A total of 130 Swiss mice (male, 65; female, 65; age, 8-12 weeks; weight, 20-25 g) were purchased from Kobay D.H.L. A.S. and were used in all experiments. Equal numbers of male and female mice were used in each experimental group. The mice were housed in a room at constant humidity and temperature (22±1˚C) with 12 h light/dark cycles and provided with food and water ad libitum. All experiments were carried out according to the EU Directive 2010/63/EU (23) for animal experiments and approved by the Animal Experimentations Local Ethics Board of Hacettepe University, Ankara, Turkey (approval no. 2018-3/2; updated by committee in the meeting commencing in 2020 for in vivo analysis of airway function, approval no. 2020-3/12).

The mice were divided into four groups for the experimental asthma and forced swim test (FST) as follows: i) Control group (n=24 for 1st, n=18 for 2nd and n=12 for 3rd FST); ii) ovalbumin (OVA; n=24 for 1st, n=18 for 2nd and n=12 for 3rd FST); iii) montelukast-treated controls (n=18 for 2nd and n=12 for 3rd FST); and iv) montelukast-treated OVA (n=18 for 2nd and n=12 for 3rd FST). Montelukast treatment was started after inflammation was occurred with the first challenge. In the first FST, mice were administrated montelukast for only 1 day, as the primary aim of the current study was to investigate the chronic effects of montelukast. The first FST was not performed in montelukast-treated groups. (Fig. 1). Following each FST, 6 mice were euthanized for histopathological analysis. In the treatment groups (montelukast-treated OVA and montelukast-treated control groups), montelukast (Abdi Ibrahim Pharmaceuticals; 20 mg/kg/day) was administrated orally through the drinking water every day at the same hour, starting on the first day after the first OVA challenge and montelukast treatment was continued for 20 days for 2nd challenge and 40 days for 3rd challenge (Fig. 1). Additional groups were used for the open field test (n=10/group) and the in vivo assessment of airway function (n=8/group). Open field tests were performed using three other groups of mice: i) The OVA group; ii) the montelukast-treated (20 days) control group; and iii) the control group. In vivo assessment of airway function was performed in another set of OVA and control groups.

An experimental asthma model was established using OVA (Sigma-Aldrich; Merck KGaA) in the OVA and montelukast-treated OVA groups (24). The OVA-induced asthma model was modified to extend the duration of asthma to two months. The mice were sensitized on days 1 and 8 by an intraperitoneal injection of 10 µg OVA dissolved in 200 µl PBS (Sigma-Aldrich; Merck KGaA) containing 1 mg aluminum hydroxide (Sigma-Aldrich; Merck KGaA). Airway inflammation was induced by intranasal OVA-challenges (0.25 µg OVA in 25 µl PBS) on days 15, 16 and 17 (1st challenge), 34, 35 and 36 (2nd challenge) and 53, 54 and 55 (3rd challenge) to induce and maintain chronic airway inflammation. The control groups were sensitized with OVA (0.25 µg OVA in 25 µl PBS) and challenged with intranasal PBS (25 µl PBS; Fig. 1). A stratified design was used for the experiments and, therefore, equal numbers of animals from each group were used on the same days for all experiments.

Airway function was assessed in the OVA and control groups by evaluating the changes in airway resistance using the Buxco Finepoint Resistance and Compliance System (Buxco Electronics, Inc.). Testing was performed one day following the 1st OVA challenge. Mice were anesthetized with 100 mg/kg ketamine/10 mg/kg xylazine intraperitoneally, tracheally cannulated and placed into individual plethysmography chambers (Buxco Electronics, Inc.). The lungs of the mice were mechanically ventilated at a respiratory rate of 150 breaths/min with a tidal volume of 200 µl and a positive end expiratory pressure of 2 cmH₂O. The basal airway resistance was recorded for 2 min and PBS was used for control responses. Methacholine (1.5-48 mg/ml) was administered intratracheally to measure the dose-dependent increase in respiratory resistance using an ultrasonic nebulizer (Buxco Electronics, Inc.). The maximum value measured following each dose for respiratory resistance was used as the measure of response. Airway resistance (cmH₂O.s/ml) results were normalized to values measured following PBS nebulization.

A modified version of Porsolt's FST was performed to assess the effect of montelukast treatment on depression-like behavior and repeated following each challenge (25,26). Briefly, glass cylinders (height, 19 cm; diameter, 12 cm) were filled with 25±1˚C water to a 15 cm depth. The mice were placed into the cylinders for 8 min and the immobility time of the last 6 min was determined. Immobility, in which the mice remained immobile or made only small limb movements necessary to float, were regarded as depression-like behavior. Following FST, 6 mice from each group were euthanized by cervical dislocation after each FST. Blood samples were collected by cardiac puncture for the determination of plasma montelukast concentration. The lungs were isolated and fixed in 10% formaldehyde solution for histopathological analysis (Fig. 1).

Isolated lungs, following each FST were fixed in 10% formaldehyde at room temperature for ≥48 h. Slides (thickness, 0.4 µm) were prepared from paraffin embedded sections and were stained with hematoxylin and eosin at room temperature for 1 h with an automated slide stainer (Thermo Shandon Varistain Gemini™; Thermo Fisher Scientific, Inc.). Histopathological analysis was performed for the OVA, control and montelukast-treated OVA groups to confirm the inflammation in the lungs following OVA and to examine whether there is a reduction in the inflammation following montelukast treatment. A total of 6 mice from each group were analyzed. Histopathologically, interalveolar (interstitial) thickening, peribronchial and perivascular inflammation were evaluated and each of these parameters were graded semiquantitatively in a blinded manner. The inflammation parameters were individually scored from 0-4 (0, no inflammation; 1, mild inflammation; 2, moderate inflammation; 3, severe inflammation; 4, very severe inflammation) by evaluating the extent of inflammatory cells, tissue injury and interalveolar (interstitial) thickening (Leica Microsystems GmbH) Final scores were defined as the sum of peribronchial (0-4) and perivascular (0-4) inflammation.

At the end of the experimental protocol, montelukast concentrations were confirmed in mice that had been treated with montelukast 40 days orally through drinking water. Blood samples were collected in heparinized tubes and centrifuged at 3.305 x g for 15 min to obtain plasma. The proteins in the plasma samples (100 µl) were crushed with 100 µl of acetonitrile (Sigma-Aldrich; Merck KGaA). The amount of montelukast in the plasma samples was quantified by liquid chromatography-electrospray ionization-mass spectrometry (LCMS-8030; Shimadzu Corporation). The mass spectrometric detection of montelukast was undertaken in the positive electrospray ionization and multiple reaction-monitoring mode using 586.0-422.10 m/z transitions using 19 eV of collision energy. The nebulizer gas flow and drying gas flow were 3 ml/min and 15 ml/min, respectively. The heat blocking temperature, desolvation line temperature and collision-induced dissociation gas pressure were 400˚C, 250˚C and 15 kPa, respectively. Chromatographic separation was achieved on the C₁₈ column (Hypersil ODS-4; GL Sciences; 50x2.1 mm; 3 µm) using a mobile phase consisting of acetonitrile containing 0.1% formic acid and water with 0.1% formic acid (70:30; v/v) at a flow rate of 0.3 ml/min.

Open field tests were used to assess any changes in the locomotor activity of the mice following the administration of OVA or montelukast. The test was performed according to previous studies (27,28). Briefly, black locomotor activity cages (40x40x40 cm) equipped with an infrared sensor system (Activity Meter; Commat, Ltd.) were used. The mice were individually placed in the center of the cages and tracked for 5 min. The locomotor activities of the mice were assessed based on the total distance travelled in the cage as measured by the analyzing system. The test was performed in the OVA group 1 day after the completion of one OVA-challenge, a separate group that had been administered only montelukast (20 mg/kg/day) for 20 days and the control group.

Data are expressed as mean ± standard error of the mean. Statistical analysis was performed using unpaired Student's t-test for in vivo assessment of airway function and one-way ANOVA followed by Tukey's post hoc test or the Kruskal Wallis test followed by Dunn's test for other experiments. Data were analyzed using GraphPad Prism software (version 5.0; GraphPad Software Inc.). P<0.05 was considered to indicate a statistically significant difference.

In the current study, the relationship between airway inflammation, chronic montelukast treatment and depression-like behavior was investigated. The induction of experimental asthma was confirmed by histopathological changes in lung segments and development of airway hyperresponsiveness to methacholine challenge. In order to maintain the allergic airway inflammation throughout the experimental protocol, the OVA-challenges were performed in triplicate as described in Fig. 1. This protocol allowed for the investigation of the effect of chronic allergic inflammation and chronic montelukast treatment on depression-like behavior. The occurrence of inflammation was presented as histopathological images (Fig. 2A and B). Total inflammation scores (Fig. 2C) and airway resistance (Fig. 2D) were significantly increased following the first OVA challenge in the OVA group compared with controls. Immobility time was significantly higher in the OVA group compared with controls (Fig. 2E). Furthermore, open field tests were performed to eliminate the possible effects of OVA and montelukast treatment on the locomotor activities of the mice. Total distances travelled in the cages in the OVA group were not significantly different compared with controls (Fig. 2F). Similarly, the montelukast-treated OVA group (20 days of treatment) did not exhibit a significant difference in total distances travelled, which was indicated the locomotor activity. Additionally, montelukast concentrations at a steady state were determined to be 226.40±34.59 ng/ml in mice administrated montelukast for >40 days (data not shown).

Airway inflammation observed in the OVA group compared with control group was slightly reduced in the montelukast-treated OVA group (20 days of treatment) following the 2nd FST (Fig. 3A-C). The total inflammation score of the OVA group was statistically increased compared with the control group (Fig. 3D). However, the decrease in the total inflammation score of the montelukast-treated OVA group was not statistically significant compared with OVA group. Furthermore, there was no statistical differences between the montelukast-treated OVA and control groups.

FST was performed following each OVA challenge phase to examine the short- and long-term effects of asthma on depression-like behavior. Immobility times were significantly increased in the OVA group compared with the control group following the second challenge (Fig. 4). Furthermore, immobility times were significantly decreased in the montelukast-treated OVA group compared with the OVA group. Immobility times in the montelukast-treated control group were not significantly different between control and montelukast-treated OVA groups. Furthermore, the immobility times of montelukast-treated control group were significantly lower compared with the OVA group.

Following the third challenge, inflammation in OVA-treated lungs was reduced followed by ongoing montelukast treatment compared with the control group (5A-C) and total inflammation scores of the montelukast-treated OVA group were significantly decreased compared with the OVA group (Fig. 5D). Following the third challenge, chronic montelukast application to the OVA-treated group (40 days of treatment) significantly decreased immobility times compared with the OVA group (Fig. 6). Immobility times of the montelukast-treated control group were not statistically different compared with controls; however, they were significantly decreased compared with the OVA group (Fig. 6). Furthermore, there were no significant differences between the immobility times of three time points in the OVA-treated groups (data not shown).