought from pasture, bedded in boxes with closed windows, on mold straw and fed hay of poor quality, to increase the dustiness of the environment and induce lower airway obstruction. Straw was not changed; it was rather raised twice a day to increase the dustiness of the environment. On day 2 (T1, early exacerbation phase) and 7 (T2, late exacerbation phase) of the challenge, bronchoscopy with bronchial biopsies was repeated with the same procedures described above. On day 8 the horses were put back to pasture after administration of dexamethasone (0.1 mg/kg) [25]. Four days later the biopsies were repeated (T3, remission phase) (Figure 2).

**Figure 2.** Timeline flow chart of trial.

For the control horses, the lung was removed as soon as the horses were slaughtered and placed on a flat surface. The presence of macroscopic lung changes was evaluated; the carina was opened to the bronchial branches, to acquire biopsy samples by using a biopsy punch in the same site chosen for EA-affected horses.

#### *2.3. Bronchial Biopsy Histology*

Immediately after biopsy collection, the samples were fixed in 10% neutral buffered formalin. Within 24–48 h, all samples were processed as routine for histology, embedded in paraffin, cut into 5 μm-thick sections and stained with hematoxylin and eosin (HE).

The observation of histological samples provided for an evaluation according to the standardized 14-point semiquantitative grading scoring system proposed by Bullone et al., 2016 for lung pulmonary biopsies based on histological features considered important in heaves [17]. The variables evaluated and scored were: (a) epithelial hyperplasia; (b) presence of epithelial inflammatory infiltrate; (c) goblet cells hyperplasia; (d) epithelial desquamation; (e) thickening of the basal membrane; (f) submucosal inflammation; (g) the presence of mucous glands within the lamina propria; (h) the presence of mucous gland among smooth muscle bundles; (i) airways smooth muscle fibrosis; and (j) presence of the end of the smooth muscle. Each variable was measured as 0 = absent and 1 = present, and the total (0–10) was calculated for each biopsy sample.

The biopsy specimens were scored by two veterinary pathologists at high and small magnification under a light microscope (NiKonEclipse 80, Nikon Corporation, Konan, Minato-ku, Tokyo, Japan). The microphotograph images were captured using a Nikon Digital Sight SD-MS camera (Nikon Corporation, Konan, Minato-ku, Tokyo, Japan) connected to the optical microscope.

#### *2.4. Immunohistochemical Analysis for Neurokinin A and IL-8*

Formalin fixed paraffin embedded samples were cut into 3–5 μm sections, mounted onto poly-L-lysine coated slides, dewaxed, rehydrated, and rinsed with tap water at room temperature. Immunolabelling was performed with a streptavidin–biotin–peroxidase technique (BIO SPA, Milan, Italy). The antibodies used and their dilutions were the following:NKA (1:400, polyclonal, Biorbyt, St. Louis, MO, USA) and IL-8 (1:100 dilution, monoclonal, Cloud Clone Corporation, Katy, TX, USA). The incubation was carried out with 3% hydrogen peroxide in methanol for 30 min (to block endogenous peroxidase activity) and microwave treatment (750 W) for antigen retrieval in citrate buffer solution at pH 6.0 (one burst of 5 min and five bursts of 2 min and 30 sec, replacing the evaporated buffer after each heating session). After that, the sections were incubated overnight at 4 ◦C in a humid chamber with the primary antibody diluted, according to the appropriate dilutions, in PBS (0.01 M, pH 7.4). The sections, subsequently washed in PBS, were incubated first with the secondary antibody (anti-rabbit IgG conjugated with biotin) for 30 min at room temperature, and then with the streptavidin-peroxidase complex for 25 min at room temperature. After a 12-min passage in the chromogenic DAB solution (diaminobenzidine 0.02%, and H2O2 0.001% in PBS), the sections were immediately rinsed in PBS, then in running water, stained with a contrast coloring (hematoxylin), dehydrated and assembled with DPX (Fluka, Riedel-de Häen, Germany).

NKA immunohistochemistry results were graded as follows: intensity of positivity (0, negative reaction; (1) weak intensity; (2) moderate intensity; (3) intense positivity; (4) very strong positivity); signal distribution (1, diffuse cellular, >50% of cells immunopositive; 2, outbreaks of positivity, <50% of the cells with a positive reaction), cell localization (1, cytoplasmic; 2: nuclear; 3: both cytoplasmic and nuclear).

IL-8 immunohistochemistry results were graded as follows: intensity of positivity (0, negative reaction; 1, weak intensity; 2, moderate intensity; 3, intense positivity; 4, very strong positivity); signal distribution (1, diffuse cellular, >50% of cells immunopositive; 2, outbreaks of positivity, <50% of the cells with a positive reaction).

Appropriate positive controls were used in order to evaluate the specificity of the reactions and ascertain the proper cross-reactivity in the horse tissue. Brain and normal horse lung were used as positive controls for NKA, horse lymph node as IL-8 control. As a negative control for the immunohistochemical procedure, 10% normal mouse serum was used in replicate sections instead of the primary antibody.

All the markers were evaluated through blinded observations by semiquantitative analysis of five representative high-power fields at the optical microscope (NiKonEclipse 80, Nikon Corporation, Japan). The microphotograph images were captured using a Nikon Digital Sight SD-MS camera (Nikon Corporation, Konan, Minato-ku, Tokyo, Japan) connected to the optical microscope.

#### *2.5. Statistical Analysis*

Statistical analysis was performed with a commercially available program (GraphPad Prism 5, GraphPad Software, San Diego, CA, USA). Assessment of data for normality was calculated by applying the D'Agostino-Pearson test. Data were expressed as median (minimum-maximum).

Differences in histological score, NKA and IL-8 immunohistochemistry between four bronchial sampling sites were analyzed by a Kruskal–Wallis one-way analysis of variance, both in EA-affected horses (at each experimental time), and in control horses.

For each parameter examined in each experimental time, the median value of variables of four biopsy samples was used for the subsequent statistical analysis.

A Mann–Whitney test (two tail *p* value) was applied to perform a comparison between control horses and EA-affected horses, at each experimental time, for the following variables: (1) Histological score; (2) NKA immunohistochemistry score; and (3) IL-8 immunohistochemistry score. Similarly, a Friedman test with Dunn's multiple comparison test, as post hoc test, was applied to perform a comparison of EA-affected horses at different experimental times, for the following variables: (1) Histological score; (2) NKA immunohistochemistry score; and (3) IL-8 immunohistochemistry score.

The significance was set for *p* < 0.05.
