Methacholine hyperresponsiveness in mice with house dust mite‐induced lung inflammation is not associated with excessive airway constriction ex vivo

Abstract

The role of excessive airway constriction in the hyperresponsiveness to nebulized methacholine in mice with experimental asthma is still contentious. Yet, there have been very few studies investigating whether the increased in vivo response to methacholine caused by experimental asthma is associated with a corresponding increase in ex vivo airway constriction. Herein, the responses to nebulized methacholine in vivo and airway constriction in lung slices ex vivo were studied in 8‐ to 10‐week‐old male mice of two strains, BALB/c and C57BL/6. Experimental asthma was induced by administering house dust mites (HDM) intranasally, once daily, for 10 consecutive days. Complementary ex vivo studies were conducted with excised tracheas to measure and compare isometric force. As expected, the in vivo response to methacholine, and especially the hyperresponsiveness caused by HDM, was greater in BALB/c than in C57BL/6 mice. In contrast, there were no differences in maximal airway constriction between mouse strains, and the hyperresponsiveness to nebulized methacholine caused by HDM in both mouse strains was not associated with a corresponding increase in ex vivo airway constriction. The experiments with excised tracheas demonstrated no differences in isometric force between strains and between mice with and without experimental asthma. It is concluded that the hyperresponsiveness to nebulized methacholine in an acute mouse model of asthma induced by repeated HDM exposures is not associated with excessive airway constriction ex vivo.

Full text

Received: 16 December 2024 Accepted: 19 February 2025
DOI: 10.1113/EP092522
RESEARCH ARTICLE
Methacholine hyperresponsiveness in mice with house dust
mite-induced lung inflammation is not associated with
excessive airway constriction ex vivo
Andrés Rojas-Ruiz1Magali Boucher1Cyndi Henry1Louis Gélinas1
Rosalie Packwood1Percival Graham2Jorge Soliz1Ynuk Bossé1
1Institut Universitaire de Cardiologie et de
Pneumologie de Québec (IUCPQ) – Université
Laval, Québec, Quebec, Canada
2SCIREQ Inc., Montreal, Quebec, Canada
Correspondence
Ynuk Bossé, Institut Universitaire de
Cardiologie et de Pneumologie de Québec
(IUCPQ) – Université Laval, IUCPQ, Pavillon A,
room 2089, 2725, chemin Sainte-Foy,Québec,
QC G1V 4G5, Canada. Email:
ynuk.bosse@criucpq.ulaval.ca
Funding information
Canadian Government | Natural Sciences and
Engineering Research Council of Canada
(NSERC), Grant/Award Numbers:
RGPIN-2020-06355, ALLRP-570485-2021;
Canadian Government | Canadian Institutes of
Health Research (CIHR), Grant/Award
Number: 508356-202209PJT; Fondation de
l’Institut Universitaire de Cardiologie et de
Pneumologie de Québec
Handling Editor: Lee Romer
Abstract
The role of excessive airway constriction in the hyperresponsiveness to nebulized
methacholine in mice with experimental asthma is still contentious. Yet, there have
been very few studies investigating whether the increased in vivo response to
methacholine caused by experimental asthma is associated with a corresponding
increase in ex vivo airway constriction. Herein, the responses to nebulized
methacholine in vivo and airway constriction in lung slices ex vivo were studied in
8- to 10-week-old male mice of two strains, BALB/c and C57BL/6. Experimental
asthma was induced by administering house dust mites (HDM) intranasally, once
daily, for 10 consecutive days. Complementary ex vivo studies were conducted with
excised tracheas to measure and compare isometric force. As expected, the in vivo
response to methacholine, and especially the hyperresponsiveness caused by HDM,
was greater in BALB/c than in C57BL/6 mice. In contrast, there were no differences
in maximal airway constriction between mouse strains, and the hyperresponsiveness
to nebulized methacholine caused by HDM in both mouse strains was not associated
with a corresponding increase in ex vivo airway constriction. The experiments with
excised tracheas demonstrated no differences in isometric force between strains and
between mice with and without experimental asthma. It is concluded that the hyper-
responsiveness to nebulized methacholine in an acute mouse model of asthma induced
by repeated HDM exposures is not associated with excessive airway constriction ex
vivo.
KEYWORDS
animal model, methacholine response, smooth muscle contraction
1INTRODUCTION
Hyperresponsiveness to inhaled methacholine is a hallmark of human
asthma (Nair et al., 2017). It is thus generally accepted that an
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
© 2025 The Author(s). Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.
appropriate animal model of asthma should exhibit this feature.
As a result, most, if not all, mouse models of asthma are hyper-
responsive to nebulized methacholine. It is often assumed that this
hyperresponsiveness caused by experimental asthma is mediated,
Experimental Physiology. 2025;1–13. wileyonlinelibrary.com/journal/eph 1

2ROJAS-RUI Z ET AL.
at least in part, by excessive airway constriction. Yet, there is a
dearth of studies investigating whether the increased in vivo response
to methacholine in specific models of asthma is associated with a
corresponding increase in ex vivo airway constriction. In the present
study, we investigated the association between the in vivo response to
nebulized methacholine and the ex vivo constriction of airways in two
mouse strains with and without experimental asthma.
BALB/c and C57BL/6 were chosen not only because they are the
most widely used mouse strains for modelling human asthma (Carroll
et al., 2023), but also because their innate response to nebulized
methacholine (i.e., in the absence of experimental asthma) is vastly
different, with BALB/c mice being more responsive than C57BL/6 mice
(Berndtetal.,2011; Duguet et al., 2000; Held & Uhlig, 2000; Leme
et al., 2010; Levitt & Mitzner, 1989). The BALB/c mice are also generally
more susceptible than C57BL/6 mice to acquire hyperresponsiveness
(i.e., induced by experimental asthma) (Adler et al., 2004; Boucher
et al., 2021; De Vooght et al., 2010; Evans et al., 2015; Ewart et al.,
2000; Gueders et al., 2009; Hirota, Ask et al., 2009; Kelada et al.,
2011; Kenyon et al., 2003; Koya et al., 2006; Li et al., 2017; Sahu
et al., 2010; Shinagawa & Kojima, 2003; Takeda et al., 2001; Van Hove
et al., 2009; Zhang et al., 1997; Zhu & Gilmour, 2009). Given that many
early studies, conducted with both mice without experimental asthma
(Weinmann et al., 1990) and non-asthmatic humans (Armour, Black
et al., 1984; Armour, Lazar et al. 1984; Cerrina et al., 1986; de Jongste
et al., 1988; Roberts et al., 1987; Taylor et al., 1985; Thomson, 1987),
have demonstrated that the level of airway responsiveness in vitro
rarely matches the degree of in vivo responsiveness, it is hypothesized
that the in vivo hyperresponsiveness caused by experimental asthma
will not be associated with changes in ex vivo airway constriction in
either mouse strain.
2MATERIALS AND METHODS
2.1 Ethical approval
All methods were approved by the Committee of Animal Care of
Université Laval, following the guidelines from the Canadian Council on
Animal Care (2020-652-4). The research also adhered to Experimental
Physiology’s policies regarding animal experiments.
2.2 Mice
Forty-eight male BALB/c mice (Charles River, Saint-Constant, QC,
Canada) and 48 male C57BL/6 mice (The Jackson Laboratory, Bar
Harbor, ME, USA) were studied between the ages of 8 and 10
weeks. Males were used because they have more airway smooth
muscle (ASM) (Gill et al., 2023) and their response to nebulized
methacholine is markedly greater than that of females (Card et al.,
2006; Gill et al., 2023). They were provided with food and water ad
libitum.
Highlights
∙What is the central question of this study?
In this study, we investigated the association
between the in vivo response of the respiratory
system to nebulized methacholine and the ex vivo
responsiveness of airways in two mouse strains
with and without experimental asthma induced by
repetitive intranasal exposures to house dust mites.
The ex vivo assays included measurements of air-
way constriction in lung slices and measurements of
isometric force with excised tracheas.
∙What is the main finding and its importance?
Although striking differences in the in vivo response
to methacholine were observed between mouse
strains and between mice with and without
experimental asthma, these changes were not
matched by corresponding changes in ex vivo
airway responsiveness.
2.3 Experimental model of asthma
Experimental asthma was induced as previously described (Boucher
et al., 2021; Rojas-Ruiz et al., 2024). Briefly, mice were exposed to
either 25 µL of 0.9% sterile saline or 25 µL of 2 mg/mL house-dust mite
(HDM) extract (Dermatophagoides pteronyssinus, lot number 360923;
Greer Laboratories, Lenoir, NC, USA) diluted in 0.9% sterile saline to
induce allergic lung inflammation. The endotoxin concentration was
47.3 EU/mg of HDM extract. The exposure occurred once daily via
an intranasal instillation for 10 consecutive days under isoflurane
anaesthesia. All measurements were made the day after the last
expos ure.
2.4 Mechanical ventilation
Twelve mice per group were anaesthetized by administering
ketamine (100 mg/kg) and xylazine (10 mg/kg) (Dechra Veterinary
Products, Pointe-Claire, Canada) intraperitoneally. They were then
tracheotomized and connected to the flexiVent (FX Module 2, SCIREQ,
Montreal, QC, Canada) through an 18-gauge cannula in a supine
position. To prevent leakage, a surgical thread was used to secure and
seal the trachea on the cannula. They were ventilated mechanically
with air at a tidal volume of 10 mL/kg with an inspiratory-to-expiratory
time ratio of 2:3 at a breathing frequency of 150 breaths/min and with
a positive end-expiratory pressure of 3 cmH2O. Once the ventilation

ROJAS-RUI Z ET AL.3
was underway, muscle relaxation was induced by injecting 100
and 300 µL of pancuronium bromide (0.12 mg/mL) intramuscularly
and intraperitoneally, respectively, to avoid spontaneous breathing
during the experiments. Heart rate was monitored continuously by
electrocardiography throughout the experiment to ensure proper
anesthesia.
2.5 The in vivo methacholine challenge
While on the flexiVent, mice were subjected to a multiple-
concentration challenge with methacholine. The concentrations
used were 0, 10 and 30 mg/ml, all diluted in PBS. They were nebulized
at 5 min intervals. For each concentration, the nebulizer for small
particle size (Aeroneb Lab, Aerogen, Galway,Ireland) was operating for
a duration of 10 s at a duty cycle of 50% under regular ventilation. Two
lung recruitment manoeuvres to 30 cmH2O were performed before
the start of the methacholine challenge and after each subsequent
concentration to avoid alveolar collapse, in line with an optimized
protocol of mechanical ventilation (Reiss et al., 2011).
To monitor the changes in respiratory mechanics during the
methacholine challenge, two short oscillometric perturbations of small
amplitude, called the SnapShot-150 (1.25 s) and the Quick Prime-3 (3
s) were used. The former consists of a single sine-wave oscillation at
2.5 Hz that allows resistance (Rrs) and elastance (Ers ) of the respiratory
system to be calculated based on the linear single-compartment
model (Bates, 2009). The latter is an oscillometric perturbation
composed of an input flow signal made of 13 sine waves of mutually
prime frequencies with different amplitudes and phases, allowing the
impedance spectrum of the respiratory system from 1 to 20.5 Hz to
be calculated (Bates et al., 2011). The impedance spectrum was then
fitted to the constant phase model for computing three parameters
(Hantos et al., 1992). One is airway resistance (Raw), which reflects the
resistance to airflow in conducting airways, although it can sometimes
be influenced by the chest wall (Hirai et al., 1999; Ito et al., 2007;Sudy
et al., 2019). Another one is tissue resistance (G), which reflects the
tissue resistance of the lung and the chest wall (Hirai et al., 1999;Ito
et al., 2007; Sudy et al., 2019) but is also sensitive to small airway
narrowing heterogeneity (Lutchen et al., 1996). The final one is tissue
elastance (H), which reflects the elastance of the whole lung and is thus
sensitive to both the accessible (i.e., reachable from the mouth) volume
of the lung and the tissue stiffness of the lung and the chest wall (Hirai
et al., 1999; Sudy et al., 2019). The hysteresivity (η),whichistheratioof
Gover H, was also determined.
The SnapShot-150 and the Quick Prime-3 were each actuated twice
at baseline in an alternating fashion. They were then each actuated
10 times, again in an alternating fashion, after each methacholine
concentration, starting 10 s after the nebulization. Eight seconds of
tidal breathing was intercalated between each actuation to avoid
desaturation. The changes in oscillometric readouts described above,
namely Rrs,Ers ,H,G,Raw and η, from their baseline values to their peak
values following each concentration of methacholine were used to
trace the concentration–response curve. The methacholine response
was then quantified by measuring the area under the curve for each
oscillometric readout.
2.6 Euthanasia
The mice undergoing the methacholine challenge, representing half
of the mice, were killed by exsanguination after the last flexiVent
measurement. The other half were killed by lethal intraperitoneal
injection of ketamine (200 mg/kg) and xylazine (20 mg/kg). The latter
mice were required for collection of tracheas and lungs for the ex vivo
assays (described below). It was not possible to conduct the in vivo
and ex vivo experiments on the same mice, because the cannulation
required for the flexiVent alters tracheal contraction, and the in vivo
exposure to methacholine compromises airway constriction in lung
slices because it prevents a smooth and homogeneous filling of the
lungs with agarose.
2.7 Wet lung weight
The lungs were surgically removed, cleaned, and weighed using a
laboratory digital scale (Mettler Toledo, Mississauga, ON, Canada).
2.8 Histology
Histology was performed on the left unilobed lung in a total of 12 mice
per group. The lobe was excised and immersed in formalin for 24 h
for fixation. The tissue was subsequently dehydrated by substituting
the formalin with increasing concentrations of ethanol. The lobe
was then embedded in paraffin and cut transversely into 5-µm-thick
sections. Sections were deposited on microscope slides, stained, and
scanned with a NanoZoomer Digital scanner (Hamamatsu Photonics,
Bridgewater, NJ, USA) at ×40 magnification.
Histological alterations seen in this specific model of experimental
asthma were characterized previously (Boucher et al., 2021; Gill et al.,
2023; Rojas-Ruiz et al., 2024). It was repeated herein to confirm
the establishment of allergic lung inflammation. Lung sections stained
with Haematoxylin and Eosin were used to quantify tissue infiltration
with inflammatory cells. Sixteen non-overlapping photomicrographs
(1440 ×904 pixels) from four non-contiguous lung sections were
scored blindly from zero (no inflammation) to five (very severe
inflammation). Lung sections stained with Periodic Acid–Schiff (PAS)
and Alcian Blue were used to count the number of goblet cells. All
airways cut transversely in four non-contiguous lung sections were
analysed, representing 9–38 airways per mouse (average of 23.2 ±6.8).
Lung sections stained with Masson Trichrome were used to quantify
the content of ASM and the thickness of the epithelium. All airways
cut transversely in four non-contiguous lung sections were analysed,
representing 10–34 airways per mouse (average of 18 ±5.6). The
content of ASM in each airway was calculated by measuring the
area occupied by the muscle divided by the square of its basement

4ROJAS-RUI Z ET AL.
membrane perimeter (Bullone et al., 2014). The thickness of the
epithelium was also analysed for the same airways by measuring the
area occupied by the epithelium divided by the basement membrane
perimeter.
2.9 Constriction of airways in lung slices
Airway constriction in lung slices was measured in 12 mice per group.
After euthanasia, thoracotomy was performed, and the chest was
opened. While the lungs were still resting inside the thorax, the right
lobe was ligated, and a tracheotomy was performed in the lower part of
the trachea. Then, 350 µL of agarose (catalogue no. 1613112; Bio-Rad
Laboratories, Redmond, WA, USA) at 40◦C was infused through the
trachea using an 18-gauge syringe. The left lung was then plunged into
ice-cold Hank’s balanced salt solution (HBSS) for 30 min to polymerize
the agarose. Slices of 200 µm thick were cut in the sagittal orientation
using the Compresstome VF-310 (Precisionary Instruments, Ashland,
MA, USA). Slices were selected more precisely in the middle part of
the lobe, avoiding the first and last 20% edges. Finally, lung slices
were placed into a six-well plate containing Dulbecco’s modified
Eagle’s medium supplemented with 1% penicillin, streptomycin and
amphotericin B and kept at 37◦C for 24 h. Fourslices were put into each
well.
Airway constriction was measured by the physioLens (SCIREQ,
Montreal, QC, Canada) as recently described (Boucher et al., 2024).
This was done one well at a time. The first step consisted of removing
the Dulbecco’s modified Eagle’s medium and washing the slices twice
with warm HBSS using a pipette. The slices were then fixed in the
middle of the well with a SCIREQ slice holder (physioMesh) in 6 mL of
warm HBSS and transferred to the physioLens. The experimenter sub-
sequently selected on the computer screen all clearly visible airways
with a perpendicular orientation in relationship to the field of view (i.e.,
circular airways), with no apparent damage and with clear beating of
the cilia. The following steps were then executed automatically by the
physioLens. First, the lung slices were washed again with HBSS and
left untouched for 3 min. This washing step allowed many airways to
dilate fully. Then, the smooth muscle was stimulated to contract with
methacholine dissolved in HBSS warmed to 37◦C at four incremental
concentrations: 10−7,10
−6,10
−5and 10−4M. Each concentration was
replaced at 3 min intervals. Images of the luminal area were collected
in all preselected airways within the well every 1 min. Constriction,
expressed as a percentage reduction in relationship to the initial
luminal area (the one at the end of the last washing step with HBSS),
was also calculated automatically. The maximal constriction obtained
during the 3 min of recording at each methacholine concentration was
used to generate the concentration–response curve. These procedures
were then repeated for each of the other wells.
2.10 Contraction of excised tracheas
Tracheal contraction was measured in 12 mice per group (in the same
mice as for the airway constriction in lung slices). The whole trachea
was collected and immersed in Krebs solution (pH 7.4, 111.9 mM NaCl,
5.0 mM KCl, 1.0 mM KH2PO4, 2.1 mM MgSO4, 29.8 mM NaHCO3,
11.5 mM glucose and 2.9 mM CaCl2). It was then mounted horizontally
in a 5 mL organ bath containing Krebs solution maintained at 37◦C. The
preparation was coupled to a force transducer (Harvard Apparatus,
St Laurent, QC, Canada) for measuring isometric force. A resting
distending force of 5 mN was applied. Before the contractile assays,
the trachea was subjected to a period of conditioning, during which
time it was stimulated to contract repeatedly for 5 min at 10 min
intervals with 10−5M methacholine until a reproducible force was
recorded.
Cumulative concentration–response curves were then generated
with methacholine and KCl. Methacholine was added in logarithmic
increments at 5 min intervals from 10−7to 10−4M. The concentration
of KCl was doubled at 5 min intervals from 20 to 160 mM. The
peak force obtained at each concentration was used to generate
the concentration–response curves. The trachea was washed
repeatedly with fresh Krebs for ≥30 min between methacholine
and KCl. To measure potency, the EC50 (the concentration causing
half of the maximal response) was also calculated by fitting the
data by the least-squares method to the following equation:
y=bottom +x(top −bottom)/(EC50 +x).
2.11 Data analysis
Two-way ANOVAs were used to assess the effect of experimental
asthma, the mouse strain and their interaction on each measured
readout. When the interaction was significant, it was followed by
Sidak’s multiple comparisons test specifically to compare mice with
and without experimental asthma in each mouse strain. Three-way
ANOVAswere also used for the ex vivo contractile assays to assess the
effect of experimental asthma, the mouse strain, the concentration–
response to methacholine and their interactions. Statistical analyses
were performed with Prism (v.10.2.1; GraphPad, San Diego, CA, USA).
3RESULTS
The body weight was similar in both mouse strains and was not affected
by HDM (Figure 1a). The wet weight of the lungs was also not different
between BALB/c and C57BL/6 mice. However, HDM-exposed mice had
heavier lungs (P<0.0001; Figure 1b).
Lung tissue alterations caused by HDM were confirmed by histology
(Figure 2). Lungs of mice exposed to HDM exhibit a greater tissue
infiltration with inflammatory cells (Figure 2a), no change in the
content of ASM (Figure 2b), an increased thickness of the airway
epithelium (Figure 2b) and a higher number of goblet cells (Figure 2c).
The histological alterations caused by HDM were similar between the
two mouse strains; however, C57BL/6 mice exhibited a higher tissue
infiltration with inflammatory cells and a thicker epithelium compared
with BALB/c mice.
The response to nebulized methacholine is depicted in Figure 3.As
expected, HDM enhanced the methacholine response irrespective of

ROJAS-RUI Z ET AL.5
FIGURE 1 Body weight (a) and total lung weight (b) in BALB/c
mice (open circles) and C57BL/6 mice (filled squares) that were
exposed to either saline or HDM. Individual results are presented,
together with means ±SD. Results of two-way ANOVAs are provided
underneath the graphs. n=12 mice per group. Abbreviation: HDM,
house dust mite.
the parameter used to measure the response (∆Rrs,∆Ers ,∆H,∆G,∆Raw
and ∆η;Figure3b–g). The mouse strain also had a significant effect
on all parameters except ∆η, indicating that C57BL/6 mice are less
responsive in vivo than BALB/c mice. There were also significant inter-
actions between HDM and the mouse strain for all parameters except
∆η, suggesting that the in vivo hyperresponsiveness caused by HDM
was greater in BALB/c than in C57BL/6 mice.
Airway constriction in response to methacholine in lung slices
is depicted in Figure 4. Twenty-five to 62 airways were analysed
per mouse (average of 44.8 ±7.1). The HDM significantly affected
airway constriction (Figure 4a). There was also a significant inter-
action between HDM and the concentration response to methacholine,
suggesting that constriction was decreased by HDM at certain
methacholine concentrations. In addition, there was a significant three-
way interaction between HDM, the mouse strain and the concentration
response to methacholine, suggesting that the decreasing effect
of HDM on the methacholine response at certain concentrations
was greater in C57BL/6 than in BALB/c mice. Accordingly, the
maximal constriction in response to methacholine was affected by
neither HDM nor the mouse strain (Figure 4b), but the EC50 was
increased by HDM and was higher in C57BL/6 than in BALB/c mice
(Figure 4c).
The isometric force generated by excised tracheas in response to
increasing concentrations of methacholine is depicted in Figure 5.The
concentration–response curves were affected by neither HDM nor the
mouse strain (Figure 5a). The maximal active force (Figure 5b)and
the methacholine potency (EC50)(Figure5b) were also not affected
by HDM or the mouse strain. Similar results were obtained with KCl
(Figure 6).
4DISCUSSION
This study confirmed that BALB/c mice are more responsive to
nebulized methacholine than C57BL/6 mice (Berndt et al., 2011;
Duguet et al., 2000; Held & Uhlig, 2000; Leme et al., 2010;Levitt&
Mitzner, 1989). This interstrain difference was especially striking in a
model of experimental asthma induced by repeated exposuresto HDM,
which is also in line with results of previous studies (Adler et al., 2004;
Boucher et al., 2021; De Vooght et al., 2010; Evans et al., 2015;Ewart
et al., 2000; Gueders et al., 2009; Hirota, Ask et al., 2009; Kelada et al.,
2011; Kenyon et al., 2003; Koya et al., 2006; Li et al., 2017; Sahu et al.,
2010; Shinagawa & Kojima, 2003; Takeda et al., 2001; Van Hove et al.,
2009; Zhang et al., 1997; Zhu & Gilmour, 2009). The novelty in the
present study is that the in vivo hyperresponsiveness acquired in this
specific, widely used (Carroll et al., 2023) model of asthma was not
associated with a corresponding increase in ex vivo airwayconstriction.
This dissociation between the in vivo and the ex vivo response to
methacholine was shown in two mouse strains.
4.1 Interstrain difference in responsiveness
As already mentioned, it is well established that BALB/c mice are more
responsive to nebulized methacholine than C57BL/6 mice (Berndt
et al., 2011; Duguet et al., 2000; Held & Uhlig, 2000; Leme et al., 2010;
Levitt & Mitzner, 1989). The in vivo results in the present study are thus
consistent with these previous findings.
Comparisons of in vitro airway responsiveness between these
two mouse strains were also undertaken previously. However, the
results were inconsistent. Some studies with excisedtracheas (Boucher
et al., 2021) and lungs (Landgraf & Jancar, 2008) suggested that air-
way responsiveness in C57BL/6 mice is greater than that of BALB/c
mice. Another study demonstrated no interstrain differences in iso-
metric contraction of excised tracheas in response to three different
spasmogens (carbachol, bradykinin and serotonin; Safholm et al.,
2011). In contrast, some studies with lung slices suggested that airway
constriction in BALB/c mice is not only greater (Duguet et al., 2000;
Zeng et al., 2023) but also faster (Duguet et al., 2000) than in C57BL/6
mice. The reason why these results were not reproduced in the pre-
sent study is not entirely clear. It is worth mentioning that the initial
calibre of airways was greater in lung slices derived from BALB/c mice
compared with the ones from C57BL/6 mice (data not shown). Given
that constriction in the present study was expressed as a percentage
of the initial calibre, it is likely that the absolute amount of airway
constriction in BALB/c mice was greater than in C57BL/6 mice, which
would then be consistent with the study by Duguet et al. (2000).
4.2 Responsiveness in experimental asthma
Interestingly, some investigators could induce ex vivo hyper-
responsiveness repeatedly in a mouse model of allergic inflammation

6ROJAS-RUI Z ET AL.
FIGURE 2 Histological analyses on lung sections of BALB/c mice (open circles) and C57BL/6 mice (filled squares) that were exposed to either
saline or HDM. Representative images are shown at the top, and individual results, together with means ±SD, are shown at the bottom. Lung
sections were stained with Haematoxylin and Eosin to quantify tissue infiltration with inflammatory cells (a), Masson Trichome to quantify the
content of airway smooth muscle and the thickness of the epithelium (b) or Periodic Acid–Schiff and Alcian Blue to quantify the amount of goblet
cells in the airway epithelium (c). Results of two-way ANOVAs are provided underneath the graphs. n=12 mice per group. Abbreviation: HDM,
house dust mite.
(Chiba et al., 2005, 2020). They used the main bronchi of male BALB/c
mice sensitized and challenged with ovalbumin. Given that many
early studies conducted with both mice without experimental asthma
(Weinmann et al., 1990) and non-asthmatic humans (Armour, Black
et al., 1984; Armour, Lazar et al. 1984; Cerrina et al., 1986; de Jongste
et al., 1988; Roberts et al., 1987; Taylor et al., 1985; Thomson, 1987)
have demonstrated that the level of in vitro airway responsiveness
rarely matches the degree of in vivo responsiveness, these results are
surprising. They suggest that the choice of the mouse model of asthma
(HDM vs. ovalbumin) or tissue preparation (trachea vs. bronchi) might
influence the presence or absence of ex vivo hyperresponsiveness
(Chiba et al., 2004).
In the present study, the hyperresponsiveness to nebulized
methacholine was not associated with a corresponding increase in ex
vivo airway constriction. This was true for both strains of mice. In fact,
experimental asthma was associated with a decreased sensitivity to
methacholine (i.e., an increased EC50) in airways of lung slices from
both mouse strains. This is consistent with our recent study (Boucher
et al., 2024). The origin of this reduced sensitivity is uncertain. It is
potentially attributable to a real decrease in the sensitivity of the
ASM. Another possibility is that the lung tissue (and/or the airway
wall) might be stiffer in HDM- versus saline-exposed mice (Gill et al.,
2023; Rojas-Ruiz et al., 2024). More force (and a higher concentration
of methacholine) would thus be required to reach any level of
constriction, explaining the decreased sensitivity. Another possibility
might be related to a technical artefact. Given that there is already
more fluid in the lungs of mice with experimental asthma (confirmed
herein by heavier lungs; Figure 1B), pushing the same amount of
agarose into their lungs might yield greater filling (agarose plus the
exudative, inflammatory and mucosal fluids). This might then stiffen
the lung tissue surrounding airways in lung slices and, consequently,
decrease sensitivity by increasing the load, impeding ASM shortening.
In other mouse models of asthma, ex vivo airway constriction in lung
slices was decreased (Donovan et al., 2013), not affected (Bourke et al.,
2019; Kim et al., 2015) or increased (Liu et al., 2017). More studies will
be needed, perhaps combined with traction force microscopy to track
the changes in stress and strain within and around the airway wall
during constriction.
Complementary ex vivo studies were conducted with excised
tracheas. Given that the trachea contracted in isometric conditions in
these assays, its responsiveness cannot be influenced by lung tissue
stiffness (unlike airway constriction in lung slices). These experiments
were especially important to tease out whether a change in ASM force
or a change in lung tissue mechanical properties is at the origin of
the decreased methacholine sensitivity seen in airways from HDM-
exposed mice. The results showed no signs of excessive isometric force
from tracheas isolated from HDM-exposed mice in both mouse strains.
This was shown in response to the classical spasmogen methacholine,
which signals via its cognate muscarinic 3 receptor (M3R). It was also
shown in response to KCl, a spasmogen acting independently from G
protein-coupled receptor signalling. Inasmuch as the contraction of the
ASM from the trachea is representative of ASM contraction in smaller
airways (Gunst & Stropp, 1988), this suggests that an increased lung
tissue stiffness (rather than a defect in ASM force) is likely to account

ROJAS-RUI Z ET AL.7
FIGURE 3 The response to nebulized methacholine in BALB/c mice (open circles) and C57BL/6 mice (filled squares) that were exposed to
either saline or HDM. (a) The response was quantified by measuring the AUC of the changes (Δ) in each parameter of respiratory mechanics caused
by methacholine. (b–g) Individual results are shown, together with means ±SD for respiratory system resistance (ΔRrs; b), respiratory system
elastance (ΔErs; c), airway resistance (ΔRaw ; d) tissue resistance (ΔG; e), tissue elastance (ΔH; f) and hysteresivity (η; g). Results of two-way ANOVAs
are provided underneath the graphs. When the interaction was significant, Sidak’s multiple comparisons test was conducted, and asterisks indicate
significant differences (**P<0.01 and **** P<0.0001). n=12 mice per group. Abbreviations: AUC, area under the curve; HDM, house dust mite.
for the decreased methacholine sensitivity of airway constriction seen
in lung slices of HDM-exposed mice. Another hint supporting this
conjecture is the significant difference in the sensitivity of airway
constriction between mouse strains. Indeed, airways from C57BL/6
mice were less sensitive to the constricting effect of methacholine than
the ones from BALB/c mice (Figure 4C;P=0.046), and it was recently
shown that the former have stiffer lung tissue than the latter (Rojas-
Ruiz et al., 2023). One downside of the experiments conducted with
isolated tracheas in the present study is that the level of inflammation
was not quantified specifically in the tracheal tissue, and the presence
versus the absence of inflammation in these types of preparations
was previously associated with ex vivo hyperresponsiveness, both in
humans (Ijpma et al., 2020) and in a different mouse model of asthma
(Chiba et al., 2004).
The most likely cause of in vivo hyperresponsiveness in the mouse
model of asthma presented herein is small airway closure. In another
acute mouse model of asthma induced by ovalbumin sensitization and
challenge, computational modelling has clearly demonstrated that an
increased propensity for small airway closure, attributable to a thicker
airway wall (caused by oedema, for example) and the accumulation
of inflammatory fluid within the airway lumen, is sufficient to
explain hyperresponsiveness to nebulized methacholine (Wagers et al.,
2004). In other words, a normal level of methacholine-induced ASM
contraction in inflamed lungs with an increased propensity for air-
way closure was sufficient to provoke exaggerated changes in lung
mechanics (Wagers et al., 2004). Although ASM contraction was not
measured directly in that study, it was subsequently shown that air-
way closure caused by nebulized methacholine was manifestly greater
in this mouse model of asthma (Lundblad et al., 2007). Based on these
findings, we surmise that in vivo hyperresponsiveness in the mouse
model presented in this study is mainly attributable to excessiveairway
closure.
The dissociation between the effect of experimental asthma on the
in vivo and ex vivo response to methacholine in the present study is
reminiscent to findings in humans with and without asthma. Despite
methacholine hyperresponsiveness in vivo being a pathognomonic

8ROJAS-RUI Z ET AL.
FIGURE 4 (a) Airway constriction in lung slices of BALB/c mice (circles) and C57BL/6 mice (squares) that were exposed to saline (black) or
HDM (red) in response to increasing concentrations of methacholine. Each point on the curve represents the mean ±SD per group. The results of
the three-way ANOVA are provided below the graph. (b, c) The maximal airway constriction (b), representing the constriction in response to the
highest concentration of methacholine tested (10−4M), and the EC50 (c), representing the concentration of methacholine causing 50% of the
maximal response, are shown as individual results, together with the mean ±SD in each group. The results of two-way ANOVAs are also shown
underneath each graph. n=12 mice per group. Abbreviation: HDM, house dust mite.
feature of human asthma, there is a dearth of evidence, as in mice,
to support its association with an enhanced ex vivo contractility of
the ASM (Chin et al., 2012; Ijpma et al., 2015, 2020; Noble et al.,
2013; Whicker et al., 1988; reviewed by Wright et al., 2013). There
are a few reports showing significant differences in some ASM contra-
ctile properties of asthmatics compared with non-asthmatics, such
as a decreased sensitivity (Ijpma et al., 2015, 2020; Whicker et al.,
1988), an increased stress-generating capacity (Ijpma et al., 2020),
increased airway narrowing (Noble et al., 2013) and an improved ability
to tolerate the decline in contractility caused by oscillating strains
simulating breathing manoeuvres (Chin et al., 2012). However, the
reported alterations are inconsistent between studies. Notably, most
of these studies were testing several contractile properties with small
sample sizes, which are the perfect ingredients for type 1 errors,
especially while considering the inherent variability observed with
these types of preparations. In our opinion, and consistent with the
present mouse study, the bulk of studies published so far with human
tissues suggest that excessive ASM contraction is unlikely to be an
important or common contributor to in vivo hyperresponsiveness. As
in mice (Lundblad et al., 2007; Wagers et al., 2004) and as reported
in several studies (Chapman et al., 2008; Dame Carroll et al., 2015;
Farrowetal.,2012), a main cause of in vivo hyperresponsiveness in
human asthma is airway closure.
One possibility in human asthma is that contraction might be
excessive owing to ASM enlargement. This was the conclusion drawn
by Noble et al. (2013) in a study with human airways. More precisely,
they have shown that the stress-generating capacity of asthmatic
ASMs are not different from ASMs derived from non-asthmatics,
but that the increased amount of ASM still leads to more airway
narrowing (Noble et al., 2013). This is a very important finding
that needs to be investigated further, because a larger amount of
ASM is a common feature of asthmatic airways (Elliot et al., 2015).
Given that our acute mouse model of asthma does not exhibit ASM
enlargement, this might be an additional reason why there was no
excessive airway constriction in the present study. Yet, these models
of acute allergic lung inflammation induced by HDM are widely used
to study experimental asthma (Carroll et al., 2023). We think it is
important to be aware that these models do not exhibit excessive
airway constriction (at least not ex vivo).
In addition, the results of the present study do not preclude the
existence of in vivo factors that might render the ASM hypercontractile
when it operates in an inflamed, ‘asthmatic’ milieu. Owing to the
phenomenal contractile plasticity of the ASM (Auger et al., 2016;Black
et al., 2012; Bossé et al., 2008;Gunstetal.,1995; Halayko et al., 2008;
Hirota, Nguyen et al., 2009), it is possible that the tissue reverts to
a normal contractile state when isolated and studied ex vivo (away

ROJAS-RUI Z ET AL.9
FIGURE 5 (a) The isometric force generated by excised tracheas of BALB/c mice (circles) and C57BL/6 mice (squares) that were exposed to
either saline (black) and HDM (red) in response to increasing concentrations of methacholine. A representative trace (top), the mean ±SD in each
group (middle) and the results of the three-way ANOVA (bottom) are shown. (b, c) The maximal active force (b) and the EC50 (c), representing the
concentration of methacholine causing 50% of the maximal response, are also shown as individual results, together with the mean ±SD in each
group. The results of two-way ANOVAs are also shown underneath each graph. n=12 mice per group. Abbreviation: HDM, house dust mite.
from all in vivo asthmatic factors that might be enhancing its contra-
ctility). Safholm et al. (2011), for example, have shown that incubating
tracheal rings from BALB/c mice with some inflammatory mediators
increases the contractile response to certain spasmogens, which was
not the case for tracheal rings from C57BL/6 mice. They suggested
that the greater susceptibility of BALB/c mice to the development of in
vivo hyperresponsiveness in the context of experimental asthma might
stem, at least in part, from this acquired hypercontractility (Safholm
et al., 2011). Notably, nearly a hundred different molecular mediators
have been shown to alter contractility in other preparations of ASM
(Gazzola et al., 2020).
The dissociation between the in vivo and the ex vivo response to
methacholine can also rely on an interplay between the ASM and other
lung tissues. For example, Der p 1, the immunodominant allergen from
D. pteronyssinus species found in HDM, was shown to reduce the lung
expression of several G protein-coupled receptors involved in ASM
contraction, including the muscarinic 2 receptor (M2R) (Kelada et al.,
2011). Although airway constriction is mediated mainly by the M3R
(Fisher et al., 2004; Roffel et al., 1990; Struckmann et al., 2003), M2R
is required for optimal contraction of murine airways (Alkawadri et al.,
2022; Struckmann et al., 2003). Counterintuitively,M2R-deficient mice
are hyperresponsive to intravenous administration of methacholine
(Fisher et al., 2004). This has been attributed to the prejunctional
role of the M2R in limiting acetylcholine release from the nerves
(Fryer & Jacoby, 1998). In fact, a dysfunctional M2R was repeatedly
shown to contribute to in vivo hyperresponsiveness in animal models
of asthma induced by a variety of offending triggers, such as antigens,
viruses, ozone and others (Fryer & Jacoby, 1998; Rynko et al., 2014).
An unfettered release of acetylcholine is likely to increase ASM tone
and, consequently, ASM force (Bossé et al., 2009), and this effect might
then be greater than the postjunctional role of the M2R in potentiating
the contraction of ASM (Alkawadri et al., 2022; Struckmann et al.,
2003). Therefore, the decreased expression of M2R alone caused by
HDM (Kelada et al., 2011) might be one putative molecular mechanism

10 ROJAS-RUI Z ET AL.
FIGURE 6 (a) The isometric force generated by excised tracheas of BALB/c mice (circles) and C57BL/6 mice (squares) that were exposed to
either saline (black) and HDM (red) in response to increasing concentrations of KCl. A representative trace (top), the mean ±SD in each group
(middle) and the results of the three-way ANOVA (bottom) are shown. (b, c) The maximal active force (b) and the EC50 (c), representing the
concentration of KCl causing 50% of the maximal response, are also shown as individual results, together with the mean ±SD in each group. The
results of two-way ANOVAs are also shown underneath each graph. n=12 mice per group. Abbreviation: HDM, house dust mite.
whereby mice with experimental asthma in our study are hyper-
responsive in vivo, whereas their airway constriction in lung slices is
attenuated at low methacholine concentrations. However, unless the
downregulation of M2R (Kelada et al., 2011) or the potentiation of
ASM contraction by the M2R (Alkawadri et al., 2022; Struckmann et al.,
2003) is specific to peripheral airways (and not the trachea), this would
not explain the lack of differences in methacholine sensitivity between
excised tracheas of HDM- and saline-exposed mice.
5CONCLUSION
It is concluded that, at least in the specific mouse model used in the pre-
sent study, the hyperresponsiveness to nebulized methacholine caused
by experimental asthma is not matched by a corresponding increase in
ex vivo airway constriction. In our opinion, the most likely explanation
is that a normal level of ASM shortening is sufficient to trigger in vivo
hyperresponsiveness when it operates in combination with other lung
alterations seen in mice with experimental asthma (Bossé, 2021; Bossé
et al., 2010;Wagersetal.,2004).
AUTHOR CONTRIBUTIONS
Andrés Rojas-Ruiz, Magali Boucher, Cyndi Henry, Percival Graham,
Jorge Soliz and Ynuk Bossé contributed to the development of
the experimental design. Andrés Rojas-Ruiz, Magali Boucher, Cyndi
Henry, Rosalie Packwood and Louis Gélinas performed laboratory
experiments. Andrés Rojas-Ruiz, Magali Boucher and Ynuk Bossé
analysed the data. Ynuk Bossé wrote the first version of the
manuscript. All authors edited the manuscript, read and approved the
final version of the manuscript and agree to be accountable for all
aspects of the work in ensuring that questions related to the accuracy
or integrity of any part of the work are appropriately investigated and
resolved. All persons designated as authors qualify for authorship, and
all those who qualify for authorship are listed.

ROJAS-RUI Z ET AL.11
CONFLICT OF INTEREST
P.G. is employed by SCIREQ Inc., a commercial entity with interests
in topics related to the content of the present work. Y.B. holds an
operating grant in partnership with SCIREQ Inc. A.R.R., M.B., C.H., L.G.,
R.P. and J.S. have no conflict of interest.
DATA AVAILABILITY STATEMENT
The datasets used and analysed during the present study are available
from the corresponding author on reasonable request.
ORCID
YnukBossé https://orcid.org/0000-0002-2023-3130
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How to cite this article: Rojas-Ruiz, A., Boucher, M., Henry, C.,
Gélinas, L., Packwood, R., Graham, P., Soliz, J., & Bossé, Y.
(2025). Methacholine hyperresponsiveness in mice with house
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