Establishment of a novel severe asthma canine model: Feasible research platform of respiratory intervention therapies

Research on interventional pulmonological therapy for asthma via bronchoscopy could only be done through large animal experiments or clinical trials. However, the absence of large animal models of asthma and the heterogeneity among asthma patients have hindered researchers from thoroughly exploring its therapeutic mechanisms. The current use of large animal asthma models is limited by lengthy modeling time, high cost, low success rate, and atypical asthma clinical signs, rendering them impractical.1 Toluene diisocyanate (TDI)-induced asthma, a common form of occupational asthma, displays characteristic features including hyperresponsiveness, airway inflammation, and remodeling, having a propensity to progress to severe asthma.2 Based on these considerations, we aimed to establish a canine model exhibiting typical asthmatic features by exposing the animals to TDI via aerosol inhalation. A total of eight adult beagles were treated with 0.3% TDI on the dorsum of both ears once per week (200 μL/ear) 2 weeks before airway challenge. TDI challenge was subsequently conducted twice per week for 8 weeks. The beagles were placed in a transparent rectangular chamber (60 cm × 75 cm × 60 cm) and exposed to TDI aerosol produced by compressed air nebulization (NE-C28; Omron, Tokyo, Japan) for 2 h per session (Figure S1). The concentration of TDI in the airway challenge reagents was gradually increased from 3% to 5% over time. Detail method was shown in supplementary material. All 8 beagles received 8 weeks of TDI challenge and exhibited pronounced asthma clinical signs, including wheeze, cough, and breathlessness. Blood and bronchoalveolar lavage fluid samples indicated that a mixed granulocytic inflammation characterized by neutrophils systemically and in the lungs, accompanied by significant increase in total immunoglobulin E, immunoglobulin G, interleukin-13, and interleukin-17 levels (Figure 1). Airway hyperresponsiveness was assessed by changes in airway resistance in response to methacholine at different concentrations. After 8 weeks of TDI exposure, there was a significant increase in airway resistance at each concentration of methacholine tested (Figure S2). The application of optical coherence tomography (OCT) provides a non-invasive method to evaluate the airway structure, airway mucus retention and mucosal thickening. A trend toward increasing airway wall area in airway segments of different sizes were shown by OCT after 8 weeks of modeling (Figure S2). In terms of histopathology analysis, the asthma model beagles presented with a significant airway remodeling, including goblet cell metaplasia, infiltration of inflammatory cells and significant thickened airway smooth muscle. As the markers of cholinergic nerve tension increase and airway neuronal remodeling, immunohistochemistry showed the high expression of muscarinic 3 (M3) receptor and protein gene product 9.5 (PGP9.5) (Figure 2). Additionally, we randomly selected four modeling beagles to perform clinically standard bronchial thermoplasty (BT) using the Alair system. After three sections of BT treatment, the canines exhibited significant ablation of airway smooth muscle, resulting in decreased airway hyperresponsiveness. (Figure S3). The decrease in M3 receptor and PGP9.5 expression indicated that this model could reflect the denervation effect of BT3 (Figure 2). Therefore, our study demonstrates that the TDI-induced beagle model exhibited typical manifestations of severe asthma and effectively reflected the efficacy of respiratory intervention therapy, suggesting its potential as a valid candidate for experiment research in this field. The latest research in interventional pulmonological therapy for severe asthma, such as targeted denervation or cryoablation, typically involves experiments conducted on healthy animals or directly recruits severe asthma patients for clinical studies, with symptom scores as the primary endpoints.4, 5 However, both types of studies failed to explore the mechanisms behind interventional respiratory therapies, thus leaving them as effective treatments without understanding the underlying reasons. Establishing a rapid severe asthma large animal model with typical clinical signs allows us to analyze the therapeutic effects of interventional pulmonological therapy from a perspective that closely mirrors clinical reality.6 It also allows researchers to delve deeper into its specific mechanisms of action, further clarifying its appropriate patient population and go further for the novel therapeutic strategy. All authors had full access to the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Conceptualization: DC, ZS, YL, SL; Project administration: DC, ZS, YL, SL; Methodology: DC, ZS, YL, KC, SL; Data curation: DC, YL, ZZ, ZG, LY, JL; Formal analysis: DC, ZS, YL, YC, KC, CZ, XC, CT; Funding acquisition: YL, SL; Resources: YL, SL; Supervision: SL; Validation: DC, ZS, YL, SL; Visualization: DC, ZS, YL, SL; Roles/Writing—original draft: DC, ZS, YL, SL. The authors have nothing to report. Shi-Yue Li reports grants from National Natural Science Foundation of China (grant number 81770017); Yu-Long Luo reports grants from Basic Research Foundation of Guangzhou (202102020916) and Plan on Enhancing Scientific Research in Guangzhou Medical University. The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The authors have no conflicts of interest to declare. The data used and analysed during the current study are available from the corresponding author on reasonable request. Data S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.