This protocol describes the development of a mouse model with cough hypersensitivity, which can serve as an ideal model for studying the mechanisms of chronic cough.

Cough is one of the most common symptoms of many respiratory diseases. Chronic cough significantly impacts quality of life and imposes a considerable economic burden. Increased cough sensitivity is a pathophysiological hallmark of chronic cough. It has been observed that cough hypersensitivity is related to airway inflammation, remodeling of airway sensory nerves, and alterations in the central nervous system. However, the precise molecular mechanisms remain unclear and require further elucidation using suitable animal models. Previous studies have utilized guinea pigs as models for studying cough, but these models present several experimental limitations, including high costs, a lack of transgenic tools, and a scarcity of commercial reagents. In addition, guinea pigs typically exhibit poor environmental tolerance and high mortality when exposed to stimuli. In contrast, mice are smaller, easier to maintain, more cost-effective, and amenable to genetic manipulation, making them more suitable for mechanistic investigations. In this study, we established a mouse model with cough hypersensitivity via continuous inhalation of citric acid (CA). This model is straightforward to operate and yields reproducible results, making it a valuable tool for further studies on the mechanisms and potential novel treatments for chronic cough.

Cough is a crucial defensive reflex that helps clear respiratory secretions or foreign materials from the airway. However, it is also one of the most common symptoms of many respiratory diseases, often prompting patients to seek medical attention¹. Chronic cough, defined as a persistent cough lasting more than 8 weeks in adults, significantly impacts the quality of life, causing issues such as incontinence, insomnia, reflux, and other unpleasant experiences, along with a substantial economic burden^2,3,4. It is widely believed that increased cough sensitivity is a pathophysiological hallmark of chronic cough, where low levels of thermal, mechanical, and chemical irritants can trigger coughing⁵. Cough hypersensitivity is associated with airway inflammation⁶, remodeling of airway sensory nerves⁷, and alterations in the central nervous system⁸, though the precise molecular mechanisms remain unclear and require further elucidation through suitable animal models.

Various animals, including guinea pigs, cats, rabbits, dogs, and pigs, have been used to study the mechanisms of cough⁹. Guinea pigs have traditionally been recognized as the most suitable model for studying cough mechanisms and the efficacy of antitussive drugs^9,10,11,12. However, these models have several experimental limitations, including high costs, a lack of transgenic tools, and a scarcity of commercial reagents. Additionally, guinea pigs often exhibit poor environmental tolerance and high mortality when exposed to stimuli. In contrast, mice are smaller, easier to maintain, more cost-effective, and amenable to genetic manipulation, making them more suitable for mechanistic investigations. Previous studies on cough models have primarily focused on cough induced by airway inflammation, mainly used to evaluate the efficacy of antitussive drugs and peripheral mechanisms^13,14. There is currently a lack of animal models for cough hypersensitivity.

In response, we introduce a method for establishing a mouse model of cough hypersensitivity via continuous inhalation of citric acid (CA). This model is simpler, easier to construct, and more feasible compared to other animal models.

All animal experiment procedures were approved by the Laboratory Animal Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University (20230656). Adult male specific-pathogen-free C57BL/6 mice, aged 8-10 weeks and weighing 20-25 g, were used in this study. The details of the reagents and equipment used are listed in the Table of Materials.

1. Chemical reagent preparation

1. Citric acid (CA) solution preparation
   1. For cough assessment, prepare a 0.4 M solution by dissolving 3.84 g of citric acid powder in normal saline to a final volume of 50 mL.
   2. For animal treatment, prepare a 0.1 M solution by dissolving 9.6 g of citric acid powder in normal saline to a final volume of 500 mL.
2. Capsaicin solution preparation
   1. Dissolve 30.5 mg of capsaicin powder in PBS containing 10% ethanol and 10% Tween-80 to make a 10 mM stock solution.
   2. Dilute the stock solution 1:100 in normal saline to make a 100 µM working solution.
3. Methacholine solution preparation
   1. Dissolve 100 mg of methacholine powder in PBS to a final volume of 2 mL to make a 50 mg/mL solution.
   2. Dilute this solution with PBS to obtain concentrations of 25 mg/mL, 12.5 mg/mL, 6.25 mg/mL, and 3.125 mg/mL.

2. Animal preparation

1. Housing conditions
   1. House mice in cages with free access to food and water, maintained at a temperature of 22 ± 1 °C with a standard 12-h light/dark cycle. Experimental manipulations were performed after 1 week of acclimation to the feeding environment.
2. Acclimation
   1. Allow mice to acclimate to the feeding environment for 1 week before conducting experimental manipulations.

3. Development of the model

1. Group assignment
   1. Randomly divide the mice into a model group and a control group, with 8 mice in each group.
2. Exposure setup
   1. Place the mice in two independent chambers (as shown in Figure 1), each connected to an ultrasonic nebulizer.
   2. Expose the model group to 0.1 M citric acid (CA) aerosol and the control group to 0.9% normal saline (NS) aerosol at an atomization rate of 3 mL/min for 2 h per day over 2 weeks (as shown in Figure 2). Return the mice to their cages after each exposure.
3. Cough sensitivity assessment
   1. After the last exposure, assess cough sensitivity by evaluating reflexive cough (challenged with NS, CA (0.4 M), and capsaicin (100 µM)) and spontaneous cough (without any challenge).
      NOTE: To monitor dynamic changes in cough sensitivity during model development, assess cough sensitivity on days 0, 3, 7, and 10 using a CA (0.4 M) challenge.

4. Cough sensitivity assessment

1. Instrument preparation
   1. Use a non-invasive whole-body plethysmography (WBP) system to measure cough sensitivity. Connect the chambers, flow transducers, bias flow, and other components according to the application manual (see Table of Materials).
   2. Perform a calibration process to ensure accurate measurements by clicking on the Calibrate button. A status screen will display the progress, and once the status bar reaches 100%, the calibration is complete.
2. Cough detection
   1. Create a cough study for mice and configure the parameters for 1 min of acclimation, 10 min of response time, and 10 min of chemical solution delivery.
   2. Place the conscious mice into individual chambers, ensuring there is only one mouse per chamber.
      NOTE: Make sure the mouse is completely inside the chamber before closing the lid to avoid damaging its tail and digits.
   3. Add 1 mL of the chemical solutions (NS, CA, or capsaicin) to the nebulizer.
      NOTE: The number of cough events-each consisting of three phases: inspiratory, compressive, and expulsive¹⁵ (as shown in Figure 3)-will be recorded once the study begins. After 10 min of recording, cough detection will be complete. Clean the chamber after detecting each animal to avoid cross-contamination.

5. Airway Hyperresponsiveness (AHR) measurement

1. Instrument preparation
   1. Prepare instruments as described in step 4.1.
2. AHR measurement
   1. Create a dose-response study for mice, configuring the measurement types, parameters, and task sequence: 1 min for acclimation, 30 s for methacholine solution delivery, 3 min for response time, and 1 min for recovery.
   2. Place the conscious mice into individual chambers, ensuring there is only one mouse per chamber.
   3. Add 50 µL of the chemical solutions (PBS or methacholine) to the nebulizer and initiate the study.
   4. Record the value of Penh, an indicator of bronchoconstriction, in response to different concentrations of methacholine (0 mg/mL, 3.125 mg/mL, 6.25 mg/mL, 12.5 mg/mL, 25 mg/mL, 50 mg/mL).

6. Bronchoalveolar lavage collection

1. Sacrifice
   1. Sacrifice the mice using hyperanesthesia with pentobarbital sodium (50 mg/kg) administered via intraperitoneal injection (following institutionally approved protocols).
2. Sample collection
   1. Collect blood from the orbital sinus of the mouse into a 1.5 mL microcentrifuge tube, then place the tube on ice. Centrifuge the blood at 3000 x g for 10 min at 4°C. Use a pipette to collect the supernatant, aliquot it, and store it at -80 °C.
   2. Collect bronchoalveolar lavage fluid (BALF) by opening the chest to expose the trachea, inserting a 22 G indwelling needle into the trachea, then lavaging three times with 0.5 mL of pre-cooled PBS and collecting the fluid.
   3. After collection, centrifuge the BALF at 500 x g for 10 min at 4 °C. Collect the supernatant, aliquot it, and store it at -80 °C. Resuspend the pellet in 100 µL of PBS and prepare cell smears for further analysis.
   4. Collect lung tissues by opening the chest to expose the lungs, removing the lung tissues, and placing them into cryogenic vials. Store the vials at -80 °C for future studies.

7. Quantitative RT-PCR

1. Extract total RNA from lung tissue using TRIzol reagent. Perform cDNA synthesis using a First-strand cDNA synthesis kit, following the manufacturer's instructions (see Table of Materials).
2. Conduct PCR in a real-time quantitative PCR detection system using SYBR green fluorescence. Normalize the quantification of relative SP and CGRP mRNA expression to the expression of β-Actin⁹.

8. Statistical analysis

1. Present data as the mean ± SEM. Use a t-test to compare data between two groups, considering P < 0.05 as statistically significant. Conduct all statistical analyses using statistical and graphing software.

As shown in Figure 4A, cough sensitivity in the model group (CA group) significantly increased after 1 week of exposure compared to the control group (NS group), and this heightened sensitivity persisted throughout the exposure period. Neither the control group nor the model group mice experienced mortality during the modeling process (Figure 4B). Figure 4C and Figure 4D demonstrate that the number of spontaneous cough events significantly increased in the model group post-exposure. Additionally, cough sensitivity elicited by NS (Figure 4E and Figure 4F), CA (Figure 4G and Figure 4H), and capsaicin (Figure 4I and Figure 4J) was substantially higher in the model group after the exposure period.

In Figure 5A,B, the two groups had no significant difference in the total number of inflammatory cells and differential cell counts in the bronchoalveolar lavage fluid (BALF). Figure 5C,D shows BALF cells with H&E staining. As depicted in Figure 6, there was no significant difference in airway hyperresponsiveness between the model and control groups after modeling. Additionally, the expression levels of CGRP and SP, neurogenic inflammatory mediators, in lung tissue were assessed by QPCR. The results indicated a significant increase in SP expression in the model group (Figure 7A), while no significant difference in CGRP expression was observed between the two groups (Figure 7B).

Figure 1: Exposure chamber for mouse model. The exposure chamber is made of acrylic glass, with dimensions of 36 cm x 20 cm x 25 cm. Please click here to view a larger version of this figure.

Figure 2: Schematic of the mouse model with cough hypersensitivity. Schematic illustration of establishing a mouse model with cough hypersensitivity through citric acid inhalation. Please click here to view a larger version of this figure.

Figure 3: Representative images of a cough event. Representative images of a cough event recorded by a non-invasive whole-body plethysmography system. The cough event consists of three phases: (a) inspiratory, (b) compressive, and (c) expulsive. Please click here to view a larger version of this figure.

Figure 4: Cough assessment in mouse model. (A) Cough events elicited by 0.4 M citric acid during the 14-day exposure period. Cough sensitivity in the citric acid (CA) group was significantly higher than in the control group after 7 days of exposure. (B) Survival curves of control and model groups during the modeling process. (C-J) Spontaneous cough events (C,D) and cough events elicited by normal saline (NS) (E,F), citric acid (G,H), and capsaicin (I,J) before and after modeling. Data are represented as the mean ± SEM, ***P < 0.001 compared to the NS group. Please click here to view a larger version of this figure.

Figure 5: Leukocyte counts in bronchoalveolar lavage fluid (BALF). (A) Total number of leukocytes in BALF. (B) Differential cell counts in BALF. Data are represented as the mean ± SEM. (C, D) Representative images of BALF cells with H&E staining. The arrows indicate macrophages (orange), neutrophils (blue), and lymphocytes (green). Scale bars: 50 µm. Please click here to view a larger version of this figure.

Figure 6: Airway resistance in CA and NS groups. Airway resistance in the citric acid (CA) group compared to the normal saline (NS) group. Data are represented as the mean ± SEM. Please click here to view a larger version of this figure.

Figure 7: mRNA expression levels of SP and CGRP in lung tissue. (A) Relative mRNA expression levels of substance P (SP) in lung tissue. (B) Relative mRNA expression levels of calcitonin gene-related peptide (CGRP) in lung tissue. Data are represented as the mean ± SEM, *P < 0.05 compared to the NS group. Please click here to view a larger version of this figure.

This study successfully established a mouse model with cough hypersensitivity through continuous inhalation of citric acid (CA). This model demonstrated a reliable increase in cough sensitivity for both spontaneous coughs and reflexive coughs elicited by citric acid and capsaicin. Citric acid and capsaicin are widely used to assess cough reflex sensitivity¹⁶.

Several critical steps in this protocol ensure its effectiveness. Firstly, the exposure chamber used for the experiments must not be completely airtight. To ensure proper ventilation, vents should be placed at the back of the chamber. Secondly, it is important to avoid overcrowding within the chamber; limiting the number of mice to 10-12 prevents potential stampedes. Thirdly, post-exposure care is crucial: after each exposure, mice must be dried with warm air to remove any remaining fluid from their fur.

While guinea pigs are traditionally used to study cough mechanisms and evaluate antitussive drugs, mouse models offer several advantages. Previous studies have developed guinea pig models of cough hypersensitivity induced by citric acid exposure. For instance, Nakaji et al. exposed guinea pigs to 0.5 M citric acid for 10 min, three times per week for 2 weeks (eight exposures in total)¹⁷. Similarly, another study by Xu et al. established a model with increased cough sensitivity by having guinea pigs inhale 0.4 M citric acid for 25 days¹⁸. However, these guinea pig models exhibited less pronounced cough sensitivity compared to the current mouse model. Additionally, guinea pig models face limitations, such as the lack of transgenic tools and commercial reagents, which hinder the study of molecular mechanisms in cough hypersensitivity.

Mice, on the other hand, are easier to maintain, more cost-effective, and amenable to genetic manipulation. Previous studies have shown that mice are also effective models for the study of cough^15,19,20,21. Additionally, mice are commonly used in neuroscience research, providing an advantage for studying the central mechanisms of cough hypersensitivity. Thus, mice are considered more suitable for mechanistic investigations of chronic cough.

Despite the successful establishment of the model, there are some limitations to consider. Firstly, citric acid, although commonly used in the food, pharmaceutical, chemical, and metallurgical industries^22,23, does not accurately mimic real-life exposure conditions that result in cough hypersensitivity in humans. Secondly, the precise mechanism of cough hypersensitivity in this model remains unclear. While pulmonary inflammation is considered a potential mediator of increased cough sensitivity⁶, the assessment of total inflammatory cell counts and differential cell counts in bronchoalveolar lavage fluid (BALF) showed no significant changes between the two groups. Additionally, bronchial hyperresponsiveness, a known trigger of chronic cough²⁴, showed no significant difference in airway resistance between the model and control groups after 14 days of exposure.

In conclusion, a mouse model has been established with cough hypersensitivity via continuous inhalation of citric acid (CA). This model is straightforward to operate and yields reproducible results, making it a valuable tool for further studies on the mechanisms and potential novel treatments for chronic cough.

The authors have nothing to disclose.

This study was supported by the National Natural Science Foundation of China (NSFC 82100034), Guangzhou Science and Technology Planning Project (202102010168).