Vagotomy influences the lung response to adrenergic agonists and muscarinic antagonists

Journal Pre-proof Vagotomy influences the lung response to adrenergic agonists and muscarinic antagonists ´ Luiz Otavio Lourenc¸o, Ana Carolina Ramos Lopes, Bruno Zavan, Roseli Soncini

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S1569-9048(19)30246-0

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https://doi.org/10.1016/j.resp.2019.103358

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RESPNB 103358

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Respiratory Physiology & Neurobiology

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12 July 2019

Revised Date:

3 December 2019

Accepted Date:

4 December 2019

Please cite this article as: Lourenc¸o LO, Ramos Lopes AC, Zavan B, Soncini R, Vagotomy influences the lung response to adrenergic agonists and muscarinic antagonists, Respiratory Physiology and amp; Neurobiology (2019), doi: https://doi.org/10.1016/j.resp.2019.103358

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Vagotomy influences the lung response to adrenergic agonists and muscarinic antagonists Luiz Otávio Lourençoa, Ana Carolina Ramos Lopesa, Bruno Zavana,b, and Roseli Soncinia*

a

Department of Physiology, Institute of Biomedical Science, Federal University of

Alfenas, 37130-000 Alfenas, MG, Brazil; b

Integrative Animal Biology Laboratory, Institute of Biomedical Science, Federal

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University of Alfenas, 37130-000 Alfenas, MG, Brazil.

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Contact Information: Roseli Soncini, Ph.D. Institute of Biomedical Sciences Federal University of Alfenas-MG Rua Gabriel Monteiro da Silva, 700 37130-000 - Alfenas - MG Brazil Phone: +55 35 3701 9375 E-mail: [email protected]/[email protected]

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Vagus nerve influences on lung function parameters.

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Impared/mismatched lung function response to salbutamol and

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ipratropium bromide after vagotomy 

Collagen and elastic fibers deposition are also modulated by

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nerve vagus

Abstract Mammals airways are extensively innervated by the vagus nerve, which controls the airway diameter and bronchial tone. However, very few studies described the

respiratory function and lung morphology after vagal section. In the present study, we evaluated the respiratory mechanics after aerosolization of vehicle (to obtain control values), a muscarinic agonist (methacholine), a β2-adrenergic agonist (salbutamol) or a muscarinic antagonist (ipratropium bromide) in intact (Vi) and bilaterally vagotomized (Vx) Swiss male mice. Different group was established for morphometric analyze. The total lung resistance, airway resistance, elastance, compliance, lung tissue damping, lung tissue elastance, and morphological parameters (collagen and elastic fibers) were significantly different in the Vx group compared to the Vi group. Bronchoconstrictor and bronchodilators change the respiratory function of the Vx group. In conclusion, the

bronchodilation, as well as lung architecture of mice.

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vagus nerve modulates the lung function in response to bronchoconstriction and

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Keywords: Vagus nerve; bronchoconstrictor; bronchodilator; collagen; elastic

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1. Introduction

Autonomic nerves are essential for airway smooth muscle contraction and

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relaxation in most mammalian species. In addition to the airway smooth muscle, extensive autonomic innervation is observed in the airway parenchyma and is associated with epithelial and pulmonary vascularization (McGovern and Mazzone, 2010).

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Autonomic airway control includes parasympathetic (cholinergic), sympathetic (adrenergic) and peptidergic components, which are also known as the nonadrenergic/noncholinergic nervous system (Hlastala and Berger, 2001). Notably,

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peptidergic components are very important for mediating and modulating airway contractions or/and relaxation in several animal species (Canning, 2006).

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The vagus nerve, the main component of the parasympathetic system, consists of sensory and motor fibers that play a fundamental role in regulating lung function by transmitting both afferent and efferent signals (Canning, 2006). Efferent vagal nerve stimulation directly induces bronchoconstriction and mucus secretion through the activation of muscarinic receptors by acetylcholine (ACh; classical cholinergic neurotransmitter/muscarinic receptors, M). However, ACh also modulates inflammation, tissue remodelling, gene expression and cytokine production (Kistemaker and Goosens, 2015). Currently, strong evidence for functional roles only exists for M1,

M2, and M3 receptors.. Inhaled muscarinic antagonists, such as ipratropium bromide, mainly bind to M3 receptors and cause bronchodilation by competing with ACh. However, they may exert some additional effects on autoinhibitory M2 receptors expressed on cholinergic nerves or on M1 receptors expressed on autonomic ganglia (Buels and Fryer, 2012). The sympathetic system influences airway smooth muscle tone by activating βadrenergic receptors, leading to bronchodilation (Hlastala and Berger, 2001; Canning, 2006). Short-acting β2-adrenergic receptor agonists, i.e., salbutamol (known as albuterol) and terbutaline, administered by inhalation are the most effective therapy for

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the rapid reversal of airflow obstruction and prompt relief of asthmatic symptoms (Barisione et al., 2010).

The effect of the vagus nerve on respiratory system tone has been described in

guinea pigs (Kesler and Canning, 1999), rats (Sorkness et al., 1994), dogs (Delpierre et al., 1983) and sheep (Colebatch and Halmagyi, 1963). Therefore, we expect that the

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vagus nerve also plays an important role in adjusting the bronchodilatory response. In this context, we assessed the effect of the vagus nerve on the lung response to a

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bronchoconstrictor and a bronchodilator challenge. We therefore evaluated the lung mechanics after acute bilateral vagotomy and subsequent aerosolization of a

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bronchoconstrictor (methacholine) and the most frequently used bronchodilators (β2adrenergic agonists or muscarinic antagonist). In addition, the histology of the lung was

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evaluated to analyze the effect of the vagus nerve on the lung tissue.

2. Materials and Methods 2.1. Animals

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Male Swiss Webster mice aged 8 weeks (n = 40) and weighing 35-45 g were

obtained from the Central Animal Facility of the Federal University of Alfenas

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(Alfenas, MG, Brazil). The animals were maintained on a 12-h light/dark cycle at 22±1°C and were provided regular chow and water ad libitum. The animals were divided into two groups: intact vagus nerve (Vi, n = 20) and vagotomized (Vx, n = 20). The experiments were approved by the local ethics committee (protocol number 60/2017).

2.2. Respiratory Mechanics

The animals were anaesthetized with xylazine (12 mg/kg; i.p.; Syntec, Hortolândia, São Paulo, Brazil) and pentobarbital sodium (68 mg/kg, i.p.; Cristália, Itapira, São Paulo, Brazil). Under anaesthesia, the Vi group received an 18-gauge metal cannula into the trachea, and the respiratory mechanics procedures were subsequently performed. The Vx group was subjected to the same procedure, except that they were vagotomized (cervical bilateral vagotomy) after the tracheostomy. The animals in the Vi and Vx groups were placed on a heated water bag (38°C) and mechanically ventilated using a small animal ventilator (flexiVent, SCIREQ, Montreal, Quebec, Canada) with a tidal volume (10 ml/kg) calculated according to body weight at a breathing frequency of

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120 breaths/min and 3 cm of H2O-positive end expiratory pressure. Pancuronium bromide (0.5 ml/kg, i.p.; Cristália, Itapira, São Paulo, Brazil) and tramadol (50 mg/kg,

i.p.; São Paulo, São Paulo, Brazil) were administered to prevent spontaneous breathing and possible pain in the animals, respectively. The heart rate (bpm) was monitored during the entire experiment using an EKG integrated with the flexiVent. The

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parameters of total resistance (Rtot), airway resistance (Raw), elastance of the

respiratory system (E), dynamic compliance (C), tissue damping (Gtis) and tissue

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elastance (Htis) were obtained according to Bates and Allen (2006). TLC (total lung capacity) tests were performed before the administration of each substance to ensure

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that no air leaked from the equipment and to promote airway recruitment. The data were obtained from pressure and volume oscillations every 15 seconds and within 5 seconds. Rtot, E and C were measured using the SnapShot-150 perturbation, and the Quick

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Prime-3 perturbation was applied for the Raw, Gtis and Htis measurements.

2.3. Experimental Protocols

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The respiratory mechanics were evaluated after the administration of aerosolized substances (Aeroneb, Aerogen, Ireland) for both groups (n = 7-8 animals per group) and

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two protocols were used for bronchodilator substances. Therefore, the animals received aerosolized vehicle (saline, 0.9% sodium chloride; to register control value) and subsequently a cholinergic agonist (methacholine, MCh, 100 mg/ml; SIGMA, St. Louis, Missouri, USA) was aerosolized as a bronchoconstrictor (according to Teixeira et al. (2016). After this, protocol 1 or 2 was conducted. For protocol 1, a β2-adrenergic agonist was aerosolized (salbutamol, Salb, 1.5 mg/ml; GlaxoSmithKline, Rio de Janeiro, Rio de Janeiro, Brazil) as a bronchodilator (according to Teixeira et al. (2016). For protocol 2, a muscarinic antagonist was aerosolized (ipratropium bromide, Ipra,

26.7 µg/kg; Boehringer Ingelheim, Itapecerica da Serra, São Paulo, Brazil) as a bronchodilator (according to Tanaka et al. (2012). In both protocols, the respiratory mechanics records were completed after 5 minutes of aerosolization of each of the aforementioned substances, and a 2-minute interval was established between treatments. Immediately after the respiratory mechanics test, the animals were euthanized by rapid exsanguination of the abdominal aorta under anaesthesia.

2.4. Histological Procedures

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After anaesthesia and surgery, animals (not submitted to the lung mechanics evaluation) from both groups (n = 5-7 animals per group), received an 18-gauge metal cannula into the trachea to lungs perfusion with 10% formalin at a pressure of 20

mmHg. Subsequently, the lungs were collected, kept for 48-hour in formalin, embedded in paraffin and cut into four-micron-thick slices. Later, haematoxylin and eosin (H&E),

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0.1% picrosirius red and 1% orcein staining were performed. H&E staining were used to analyze the airway wall thickness and airway luminal area, while picrosirius red

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staining, which shows birefringence under polarized lens, enabled the analysis of different types of collagen in the lung. In our protocol, we analyzed collagen fibers

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(types I and III) in the airways and blood vessels. Orcein staining was performed to show the brown areas of the elastic fibers in the airways and blood vessels. Lung morphology was assessed using a conventional light microscope (Nikon Eclipse 80i,

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Tokyo, Japan) equipped with a digital camera (Digital Sigth-Fi1, Nikon, Tokyo, Japan), and images were captured from ten randomly selected areas in non-adjacent microscopic fields at 200x magnification. H&E-stained sections were analyzed using

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NIS Elements 3.1 (Nikon, Tokyo, Japan), and picrosirius red- and orcein-stained sections were analyzed using ImageJ v 1.52 (National Institutes of Health, USA). The

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percentage of stained collagen or elastic fibers (%) in the image was reported.

2.5. Data Analysis The data are presented as the means ± SEM. Statistical significance was assessed with parametric methods. Respiratory mechanics values were analyzed using two-way ANOVA, and data for other parameters were analyzed using Student’s t test. For each analysis, a value of p<0.05 was considered statistically significant. The

software used for the statistical analyzes was GraphPad Prism (version 7.0, San Diego, CA, USA).

3. Results and Discussion A bilateral vagotomy was performed immediately before the animal was attached to the ventilator. We recorded the heart rate prior to drug administration to confirm the efficiency of the vagotomy and during the experiments to assess the vital signs of the animals during respiratory mechanics procedures. Our data showed increased heart rate in the Vx (426 ± 19 bpm) compared to Vi group (358 ± 6 bpm (n =

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7; p<0.01; Table S1). The Zefirov and Sviatova (1997) observed an increase in the heart rate immediately after a vagotomy procedure (due to the loss of the parasympathetic

control that determines bradycardia). Furthermore, these authors have also reported that the rhythm rapidly returns to control values.

A bilateral vagotomy was performed to disrupt pulmonary afferent and

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bronchomotor efferent connections, e.g., receptors and reflexes of the airways and lungs (Hlastala and Berger, 2001). In this procedure, we attempted to reduce ACh signalling

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from the vagus nerve (neuronal) and to decrease the influence of the vagus nerve on the respiratory mechanics. However, the neuronal and non-neuronal (ACh sinthezis and

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release by airway epithelial and immune cells) sources co-exist in lung (Kummer et al., 2008). ACh interacts with muscarinic receptors to induce signalling in airway smooth muscle, a pathway that is essential to adjust the intracellular Ca+2 concentrations during

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direct/indirect constriction (Meurs et al., 2013).

We used different animals in the Vi and Vx groups to avoid long-term mechanical ventilation of the same animals and to minimize lung damage. The values recorded from

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the respiratory mechanics analysis are shown in Fig. 1 (for additional details, see Table S2). We observed a significant difference in control values of all evaluated parameters

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(administration of aerosolized saline) between the groups (Vi vs Vx). Rtot increased after MCh aerosolization in both group compared with their

respective control values (p<0.001 for both; Fig. 1A). However, after vagotomy the Rtot response to MCh was attenuated. McAlexander et al. (2015) observed reduced Rtot in response to MCh after bilateral vagotomy and showed that vagotomy reverses established airway hyperreactivity in inflamed mice. Similarly, the effects of vagotomy were reported by Kesler and Canning (1999), who showed that the tracheal tone in guinea pig was abolished after vagotomy (vagus nerves were cut at the laryngeal

portion). In a comparative study to investigate lung irritant receptors, Delpierre et al. (1983) observed reduced lung resistance in a segment of the cervical trachea of dogs following vagotomy. This reduction was maintained after the administration of aerosolized MCh (similar to our study). Moreover, Sorkness et al. (1994) observed potentially significant effects of changes in airway parasympathetic tone on airway responsiveness, depending on which agonist was administered during a bronchoconstriction challenge and how the responses were measured. These authors showed supra-additive interactions between intravenous MCh and parasympathetic tone in rats.

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In the present study, Raw values decreased after vagotomy compared to intact vagus after saline administration (Fig 1B, p<0.01), and no significant response to MCh was observed after vagotomy compared to the intact group. A previous study analyzed Raw in sheep after a bilateral vagotomy (Wagner and Mitzner, 1995). The authors

designed the experiments to address the functional importance of the different perfusion

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pathways of airways as applied to the smooth muscle. Vagotomy per se decreased Raw values before and after an MCh infusion into the bronchial artery.

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We observed, after saline aerosolization, significant increases of E (p<0.01) and decreases in C (p<0.05) of the vatomized animals in comparison to intact animals (Fig.

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1C and 1D; see values on Table S2). In more detail, we designated E as the reciprocal of C (dynamic compliance of the respiratory system in the chest of intact animals); C also characterized the overall elastic properties of the respiratory system to move air in and

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out of the lungs during tidal volume. According to Islam and Ulmer (1982), vagotomy minimally affects the resting tone or on change in E (dynamic elastance) in artificially ventilated dogs. However, a vagotomy substantially reduces airway tone, as evidenced

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by the decrease in E in spontaneously breathing dogs. Additionally, De Kock et al. (1966) observed a decrease in C following vagotomy as a consequence of interstitial

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oedema or a direct (non-reflex) action of the drugs on the lungs and airways. In the present study, the ratio of lung wet weight to dry weight ratio showed no changes in the Vx group compared to the Vi group (Table S1); thus, no oedema was observed. A previous study by Colebatch and Halmagyi (1963) reported contradictory results from sheep in which no changes were observed in C after a vagotomy associated with forced inflation of lung. These authors registered a similar decrease in C (about 20%) after tying and cutting the vagus nerve. In the present study, after MCh aerosolization, increases in E and C were observed compared to the control values, but a significant

difference in the response to MCh of the Vx group was not observed compared with that of the Vi group. The administration of aerosolized MCh induces bronchoconstriction and subsequent changes in E and C by increasing the resistance that restricts the airflow (Hlastala and Berger, 2001). The control values of Gtis and Htis after vagotomy were significantly different from intact animals. These parameters decreased 12% and 8%, respectively (Fig. 1E and 1F; see details Table S2) and they are correlated with lung contents of collagen and elastic fibers. The arrangement of the pulmonary parenchyma influences the mechanical proprieties under normal and pathological conditions (Faffe and Zin, 2009). After MCh

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aerosolization, Gtis values increased in both groups compared to control values, but the difference was not significant.

Bronchodilatory responses were assessed using Salb or Ipra after MCh

bronchoconstriction, i.e., the level of bronchodilation induced by Salb or Ipra were

analyzed after bronchoconstriction induced by MCh (see Fig. 1). The results recorded

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for the Vi group showed a decrease of approximately 47% in Rtot values after Salb

aerosolization. Therefore, after Salb administration, the Rtot values became similar to

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the control values (p>0.05). However, after Ipra aerosolization, a reduction of approximately 43% was observed; thus, Ipra administration resulted in Rtot values that

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were significantly different from the control values (p<0.05). In the Vx group, Salb administration produced a lower (26%) Rtot. Nevertheless, after Ipra aerosolization, a reduction of approximately 45% was observed, demonstrating that Ipra administration

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restored Rtot values to the control values. The Rtot values recorded after Ipra administration in the Vx group were different from those of the Vi group (p<0.01). After evaluating the Raw values recorded following Salb or Ipra administration, we

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observed similar patterns of the Rtot values in the Vi and Vx groups. The Raw response to Ipra in the Vx group differed from that of the Vi group (p<0.01). In this context, we

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presumed that vagotomy increased the bronchodilatory response to Ipra. Additionally, the values of E decreased and those of C increased after the administration of aerosolized Salb, and E increased and C decreased after the administration of aerosolized Ipra (both values were significantly different from the control values) in Vi group. In contrast, in the Vx group, we observed the opposite responses to the Vi group after Salb and Ipra administration: the values of E and C were different from the Vi group. Finally, the Gtis and Htis values after Salb or Ipra administration revealed a similar pattern of responses to the Rtot and Raw values in the Vi and Vx groups. The

values were not significantly different between the Vi and Vx groups. Salb (β-agonist) is considered a direct bronchodilator because it activates airway smooth muscle β2 receptors to induce relaxation, regardless of the constrictive signal. While antagonists of M3 receptors (such as Ipra, which was used in the present study) block the constriction signal, these indirect bronchodilators are not particularly effective in asthma when used as a monotherapy for bronchospasm (Camoretti-Mercado et al., 2015). A reduction in the bronchodilatory responses of almost all parameters, except for Htis, to Salb was observed after vagotomy in the present study. We considered this phenomenon rebound bronchoconstriction, in which adverse effects of bronchodilators

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were observed. The main hypothesis suggested to account for this effect was an altered regulation and/or response capacity of the physiological receptors involved in the

mechanisms of action of the drugs (Teixeira, 2013). Even immediately after bilateral

vagotomy, as observed in this study, downregulation of the muscarinic receptors and

other receptors involved in bronchoconstriction and bronchodilation may have occurred.

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Although these hypotheses have not explored in the lung, they have been explored in

gastroenterology to understand gastric cancer signalling pathways (Wang et al., 2018).

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Finally, the lung histology of animals that were not treated with any bronchoconstrictor or bronchodilator and were not subjected to mechanic ventilation

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was examined. We observed effects of the vagotomy on wall thickness in relation to airway area, collagen I and III fibers in lung vessels and elastic fiber contents in airway and lung vessels (Table 1 and images in Fig. S1). The wall thickness in relation to

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airway area was reduced in vagotomized animals (p<0.05) compared to intact animal values. Also, our results showed an increase levels of collagen fibers (I and III) surrounding lung vessels after vagotomy that would certainly limit the force transferred

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among airway muscle cells and thereby decrease bronchoconstriction (Araujo et al., 2008). In addition, increases in the number of elastic fibers were observed in lung

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vessels and airways after vagotomy. Elastic fibers are an important extracellular matrix component with a major role in regulating airway patency and lung elastic recoil (Araujo et al., 2008). The imbalance of these components indicated remodelling in the vagotomized animal and that the M3 receptors are involved (Kistemaker and Gosens, 2015). Airway remodelling is associated with ACh release in airway disease (Koarai and Ichinose, 2018) and vesicular ACh transporter (Pinheiro et al., 2015). Also, it is known that normal human lung fibroblasts cells express muscarinic receptor proteins which are involved with fibroblast proliferation (Matthiesen et al., 2006).

Conclusions The central and peripheral mechanisms associated with many modulatory substances used to control airway smooth muscle activity are still very complex. Our data reveal the evident participation of the vagus nerve in regulating respiratory mechanics (as evidenced by lung total resistance, airway resistance, elastance, complacence, lung tissue damping and lung tissue elastance) and morphological parameters (as evidenced by the ratio of wall thickness to airway area and contents of collagen and elastic fibers). Immediately after vagotomy, the MCh and subsequent Salb

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or Ipra responses verified impairments in most respiratory mechanical parameters that were associated with poor efficiency in the modulation of smooth muscle and airway remodelling. Therefore, bilateral vagotomy promotes changes in lung function

parameters induced by a muscarinic agonist, β2-adrenergic agonist and muscarinic

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antagonist and in lung morphology in male Swiss mice.

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Conflict of Interest

Author Statement

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No applicable

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The authors have no conflicts of interest to declare.

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Authors’ contributions

LOL and ACRL (graduate students) contributed to the collection of mechanical data and

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histological analyzes. BZ and RS analyzed the data and drafted the manuscript. RS designed the study, supervised the laboratory experiments and contributed to the writing of the final version of the manuscript. All authors have read the final version of the manuscript and approved its submission.

Acknowledgements

Our study was financially supported by the UNIFAL-MG (Federal University of Alfenas, Minas Gerais). We are grateful to CAPES for providing the post-doctoral

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fellowship to BZ (process number 23087.001310/2019-52).

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Zefirov, T.L., Sviatova, N.V., 1997. Effect of vagotomy on heart rate in intact and sympathectomized rats of different ages. Bull. Exp. Biol. Med. 124, 21-24.

Legends:

Jo

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Fig. 1. Changes in respiratory mechanics in vagotomized mice. The values are presented as the means ± SEM. Rtot = total resistance (A), Raw = airway resistance (B), E = elastance (C), C = compliance (D), Gtis = lung tissue damping (E), and Htis = lung tissue elastance (F) in mice in the Vi and Vx groups (n = 7-15 mice per group). * Comparison of matched aerosolized substances between Vi and Vx group (* p<0.05, ** p<0.01, and *** p<0.001), Mann-Whitney test. # Comparison of the aerosolized substances with saline in the Vi group (# p<0.05, ## p<0.01, and ### p<0.001). + Comparison of the aerosolized substances with saline in the Vx group (+ p<0.05, ++ p<0.01, and +++ p<0.001), Kruskal-Wallis test followed by Dunn’s multiple comparisons test.

A

In ta c t v a g u s 2 .5

1 .5

### +++

2 .0

**

P ro to c o l 1

++

1 .5

P ro to c o l 2

#

***

1 .0

*** 0 .5

+++

R aw ( c m H 2 O .s /m L )

R t o t ( c m H 2 O .s /m L )

B

V a g o to m y

### P ro to c o l 1

++

##

** 0 .5

0 .0

**

0 .0

S a lin e

MCh

S a lb

S a lin e

Ip r a

C

MCh

Ip r a

S a lb

D 60

ro of

+++

**

++

P ro to c o l 1

*

###

###

40

0 .0 5

P ro to c o l 2

** 20

P ro to c o l 2

0 .0 4

+

+++

0 .0 3

**

##

###

0 .0 2

-p

++

C ( c m H 2 O /m L )

P ro to c o l 1

E ( c m H 2 O /m L )

P ro to c o l 2

1 .0

0 .0 1

0

0 .0 0

MCh

S a lb

Ip r a

E

MCh

S a lb

Ip r a

F

15

50

+++

5

na

+

#

**

0

MCh

Jo

ur

S a lin e

S a lb

H tis ( c m H 2 O /m L )

###

10

P ro to c o l 1

P ro to c o l 2

lP

P ro to c o l 1

G tis ( c m H 2 O /m L )

S a lin e

re

S a lin e

P ro to c o l 2

##

40 +

30

* 20

10

0

Ip r a

S a lin e

MCh

S a lb

Ip r a

Table 1. The values are expressed as mean ± SEM (n = 5-7). *(p<0.05) significantly different from Vi, Mann Whitney test. Table 1. Lung histology Intact vagus (Vi)

Vagotomy (Vx)

6.9 ± 0.64

4.1 ± 0.7*

Airway

0.2 ± 0.03

0.17 ± 0.02

Lung vessel

2.7 ± 0.5

4.9 ± 0.7*

Wall thickness/airway area (µm.104)

Collagen III fibers content (%) Airway

0.040 ± 0.01

Lung vessel

0.7 ± 0.2

-p

Elastic fibers content (%)

ro of

Collagen I fibers content (%)

0.044 ± 0.01 1.4 ± 0.2*

22.2 ±1.3

17.5 ± 1.0*

Lung vessel

37.2 ± 2.5

27.6 ± 2.2*

re

Airway

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The values are expressed as mean ± SEM (n = 5-7). *(p<0.05) significantly different from Vi, Mann Whitney test.