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Eight pig tracheal strips were stimulated to contract with log increments of methacholine from 10 -8 to 10 -5 M. For each strip, the concentration-response was repeated four times in a randomized order to measure isometric force, isotonic shortening against a load corresponding to either 5 or 10 % of a reference force, and average force, stiffness, elastance and resistance over one cycle while the strip length was oscillating sinusoidally by 5 % at 0.2 Hz. For each readout, the logEC50 was calculated and compared. Isotonic shortening with a 5 % load had the lowest logEC50 (-7.13), yielding a greater sensitivity than any other contractile readout (p<0.05). It was followed by isotonic shortening with a 10 % load (-6.66), elastance (-6.46), stiffness (-6.46), resistance (-6.38), isometric force (-6.32), and average force (-6.30). Some of these differences were significant. For example, the EC50 with the average force was 44 % greater than with the elastance (p=0.001). The methacholine sensitivity is thus affected by the contractile readout being measured., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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 β 2 -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 vagus nerve modulates the lung function in response to bronchoconstriction and bronchodilation, as well as lung architecture of mice., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare., (Copyright © 2019. Published by Elsevier B.V.)

A certain amount of time is required to achieve a maximal contraction from airway smooth muscle (ASM) and stretches of substantial magnitude, such as the ones imparted by deep inspirations (DIs), interfere with contraction. The duration of ASM contraction without interference may thus affect its shortening, its mechanical response to DIs and the overall toll it exerts on the respiratory system. In this study, the effect of changing the interval between DIs on the dynamics of ASM was examined in vitro. Isolated bronchi derived from guinea pigs were held isotonically and stimulated to both contract and relax, in a randomized order, in response to 10 -5 M of methacholine and 10 -6 M of isoproterenol, respectively. Interference to ASM was inflicted after 2, 5, 10 and 30 min in a randomized order, by imposing a stretch that simulated a DI. The shortening before the stretch, the stiffness before and during the stretch, the post-stretch elongation of ASM and the ensuing re-shortening were measured. These experiments were also performed in the presence of simulated tidal breathing achieved through force fluctuations. The results demonstrate that, with or without force fluctuations, increasing the interval between simulated DIs increased shortening and post-stretch elongation, but not stiffness and re-shortening. These time-dependent effects were not observed when ASM was held in the relaxed state. These findings may help understand to which extent ASM shortening and the regulatory effect of DI are affected by changing the interval between DIs. The potential consequences of these findings on airway narrowing are also discussed., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Altered extracellular matrix (ECM) production by airway smooth muscle cells (ASMCs) is an important feature of airway remodeling. Muscarinic receptor agonists contribute to ECM production in vivo, but the mechanisms involved remain unclear. This study attempted to investigate the role of methacholine in promoting ECM production by human ASMCs (HASMCs) and the underlying mechanism. We found that methacholine induced the expression of collagen I protein and multiple ECM genes. β-catenin signaling was activated in this process upon GSK3β phosphorylation, leading to upregulation of total and active β-catenin. Silencing β-catenin by specific small interfering RNA (siRNA) or with the β-catenin inhibitor, PKF115-584, decreased collagen I expression. Conversely, overexpression of active β-catenin by adenoviruses carrying the S33Y-β-catenin mutant increased the methacholine-induced collagen I expression. Furthermore, methacholine induced TGF-β expression in HASMCs, while pan-TGF-β-neutralizing antibody only partially decreased collagen I expression. These findings suggest that methacholine induced ECM production through β-catenin signaling and partially through TGF-β., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The complexity of respiratory dynamics is decreased, in association with disease severity, in patients with asthma. However, the pathophysiological basis of decreased complexity of breathing pattern in asthma is not clear. In the present study, we investigated the effect of airway remodeling and hyperresponsiveness induced by repeated bronchoconstriction (using methacholine) on breathing pattern in rats with or without allergen-induced sensitization. Entropy analysis of respiratory variability showed decreased irregularity (less complexity) of respiratory rhythm in this rat model of asthma. Airway remodeling and hyperresponsiveness induced by repeated bronchoconstriction also led to increased regularity of respiratory dynamics in sensitized rats. However, these airway alterations had no significant effect on the complexity of breathing pattern in non-sensitized rats. Our results indicate that mechanical respiratory alterations cannot per se attenuate the complexity of respiratory dynamics, unless there is an underlying inflammation. We suggest further studies on underlying mechanisms of breathing variability with focus on respiratory control alterations due to airway inflammation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Ovalbumin (OVA) sensitization has limitations in modelling asthma. Thus, we examined the value of allergic sensitization using a purified natural allergen, house dust mite (HDM), over the sensitization performed with OVA. Mice were sham-treated, or sensitized with OVA- or HDM with identical chronology. Airway resistance, tissue damping and elastance were assessed under control conditions and after challenging the animals with methacholine (MCh) and the specific allergen. Inflammatory profile of the bronchoalveolar lavage fluid was characterized and lung histology was performed. While no difference in the lung responsiveness to the specific allergen was noted, hyperresponsiveness to MCh was observed only in the HDM-sensitized animals in the lung peripheral parameters. Lung inflammation differed between the models, but excessive bronchial smooth muscle remodelling occurred only with OVA. In conclusion, we demonstrate that a purified natural allergen offers a more relevant murine model of human allergic asthma by expressing the key features of this chronic inflammatory disease both in the lung function and structure., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The factors altering the bronchodilatory response to a deep inspiration (DI) in asthma are important to decipher. In this in vitro study, we investigated the effect of changing the duration between DIs on the rate of force recovery post-DI in guinea pig bronchi. The airway smooth muscle (ASM) within the main bronchi were submitted to length oscillation that simulated tidal breathing in different contractile states during 2, 5, 10 or 30min prior to a larger length excursion that simulated a DI. The contractile states of ASM were determined by adding either methacholine or isoproterenol. Irrespective of the contractile state, the duration between DIs neither affected the measured force during length oscillation nor the bronchodilator effect of DI. Contrastingly, the rate of force recovery post-DI in contracted state increased as the duration between DIs decreased. Similar results were obtained with contracted parenchymal strips. These findings suggest that changing the duration between DIs may alter the rate of ASM force recovery post-DI and thereby affect the rate of renarrowing and the duration of the respiratory relief afforded by DI., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Studying airway smooth muscle (ASM) in conditions that emulate the in vivo environment within which the bronchi normally operate may provide important clues regarding its elusive physiological function. The present study examines the effect of lengthening and shortening of ASM on tension development in human bronchial segments. ASM from each bronchial segment was set at a length approximating in situ length (Linsitu). Bronchial tension was then measured during a slow cyclical strain (0.004Hz, from 0.7Linsitu to 1.3Linsitu) in the relaxed state and at graded levels of activation by methacholine. In all cases, tension was greater at longer ASM lengths, and greater during lengthening than shortening. The threshold of methacholine concentration that was required for ASM to account for bronchial tension across the entire range of ASM lengths tested was on average smaller by 2.8 logs during lengthening than during shortening. The length-dependency of ASM tension, together with this lower threshold of methacholine concentration during lengthening versus shortening, suggest that ASM has a greater ability to resist airway dilation during lung inflation than to narrow the airways during lung deflation. More than serving to narrow the airway, as has long been thought, these data suggest that the main function of ASM contraction is to limit airway wall distension during lung inflation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The effect of remodeling on airway function is uncertain. It may affect airway compressibility during forced expirations differently than airflow resistance, providing a tool for its assessment. The aim of the current study was to compare the effects of acute and chronic antigen challenge on methacholine-induced bronchoconstriction assessed from resistance and maximal tidal expiratory flow. Balb/C mice were sensitized with ovalbumin (OVA) and challenged either daily for three days with intra-nasal OVA or daily for 5 days and three times a week for 5 subsequent weeks. Acute and chronic allergen challenge induced airway hyperresponsiveness (AHR) to methacholine. However the relationship between maximal tidal expiratory flow and resistance during methacholine challenge was different between the two conditions, suggesting that the determinants of AHR are not identical following acute and chronic allergen exposure. We conclude that the contrast of changes in maximal tidal expiratory flow and respiratory resistance during methacholine-induced bronchoconstriction may allow the detection of the mechanical consequences of airway remodeling., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Infants born very preterm are usually exposed to high oxygen concentrations but this may impair lung function in survivors in later life. However, the precise changes involved are poorly understood. We determined how neonatal hyperoxia alters lung function at mid-adulthood in mice. Neonatal C57BL/6J mice inhaled 65% oxygen (HE group) from birth for 7 days. They then breathed room air until 11 months of age (P11mo); these mice experienced growth restriction. Controls breathed only room air. To exclude the effects of growth restriction, a group of dams was rotated between hyperoxia and normoxia during the exposure period (HE+DR group). Lung function was measured at P11mo. HE mice had increased inspiratory capacity, work of breathing and tissue damping. HE+DR mice had further increases in inspiratory capacity and work of breathing, and reduced FEV100/FVC. Total lung capacity was increased in HE+DR males. HE males had elevated responses to methacholine. Neonatal hyperoxia alters lung function at mid-adulthood, especially in males., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Aspirative capnography may be of help to diagnose early childhood asthma, but clinical usefulness in young children is limited by the relatively high respiratory rate. This study aimed to characterize the [Formula: see text] time course during airway constriction in 8 anesthetized rabbits, artificially ventilated at 30, 60 and 80breaths/min. Methacholine was inhaled to double the respiratory resistance measured at 8Hz by the forced oscillation technique. The capnogram shape changed in response to both methacholine and ventilatory frequency. Slope of phase II, the peak of first-order time derivative and trough of the second-order time derivative of the [Formula: see text] signal, were significantly attenuated after methacholine compared with baseline at all breathing rates (p<0.02). Moreover, significant correlations between respiratory reactance and resistance were observed with the phase III slope and the angle described by phase II and phase III (p<0.01). It is concluded that capnography may be useful to identify acute airway changes related to bronchoconstriction, even at high breathing frequencies., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Respiratory responses to bronchoconstriction in asthma have been partially assessed and their significance is unclear. In 44 mild asthma patients we investigated respiratory responses during increasing levels of methacholine-induced bronchoconstriction. Inspiratory muscle activity, tidal volume, inspiratory and expiratory times were continuously monitored; breathing discomfort was rated. Mean inspiratory flow, respiratory frequency and ventilation were calculated. Lung function was assessed prior to and at maximum bronchoconstriction. Bronchoconstriction "dose-dependently" increased inspiratory muscle activity and breathing discomfort (P<0.01). In 37 patients (84.1%), the increase in inspiratory muscle activity was associated with increases in mean inspiratory flow and ventilation (P<0.01) because of selective rises in breathing depth (volume responders), or rate (frequency responders) or both (dual responders). In seven patients (15.9%) ventilation was unchanged. Individual respiratory responses were reproducible. With bronchoconstriction, frequency responders displayed greater hyperinflation and stronger breathing discomfort than volume responders (P<0.01). Analysis of the responses to induced bronchoconstriction disclosed distinctive and reproducible respiratory adjustments that may identify functionally different asthma subpopulations., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The explanted lung slice has become a popular in vitro system for studying how airways contract. Because the forces of airway-parenchymal interdependence are such important modulators of airway narrowing, it is of significant interest to understand how the parenchyma around a constricting airway in a lung slice behaves. We have previously shown that the predictions of the 2-dimensional distortion field around a constricting airway are substantially different depending on whether the parenchyma is modeled as an elastic continuum versus a network of hexagonally arranged springs, which raises the question as to which model best explains the lung slice. We treated lung slices with methacholine and then followed the movement of a set of parenchymal landmarks around the airway as it narrowed. The resulting parenchymal displacement field was compared to the displacement fields predicted by the continuum and hexagonal spring network models. The predictions of the continuum model were much closer to the measured data than were those of the hexagonal spring network model, suggesting that the parenchyma in the lung slice behaves like an elastic continuum rather than a network of discrete springs. This may be because the alveoli of the lung slice are filled with agarose in order to provide structural stability, causing the parenchyma in the slice to act like a true mechanical continuum. How the air-filled parenchyma in the intact lung behave in vivo remains an open question., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We studied repeatedly the development of bronchial hyperreactivity (BHR) and bronchoalveolar lavage fluid (BALF) in rats undergoing different modes of ovalbumin exposures. Treatment was two intraperitoneal injections of ovalbumin in Groups 1-3, followed by one ovalbumin aerosolization in Groups 2 and 3, while rats in Group 4 received repeated ovalbumin aerosols after one single intraperitoneal injection. BHR was assessed longitudinally on day 0 (before treatment) and on day 14 (Groups 1 and 2) or 20 (Groups 3 and 4) and cellular influx was estimated from BALF. No BHR or change in BALF cellular profile was detected in Groups 1-3. However, the infiltration of inflammatory cells, associated with BHR (PC(100) 8.9+/-1.3 microg/kg vs. 4.2+/-1.1 microg/kg), was observed in Group 4. The BHR was always associated with increased number of eosinophils in the BALF. The substantial interindividual variability confirmed the need for a technique that permits follow-up of lung responsiveness and BALF profile. This approach evidenced strong associations between the severity of BHR and the eosinophilia., (Copyright 2009 Elsevier B.V. All rights reserved.)

Airway responses to intravenous (i.v.) and inhaled (i.h.) delivery of methacholine (MCh) in BALB/c and C57BL/6 mouse strains have been compared with and without ovalbumin (OVA)-induced airway inflammation. Bronchial reactivity to MCh was assessed in anaesthetised and tracheostomised animals by using an animal ventilator (flexiVent). We partitioned the response of the lungs into airway and parenchymal components in order to compare the contributions of the airways with those of the lung parenchyma to the pulmonary mechanical responses resulting from different routes of MCh administration. Our results indicate disparate physiological responses. Intravenous MCh delivery induced a higher maximum lung resistance than i.h. MCh in OVA-treated BALB/c mice but not in C57BL/6 mice. Inhaled MCh delivery led to a significantly larger fall in lung compliance and a greater impact on peripheral airways than i.v. MCh in both strains. In conclusion, i.v. and i.h. MCh produced disparate effects in different murine strains and variant responses in inflamed airways and healthy controls. The two methods of MCh delivery have important advantages but also certain limitations with regard to measuring airway reactivity in a murine model of allergic asthma.

The airways are thought to dilate during exercise in humans but the time course and mechanisms of the response are not fully described. The aim of the study was to document changes in airway calibre during electrically induced muscular contractions (MC) in spontaneously breathing rabbits. Experiments were also performed after induced bronchoconstriction to assess the effect of change in breathing pattern on airway calibre during MC. Respiratory resistance (R(RS)) was measured in 12 rabbits using the forced oscillation technique at 20 Hz before, during and after 30s MC in control conditions and after methacholine induced bronchoconstriction (Mch). MC was associated with significant decrease in R(RS) both at control and Mch. The MC induced increase in V(E) occurred with significant albeit small change in mean tidal volume (V(T)) at Mch but not control. An augmented breath (AB) occurred in 29/35 MCs and was usually associated with an abrupt drop in R(RS). The decrease in R(RS) induced by AB was significantly larger at Mch compared with control. Passively inflating the lung after MC induced significantly larger decrease in R(RS) than AB during MC. The data indicate bronchodilation by MC in spontaneously breathing rabbits. The mechanisms appear to include AB dependent airway wall stretching as well as removal of cholinergic input to the airway smooth muscle.

We investigated whether end-expiratory lung volume (EELV) or lung mechanical parameters are more sensitive for the detection of a compromised gas exchange during bronchoconstriction and after surfactant depletion. EELV was determined via SF(6) multiple breath wash-outs in mechanically ventilated rabbits while a positive end-expiratory pressure (PEEP) of 1, 3 or 7 cm H(2)O was maintained. Airway resistance (R(aw)) and parenchymal elastance (H) were estimated from the pulmonary input impedance measured at each PEEP level by means of forced oscillations. Measurements were repeated during i.v. methacholine (MCh) infusions and following lung injury induced by saline lavage. MCh induced marked elevations in R(aw), with no significant change in EELV or H at any PEEP. After lavage, the severity of hypoxia was reflected systematically in significant decreases in EELV at all PEEP levels (-42+/-13%, -26+/-4%, and -18+/-5% at 1, 3 and 7 cm H(2)O, respectively), whereas compromised gas exchange was not associated with consistent changes in the mechanical parameters at a PEEP of 7 cm H(2)O (20+/-9% and 14+/-9% in R(aw) and H, respectively; p=0.2). We conclude that R(aw) is the only sensitive indicator for the detection of a compromised lung function during MCh infusions, whereas the estimation of EELV is necessary to follow the progression of a lung injury when a high PEEP level is applied.

The multiple breath nitrogen washout (MBNW) can be analysed to produce the parameters Scond and Sacin as measures of ventilation heterogeneity in conductive and acinar airways, respectively. The derivation of these parameters is based on a model of pulmonary ventilation and results of similar modelling suggest that respiratory system conductance (Grs) measured by forced oscillation technique (FOT) is also sensitive to heterogeneity and to airway closure. Therefore, Scond, the volume of gas trapping at FRC (VtrappedFRC) and Grs may be inter-related parameters. These relationships were examined in 12 normals under baseline and bronchoconstricted states. Specific Grs was measured at 5Hz (sGrs5=Grs5/FRC) and Scond, Sacin and VtrappedFRC by MBNW, before and after methacholine challenge. Scond was independently predicted by VtrappedFRC and FRC in a multivariate model (R2=0.68, p=0.002). Post methacholine challenge, Scond related only to VtrappedFRC (R2=0.79, p<0.0001). The absolute change in Scond induced by methacholine challenge were predicted by the changes in VtrappedFRC and sGrs5 in a multivariate model (R2=0.82, p=0.0002). Sacin was unrelated to VtrappedFRC and sGrs5 before and after methacholine challenge. In conclusion, Scond and sGrs5 are measurements that are sensitive to changes occurring to the function of peripheral conducting airways, in particular heterogeneity and airway closure, while Sacin and presumably heterogeneity in terminal airways, are independent of these. Scond is also related to lung size. We review the current state of knowledge of FOT and MBNW in obstructive lung diseases and discuss future research directions.

Efficacy of beta2-agonists (B2), anticholinergics, corticosteroids, anti-inflammatory drugs or antihistamines against methacholine, histamine, or allergen-induced lung constriction was tested in ovalbumin sensitized guinea pigs. Airway resistance (Raw) and parenchymal damping (G) and elastance (H) were determined from low-frequency forced oscillatory input impedance (ZL). ZL was measured under control conditions, during iv infusions of methacholine, and following iv boluses of histamine or ovalbumin. In decreasing potency, ipratropium, salmeterol, fluticasone or cromoglycate reduced the methacholine-induced increases in Raw and G. Only antihistamines had any effects on the histamine-induced increased in Raw, G, and H. The ovalbumin-induced increases in Raw, G and H in the control animals (120 +/- 90%, 201 +/- 126%, 86 +/- 71%) were markedly reduced by pretreatments with antihistamines (18 +/- 11%, 13 +/- 9%, 3+/- 3%) and cromoglycate (29 +/- 13%, 35 +/- 22%, 18 +/- 10%). Bronchoconstriction induced by muscarinic receptor stimulation is inhibited by anticholinergic, anti-inflammatory or beta2-agonist pretreatment; antihistaminic or anti-inflammatory premedication is beneficial if the release of histamine via an exposure to allergen is responsible for the fall in lung function.

Measurement of airway contractile responses to methacholine (MCh) is an important investigational tool in humans and mice. However, i.v. administration of MCh in murine models may suffer from potential cardiac sequelae produced by stimulation of cholinergic receptors within cardiac muscle. Therefore, we studied the i.v. (0-10,000 microg/kg) and aerosolized (inhaled; 0-25 mg/ml) administration of MCh, to determine their effects on pulmonary resistance (RL) and cardiac muscle activity (as heart rate; HR) in anesthetized, mechanically-ventilated C57Bl6 mice. MCh, i.v., increased RL but produced: (1) prolonged asystole (29-47 sec); (2) subsequent overshoot of baseline HR; and (3) prolonged HR recovery times (7-25 min), suggestive of sympathetic modulation after cholinergic stimulation. In contrast, inhaled MCh aerosol produced no change in HR, while increasing RL similar to i.v. MCh. These results suggest that, for specific instances utilizing the plethysmographic technique, inhaled MCh aerosol may be superior to i.v. administration, due to the avoidance of potential bouts of asystole that can confound experimental results and lead to premature death of mice.

Background: Our recently developed murine asthma model is capable of inducing airway-specific chronic inflammatory changes and remodeling, features of human asthma commonly missing in conventional animal models., Objectives: To validate this model by site-specific physiological evaluation of hyperresponsiveness., Methods: Non-sensitized and sensitized mice received either short-term uncontrolled or long-term controlled low-level exposures to aerosolized ovalbumin (OVA). Respiratory impedance (Zrs) was measured in response to increasing doses of methacholine (Mch). The constant-phase model was fitted to Zrs spectra to determine the specific site of hyperresponsiveness., Results: Sensitized acutely exposed mice had significantly increased tissue damping (G), tissue elastance (H) and hysteresivity (eta) in response to Mch, but no significant increase in airway resistance (Raw), indicating tissue-specific hyperresponsiveness. In contrast, sensitized chronically exposed mice had significantly elevated Raw at all concentrations of Mch but no increases in G, H or eta indicating airway-specific hyperresponsiveness., Conclusions: Chronic inhalational exposure of sensitized mice to low-mass concentrations of OVA induces airway-specific hyperresponsiveness.