Methods for Detecting Cough and Airway Inflammation in Mice
Summary
Here, we describe the measurement of cough using a noninvasive and real-time whole-body plethysmography (WBP) system and the normative procedures for harvesting tissue samples of mice and introduce some methods to assess airway inflammation.
Abstract
Chronic cough, which lasts for more than 8 weeks, is one of the most common complaints requiring medical attention, and patients suffer from a huge socioeconomic burden and a marked decrement in quality of life. Animal models can mimic the complex pathophysiology of the cough and are important tools for cough research. The detection of cough sensitivity and airway inflammation is of great significance for studying the complex pathological mechanism of cough. This article describes the measurement of cough using a noninvasive and real-time whole-body plethysmography (WBP) system and the normative procedures for harvesting tissue samples (including blood, lung, spleen, and trachea) of mice. It introduces some methods to assess airway inflammation, including pathological changes in hematoxylin and eosin (HE)-stained lung and trachea sections, the total protein concentration, the uric acid concentration, and the lactate dehydrogenase (LDH) activity in the supernatant of bronchoalveolar lavage fluid (BALF), and the leukocytes and differential cell counts of BALF. These methods are reproducible and serve as valuable tools to study the complex pathophysiology of cough.
Introduction
Cough is an important defense behavior to maintain airway patency and protect the lungs from potentially harmful substances. However, when dysregulated, cough becomes a pathological condition1. Chronic cough, usually defined as lasting eight or more weeks, is one of the most frequent symptoms requiring medical attention2. Because chronic cough frequently persists for years, patients suffer from a huge socioeconomic burden and a marked decrement in quality of life3,4,5. Chronic cough is widely considered a cough hypersensitivity syndrome and is characterized by troublesome coughing often triggered by low levels of thermal, mechanical, or chemical exposure6. The occurrence of cough hypersensitivity is closely related to airway inflammation7. However, the pathophysiological mechanisms underlying the modulation of cough sensitivity need to be further elucidated.
Animal models can mimic the complex pathophysiology of the cough and are important tools for cough research8,9. Previous studies have found that viral infection, intrapulmonary Interferon-γ (IFN-γ) instillation, esophageal perfusion of hydrochloric acid, pollutant exposure, cigarette smoke, and citric acid can induce cough in animals10,11,12,13,14,15,16,17. In order to better evaluate cough and airway inflammation, a mouse model of cough was established using a nonlethal dose of the H1N1 virus in this study. For the detection of cough, some cough measurement tools have been established clinically to measure cough, including subjective and objective methods18. The subjective evaluation tools to assess the cough severity primarily include a visual analog scale, the cough score, and quality of life questionnaires, etc19,20. However, they are unlikely to be used to assess cough in animals. In addition, cough can be objectively assessed through a cough challenge test and cough frequency monitoring. The cough challenge test using a whole-body plethysmography (WBP) system is an objective method widely used in animal studies to measure cough sensitivity and reveal the underlying mechanisms of cough13,16. Based on the neuroanatomical characteristics of the cough reflex, citric acid, capsaicin, adenosine 5'-triphosphate (ATP), allyl isothiocyanate (AITC), and inflammatory mediator bradykinin are commonly used as the tussive agents to induce cough21,22. Citric acid is one of the earliest and the most widely used tussive agents triggering cough reflexes, which has been validated for measuring cough sensitivity. Besides, the citric acid challenge has good safety, feasibility, and tolerability and is suggested to assess cough reflex sensitivity in response to cough therapies23. Therefore, this article will describe the method for measuring cough sensitivity in response to citric acid in mice using a noninvasive and real-time WBP system.
The studies of the pathophysiology of cough require test samples, including samples from the blood, bronchoalveolar lavage fluid (BALF), and lung and trachea tissues to verify changes in the levels of key factors24. Currently, there is a lack of normative procedures for harvesting tissue samples of mice, and related studies employ different approaches that complicate the assessment of airway inflammation. Bronchoalveolar lavage is an important method to evaluate airway inflammation in respiratory diseases25. Different methods of bronchoalveolar lavage will lead to a lack of comparability between related studies. Moreover, different bronchoalveolar lavage methods have an impact on inflammatory cells and inflammatory cytokines in BALF. Therefore, this article will describe the establishment of a mouse model of cough with a nonlethal dose of the H1N1 virus, the measurement of cough using a WBP system, and a reliable, safe, and highly successful bronchoalveolar lavage method of mice.
Protocol
Representative Results
Discussion
Some chronic refractory and postinfectious coughs are common conditions associated with respiratory virus infection27. In order to better evaluate cough sensitivity and airway inflammation, a mouse model of cough was established using the H1N1 virus in this study. Appropriate mouse cough models should be selected for other studies according to the purpose of the study. Most previous studies used the guinea pig as an animal model in mechanistic studies or new drug trials for cough…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Guangzhou Science and Technology Planning Project (202002030151), the Major Project of Guangzhou National Laboratory (GZNL2024A02001), and the grant of State Key Laboratory of Respiratory Disease (SKLRD-Z-202202).
Materials
| 4% paraformaldehyde | Biosharp | BL539A | |
| Buxco Small Animal Whole Body Plethysmography System | DSI | — | |
| Calcium-free and magnesium-free Hank’s Balanced Salt Solution | Beyotime | C0219 | |
| Citric acid | Sigma-Aldrich | C2404 | |
| Hematoxylin-Eosin | BASO Biotechnology | BA-4098 | |
| Heparin sodium | Alfa Aesar | A16198 | |
| Influenza A/California/7/2009 (H1N1) virus | ATCC | VR-1894 | |
| Isoflurane | RWD | R510-22 | |
| Lactate dehydrogenase assay kit | Nanjing Jiancheng Bioengineering Institute | A020-2-2 | |
| Normal saline | Guangzhou Zhongbo Biotechnology | 1234-1 | |
| Pasteur pipet | NEST | 318415 | |
| Pentobarbital sodium | Merck | P3761 | |
| Phosphate buffered saline | Meilunbio | MA0015 | |
| Total protein assay kit | Nanjing Jiancheng Bioengineering Institute | A045-3 | |
| Uric acid assay kit | Thermo Fisher Scientific | A22181 |
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