Detection of Mouse Cough Based on Sound Monitoring
and Respiratory Airflow Waveforms
Liyan Chen1,2,3, Kefang Lai1,2,3, Joseph Mark Lomask4, Bert Jiang4, Nanshan Zhong1,2,3*
1Department of Respiratory Diseases, The 1stAffiliated Hospital of Guangzhou Medical College, Guangzhou, China, 2Guangzhou Institute of Respiratory Disease,
Guangzhou, China, 3State Key Laboratory of Respiratory Disease, Guangzhou Medical College, Guangzhou, China, 4Buxco Electronics, Inc, Wilmington, North Carolina,
United States of America
Detection for cough in mice has never yielded clearly audible sounds, so there is still a great deal of debates as to whether
mice can cough in response to tussive stimuli. Here we introduce an approach for detection of mouse cough based on
sound monitoring and airflow signals. 40 Female BALB/c mice were pretreated with normal saline, codeine, capasazepine or
desensitized with capsaicin. Single mouse was put in a plethysmograph, exposed to aerosolized 100 mmol/L capsaicin for
3 min, followed by continuous observation for 3 min. Airflow signals of total 6 min were recorded and analyzed to detect
coughs. Simultaneously, mouse cough sounds were sensed by a mini-microphone, monitored manually by an operator.
When manual and automatic detection coincided, the cough was positively identified. Sound and sound waveforms were
also recorded and filtered for further analysis. Body movements were observed by operator. Manual versus automated
counts were compared. Seven types of airflow signals were identified by integrating manual and automated monitoring.
Observation of mouse movements and analysis of sound waveforms alone did not produce meaningful data. Mouse cough
numbers decreased significantly after all above drugs treatment. The Bland-Altman and consistency analysis between
automatic and manual counts was 0.968 and 0.956. The study suggests that the mouse is able to present with cough, which
could be detected by sound monitoring and respiratory airflow waveform changes.
Citation: Chen L, Lai K, Lomask JM, Jiang B, Zhong N (2013) Detection of Mouse Cough Based on Sound Monitoring and Respiratory Airflow Waveforms. PLoS
ONE 8(3): e59263. doi:10.1371/journal.pone.0059263
Editor: Andreas Meisel, Charite ´ Universitaetsmedizin Berlin, Germany
Received November 5, 2012; Accepted February 12, 2013; Published March 21, 2013
Copyright: ? 2013 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by a grant of the National Natural Science Foundation of China (No. 81100009 and No.30670934) (http://www.nsfc.gov.cn/
Portal0/default152.htm).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The Whole Body Plethysmography System (Buxco Company) is used for several years in our lab for detecting mouse lung function.
Joseph Mark Lomask and Bert Jiang are employed technicians of Buxco Electronics, Inc, Wilmington, NC, USA. The authors have good communication with them
in instrumentation and maintenance. The authors found suspicious mouse cough respiratory waveforms in the anterior experiments and conceived mouse cough
detection method. The two gentlemen gratuitously participated in software design, development and software mechanism writing, so that the authors decided
to have them both on the authorship of this article. This does not alter the authors’ adherence to all the Plos One policies on sharing data and materials. Linked to
this study, a patent named ‘‘The development of mouse cough model and the detection method of mouse cough’’ has been authorized by State Intellectual
property office of the People’s Republic of China in July 31th, 2012. The co-holders of this patent are: (1) First Affiliated Hospital of Guangzhou Medical College, (2)
Guangzhou Institute of Respiratory Disease, (3) State Key Laboratory of Respiratory Disease, and (4) Buxco Electronics, Inc, USA. The main contents of the patent
comprise: 1. 10-week old specific-pathogen free female Balb/c mice with 22–25 grams; 2. A set of Buxco non-invasive Whole Body Plethysmography System
where mice could be put in for free moving, attached with sonic audiomonitor and signal converter. 3. Mice coughs elicited by capsaicin aerosol were manually
monitored by observers and automatedly recorded and analyzed by Finepointe software. If the manual and automated cough counts were coincident, the mouse
cough could be verified. This patent is about the establishment of new method of mice cough detection process, not concerning with software development. The
authors confirm adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the PLOS ONE guide for authors.
* E-mail: firstname.lastname@example.org
Cough is one major defensive reflex that enables vital
respiratory tract. Each involuntary cough event involves a series
of activities in an integrated reflex arc. Capsaicin has been
shown to act mainly on capsaicin-sensitive fibers. A number of
authors have proposed that neurogenic inflammatory mediators
from endings of capsaicin-irritated C fibers act on rapid
adaption receptors, which in turn generate the stimuli that
ultimately give rise to cough [1–3].
Despite their widespread use in mechanistic studies or in new
drug trials for cough [4–5], guinea pig cough models have such
experimental limitations as high costs, physical weakness and a
large demand for experimental drugs. Especially, a guinea pig has
32 couples of chromosomes, which is different from the 21 couples
in a human being. In this regard, mice can be more suitable owing
to shorter reproductive cycle, prolificacy, less demand for feeding
and drugs, and readiness for genetic manipulations. Furthermore,
mice have 20 couples of chromosomes, which share 80% of
hereditary substances and 99% of genes with human beings.
Therefore, detection of cough in mice seems promising.
But there is still controversy over whether mice can cough in
response to tussive stimuli , chiefly because of their tiny
anatomic structures and weak sound signals reported in very few
studies with mixed results. In studies from India and Mexico, mice
were exposed to irritants and then placed in an up-ended filter
funnel with a stethoscope at the tip to be monitored for cough
sounds. Since the sounds were not recorded for further analysis,
conclusions of these studies appeared somewhat arbitrary [7–8]. In
experiments by Junzo Kamei who has persisted in anti-tussive
studies in mice models for more than two decades, a double-
chamber plethysmograph was employed in which the head and
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body of a consciously restrained mouse were positioned respec-
tively in each of the two separated chambers. The cough in mice
was determined by altered breaths as measured by pneumotacho-
graphy in combination with rapid abdominal twitches [9–11]. But
these studies are not convincing enough because they lacked sound
monitoring. Because detection of cough in mice has never yielded
clearly audible sounds as found in guinea pigs, it is not generally
After intensive studies on neurophysiology over the recent years,
vagal sensory neurons have been found to exist in mice, and vagus
nerve stimulation or vagotomy has been shown to increase or
reduce levels of neuropeptides [12–14]. Zhang et al recorded
single unit activities in the cervical vagus nerve stimulated with
bipolar electrodes in mice, and demonstrated the presence of
mechanosensors as well as of chemosensors . Transient
receptor potential V1 (TRPV1), the main receptor of capsaicin
that was found to mediate capsaicin-induced cough , was also
localized in mice, as indicated by Symanowicz et al who
studied respiratory reflex induced by several inhaled irritants in
TRPV1 gene knock-out mice. Based on these findings, it can be
conclusive that mice possess a similar set of airway sensors and
pulmonary reflexes as typically found in larger animals. Therefore,
we designed an approach to detecting cough based on sound
monitoring and observation of airflow signals in freely moving
Ten-week-old female BALB/c mice (22–25 g, Guangdong
Laboratory Animal Center) were housed in a specific pathogen-
free animal facility at the Guangzhou State Key Laboratory of
Respiratory Diseases. The animals had free access to food and
water, and were maintained at a 12-h light/12-h dark cycle. This
study was carried out in strict accordance with the recommenda-
tions in the Guide for the Care and Use of Laboratory Animals of
the State Key Laboratory of Respiratory Disease. All experimental
procedures were approved by the Animal Ethics Committee, The
First Affiliated Hospital, Guangzhou Medical College (Approval
For each measurement, a free-moving mouse was put alone
within a whole body plethysmography chamber (Buxco Elec-
tronics, Inc. Wilmington, NC, USA). A set of software for
automated detection and counting of cough events (Finepoin-
teTM, jointly developed by Guangzhou State Key Laboratory of
Respiratory Diseases and Buxco Electronics, Inc.) was employed
to analyze the waveforms of pressure generated from activities
of the mouse (such as breaths, coughs, and body behavior)
within the chamber. The fluctuations in pressure were reflected
on a pneumotachography as the airflow running into and out of
the plethysmograph. As such, waveforms inside the chamber
can be recorded for real-time analysis and later review (Fig. 1).
Moreover, a mini-microphone was mounted to the lateral
aperture of plethysmograph to facilitate real-time acoustic
monitoring. Sound and sound waveforms were recorded by
the software Adobe Audition (formerly Cooledit Pro, Adobe
Systems, California, USA) for further analysis.
Detection of Cough
The mouse was rendered conscious and free to move in the
chamber. The irritant for eliciting cough was prepared by
dissolving capsaicin in a solution containing 10% ethanol and
10% Tween-80, tittered at a final concentration of 100 mmol/L.
Then we exposed the mouse to aerosol of 1 ml capsaicin from a
nozzle for 3 min. The number of cough events elicited in mice was
counted during the 3 minutes of and within 3 minutes after
nebulized capsaicin stimulation. Briefly, an operator was desig-
nated to identify the cough sounds by ear and to observe mouse
body movements, in parallel with automated recognition of cough
events by Finepointe software during the same procedure.
Throughout the detection of cough in mice, raw acoustic signals
and box flows were acquired simultaneously and displayed on a
computer screen. Abnormal box flows identified as arising from
coughs were automatically displayed in white. When a cough
sound was clearly heard, the operator immediately hit a hot key
which in turn prompted red hollow dots and the word ‘cough’ on
the respiratory channel to mark a manually counted cough event.
Denoised and amplified sound waveforms through Adobe
Audition software were also analyzed to identify the correction
of manual cough counts. The operator did not know the automatic
monitoring results during the observation period. After the 6 min
acoustic monitoring, automatic and manual cough counts were
evaluated. A single cough was confirmed only with consistent
labeling by both approaches (video S1).
For antitussive pretreatment, 30 mice were randomly divided
into three groups: normal saline (NS) control group, codeine (CDI,
Qinghai Pharmaceutical Co. Ltd., China) group and capsazepine
(CPZ, a competitive antagonist of capsaicin, Sigma Chemical Co.,
USA) group (n=10 in each group). On days 22, 21 and 0, the
mice received once-daily antitussive pretreatment with gavage of
0.2 ml normal saline (NS group) or codeine (100 mg/kg, CDI
group), or intraperitoneal injection of 0.2 ml capsazepine (6 mg/
kg, CPZ group, Sigma Chemical Co., USA). Cough detection was
performed at 1 h after the last pretreatment.
Desensitization of C-fibers by Capsaicin Pretreatment
A group of mice (n=10) was assigned to receive subcutaneous
injection of capsaicin (CAP) at a total dose of 300 mg/kg,
scheduled as 50 mg/kg on day 23, 100 mg/kg on day 22,
150 mg/kg on day 21. Pentobarbital sodium (60 mg/kg, i.p.),
terbutaline (0.1 mg/kg, s.c. AstraZeneca Pharmaceutical Co. Ltd,
UK) and aminophylline (25 mg/kg, i.p. Baiyunshan Pharmaceu-
tical Co. Ltd., China) were given to counteract potential adverse
effects associated with the capsaicin injections. Cough detection
was performed at 24 h after the drug pretreatment.
The means of multiple samples were examined with one-way
analysis of variance and homogeneity of variance test, followed by
least significant difference test, Tamhane post hoc test, or
independent samples T-test. Statistical analysis was performed
with SPSS software version 12.0 (SPSS Inc., Chicago, IL, USA).
Bland-Altman analysis and consistency analysis were performed
between automatic and manual cough counts.
Airflow Signals of Respiration in Mice
By holistic evaluation of the acoustic monitoring and Fine-
pointe-based automated detection, seven types of airflow signals of
respiration in mice after capsaicin stimulation were defined and
identified as follows (Fig. 2):
Mouse Cough Detection
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1. Cough, characterized by apparently enormous amplitudes
(pressure changes) and widths (time phase) associated with
abrupt head-tossing, opened mouth, abdominal jerking and
with a clearly audible sound in mice.
2. Sneeze, characterized sometimes by acoustic waveforms and
body behavior (head-tossing, opened mouth, abdominal
jerking) similar to those in cough, but chiefly by significantly
lowered magnitude of the airflow signals, as well as by dull or
3. Eupnea, characterized by uniform frequency and depth of
airflow signals, as was often seen when mice had accommo-
dated themselves to the chamber environment or recovered
from the capsaicin stimulation.
4. Tachypnea, characterized by higher frequency and magnitude
of respiration signals, as was often seen when mice were new to
the chamber and not familiar with the internal environment, or
in rapid movements, or in the process of recovery phase after
5. Breath-holding, characterized by a significant reduction in
both frequency and magnitude of respiration signals to nearly
the baseline, as was often seen when the mice voluntarily held
back their breath to avert capsaicin inhalation.
6. Deep inspiration, characterized by airflow signals that
appeared wider during the early phase and became narrowed
later, in contrast to those produced by coughs. In some cases,
the signals showed great amplitudes associated with cough-like
body behavior (head-tossing, opened mouth and abdominal
jerking) but were not accompanied by a cough sound.
7. Head-twitch, accompanied by cough-like sound but also by
production of inverted V-shaped airflow signals that were
readily distinguishable from those of cough.
Adverse effects (such as depression and loss of appetite) were
shown among the 8 mice that survived out of 10 in the capsaicin
group but not in the other groups. Mice subjected to antitussive
pretreatment had significantly less cough in response to capsaicin
stimulation, compared with the control group (Table 1).
Comparison of Automated and Manual Cough Counts
Complete data for 31 out of 38 mice were available for statistical
processing, because computer malfunction had led to a loss of the
storeddatafor7 mice. Bland-Altman
mean+2SD=4.87 and mean–2SD=25.06. Only one pair of
Figure 1. Mouse cough detection equipments. (1) Bias flow generator; (2) Desiccant; (3) Nebuliser controller; (4) Nebuliser; (5) Plethysmograph;
(6) Amplifier; (7) Speakers; (8) Monitor display.
Mouse Cough Detection
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the total 31 manual and automatic cough counts (3.23%) had the
deviation with 8, and the other 30 pairs were in the limits of
agreement (Fig. 3A). Consistency analysis showed that the intra-
class correlation coefficient (ICC) was 0.956 (95% confidence
interval: 0.911,0.978). Errors of other sources accounted for
4.4% of the total errors (1-ICC) (Fig. 3B). Both measurements
indicated high precision of and good consistency between
automated and manual cough counts.
Criteria for Detection of Cough in Mice
Based on real-time acoustic monitoring and airflow signals in
this study, we proposed an established cough in mice should fulfill
1. automatic capture of cough airflow signals, and
2. a clear cough sound audible to the operator and consistent with
the captured cough airflow signals.
Figure 2. Seven types of mice respiratory waveforms: (1) cough; (2) sneeze; (3) eupnea; (4) tachypnea; (5) breath-holding; (6) deep
inspiration; (7) head-twitch.
Table 1. Frequency of mice cough after antitussive pretreatment compared with control group.
Groups Cough numbers (/6 min) P (compared with control group)
Capsaicin (n=8)464** 0.000
Data were expressed as mean 6 standard error of the mean (SEM). P,0.05 was considered as the level of statistical significance.
**p,0.01, antitussive pretreatment groups had statistical significance compared with the control group.
Mouse Cough Detection
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Mouse Cough Detection
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