Supplementary Material for: Efficacy of Spray Cryotherapy on Airway Secretions in Canine Models of Chronic Bronchitis and Mechanism Insights

Introduction Bronchoscopic spray cryotherapy (SCT) is a novel treatment showing promise for chronic bronchitis (CB), characterized by excessive mucus secretion and productive cough. A large animal model for preclinical research of SCT is lacking, and its treatment's efficacy and mechanisms for CB are not well understood. Methods Eight Labradors were exposed to 200 ppm SO2 for 6 months to develop a CB model. Evaluations included pulmonary resistance, bronchoalveolar lavage fluid (BALF), CT images, and pathology. After model validation, six dogs received SCT and were observed for short-term (7 days) and long-term (30 days) outcomes. Metrics included pulmonary resistance, bronchoscopy findings, and BALF analysis for inflammatory factors, acetylcholine, and mucins. Bronchial tissue was assessed via HE staining, electron microscopy, and IHC staining. BEAS-2B cells were used to study MUC5AC expression in response to LPS and acetylcholine. Results SO2 exposure led to persistent cough, increased pulmonary resistance, goblet cell hyperplasia, and inflammation. Mucin, MUC5AC, and MUC5B levels in BALF increased over time, which validated the CB model. SCT treatment reduced mucus and pulmonary resistance, improved bronchial structure, and decreased goblet cells. SCT significantly reduced BALF mucin, MUC5AC, MUC5B, acetylcholine, IL-6, INF-γ, TNF-α, and IL-10, and bronchial MUC5AC and CHRM3. In the LPS treatment BEAS-2B cells, MUC5AC expression increased when acetylcholine pretreatment concentration increased. Conclusion The SO2 inhalation protocol effectively establishes a CB model in dogs. SCT effectively treats CB by reducing mucin levels and may lower MUC5AC expression by decreasing acetylcholine and CHRM3.