Modeling Principle

The Beagle Pulmonary Fibrosis Model is a drug-induced idiopathic pulmonary fibrosis pathological model constructed by precise intratracheal puncture and instillation of bleomycin solution under general anesthesia based on SCI standardized large-animal modeling system. It accurately recapitulates the whole occurrence and progression of clinical idiopathic pulmonary fibrosis (IPF), including acute injury and apoptosis of alveolar epithelial cells, massive infiltration of inflammatory cells in lung with release of inflammatory factors, collapse and destruction of alveolar structure, abnormal activation and proliferation of fibroblasts, disordered massive deposition of extracellular matrix, thickening and occlusion of alveolar wall, pulmonary parenchymal remodeling, decreased diffusion capacity and restrictive ventilatory disorder. It remedies the experimental defects of rodents, which have obvious discrepancies with human interstitial lung diseases in tiny lung volume, airway anatomical hierarchy, alveolar arrangement, intensity of pulmonary inflammatory response, progression rate of fibrosis and variation law of respiratory mechanical parameters.

The airway branch anatomy, alveolar morphology and distribution, physiological function of type Ⅰ/Ⅱ alveolar epithelial cells, component of pulmonary interstitial cells, response pattern of pulmonary immune cells, classic TGF-β1/Smad fibrotic signaling pathway, metabolic degradation rule of pulmonary collagen and respiratory mechanical characteristics of adult Beagle dogs are highly homologous to human lung tissues. Animals are fixed in supine position after anesthesia, intratracheal puncture is carried out with laryngoscope assistance, and quantitative sterile bleomycin solution is instilled at one time. Bleomycin directly causes oxidative stress injury of alveolar epithelium. In the early stage, neutrophils, macrophages and lymphocytes are recruited to infiltrate lung tissues, and a large number of pro-inflammatory mediators such as TNF-α, IL-1β and IL-6 are released to form acute inflammatory lung injury. Sustained inflammatory stimulation induces resting pulmonary fibroblasts to transform into myofibroblasts, which continuously secrete extracellular matrix such as type Ⅰ and Ⅲ collagen and fibronectin. Massive collagen accumulates diffusely in alveolar septa and pulmonary interstitium, normal alveolar structures are replaced by fibrous tissues, lung tissue stiffness increases and lung compliance decreases, finally forming a stable pathological phenotype of chronic diffuse pulmonary interstitial fibrosis. This model fully recapitulates the classic pathogenic cascade of human idiopathic pulmonary fibrosis: toxic damage of alveolar epithelium induced by drugs → acute pulmonary inflammatory response → sustained stimulation of chronic inflammation → activation and proliferation of fibroblasts → accumulation and deposition of extracellular matrix → destruction and remodeling of alveolar structure → irreversible pulmonary fibrosis.

Beagle dogs have thick airways convenient for intratracheal administration, the dosage and concentration for modeling can be accurately quantified and regulated, the course of pulmonary inflammation and fibrosis progresses gently and gradually with low discrete degree of individual experimental data, and there is no spontaneous pulmonary inflammation or congenital pulmonary dysplasia. Three pathological stages can be clearly distinguished: early stage of acute inflammatory injury, middle stage of fibrous proliferation after inflammation subsidence, and terminal stage of mature diffuse pulmonary fibrosis. It serves as a standardized large-animal gold-standard model for translational researches on new anti-fibrotic drugs, pulmonary epithelial protective preparations, pulmonary anti-inflammatory drugs, inhaled targeted preparations and interventional intervention strategies for pulmonary fibrosis.

Modeling Success Criteria

In-vivo Pulmonary Function, Chest CT and General Lung Morphological Phenotype

The FVC, DLCO and lung compliance of animals in the sham control group were within the normal range, the lung field on chest CT was clear without abnormal high-density shadow, and the isolated lung tissue was soft and uniformly pink in color. At Week 8 terminal point, FVC and DLCO of the model group decreased significantly compared with the sham group, and lung compliance was obviously reduced. Multiple cord-like and grid-like high-density fibrotic lesions could be seen in both lungs on chest CT. Gross anatomical observation showed hardened lung tissue with shrunk volume, uneven lung surface accompanied by hyperplasia of gray-white fibrous cords. The macroscopic phenotype of diffuse pulmonary fibrosis was obvious with extremely significant statistical difference between groups, which could preliminarily confirm the successful construction of bleomycin-induced pulmonary fibrosis model.

Quantitative Biochemical Gold-Standard Serum Indexes

The serum levels of TNF-α, IL-1β and IL-6 in the acute phase of the model group were extremely significantly higher than those in the sham group; serum pulmonary injury markers KL-6 and SP-A were persistently highly expressed in the late observation period, and the ratios of TGF-β1, type Ⅰ/Ⅲ collagen and MMP-9/TIMP-1 in plasma and lung tissues increased significantly, which fully conforms to the biochemical diagnostic characteristics of clinical IPF: release of markers due to alveolar epithelial injury, persistent existence of chronic low-grade inflammation, massive collagen deposition mediated by TGF-β1 and imbalance of matrix metalloproteinase system.

Lung Histopathological Characteristics

Sequential pathological injury changes of pulmonary fibrosis can be observed via combined staining of lung tissues with HE, Masson, Sirius red and TUNEL:

1. Acute stage at Week 2 after modeling: a small amount of exudate in alveolar cavity, thickened alveolar wall, massive inflammatory cell infiltration in pulmonary interstitium, and significantly increased number of TUNEL-positive apoptotic alveolar epithelial cells;
2. Middle proliferation stage at Week 5 after modeling: alleviated inflammatory cell infiltration, disordered alveolar structure, increased pulmonary interstitial fibroblasts, and a small amount of collagen fiber deposition observed by Masson and Sirius red staining;
3. Terminal fibrosis stage at Week 8 after modeling: massive destruction and disappearance of normal alveolar structure, significantly widened alveolar septa, and diffuse accumulation of large-area blue-stained collagen fibers in pulmonary interstitium to form typical fibrotic lesions, which perfectly recapitulates the progressive pathological evolution law from acute inflammatory injury to extensive interstitial fibrosis in human idiopathic pulmonary fibrosis.

Gold-Standard Indexes of Fibrosis and Inflammatory Pathways

Bleomycin in the model group induced alveolar epithelial apoptosis to release damage-associated molecular patterns, activated NF-κB inflammatory pathway to continuously amplify pulmonary inflammatory response. The chronic inflammatory microenvironment up-regulated the activity of TGF-β1/Smad signaling pathway, driving resting fibroblasts to transform into myofibroblasts and excessively synthesize & secrete type Ⅰ and Ⅲ collagen. Imbalance of MMP/TIMP hindered the degradation of extracellular matrix, and continuous collagen accumulation remodeled the pulmonary parenchymal structure. It accurately conforms to the complete pathological mechanism of intratracheal bleomycin administration: epithelial oxidative damage – pulmonary inflammatory cascade – fibroblast activation – collagen accumulation – diffuse pulmonary interstitial fibrosis, serving as the core academic basis for verifying effective model formation.

Model Advantages

This model is a well-recognized large-animal gold-standard model for pulmonary fibrosis prepared by intratracheal instillation of bleomycin in respiratory medicine and pulmonary pharmacology SCI fields. The 8-week standardized modeling process can stably recapitulate the complete IPF pathological phenotypes highly homologous to humans, including alveolar injury, acute pulmonary inflammation, interstitial collagen deposition, alveolar remodeling and decreased restrictive ventilation function. The airway hierarchical structure, alveolar physiology, pulmonary immune response and fibrosis progression kinetics of Beagle dogs are highly consistent with human beings. The distribution of fibrotic lesions is uniform with mild and controllable disease progression and low intra-group data dispersion, and the experimental repeatability is obviously superior to small animals such as rodents and rabbits. The modeling method adopts local airway administration with targeted damage concentrated in lung tissues without large-scale systemic toxic and side effects, which highly matches the injury mechanism of clinical idiopathic pulmonary fibrosis. Non-invasive pulmonary function detection and blood sample collection can be carried out for many times with abundant whole lung tissue samples, which is suitable for preclinical pharmacodynamic and safety evaluation of various oral/inhaled/intravenous new anti-fibrotic drugs, pulmonary protective preparations, stem cell pulmonary repair systems and pulmonary targeted delivery carriers. Experimental data are widely accepted by high-impact SCI journals of respiratory medicine, pharmacology and pharmaceutics, which is applicable for National Natural Science Foundation projects, master & doctoral proposal writing, respiratory department graduation theses and translational medical researches on targeted intervention of pulmonary fibrosis.

Research Applications

The Beagle bleomycin-induced Pulmonary Fibrosis Model is mainly applied to analyze basic pathological mechanisms including oxidative apoptotic injury of alveolar epithelium caused by intratracheal bleomycin administration, massive release of acute pulmonary inflammatory factors, sustained stimulation of chronic inflammation leading to fibroblast activation, massive collagen synthesis & deposition mediated by activation of TGF-β1 signaling pathway, destruction and remodeling of alveolar structure, as well as initiation and progression of restrictive ventilatory dysfunction. It is specially adopted to screen and verify small-molecule anti-fibrotic drugs, active components of traditional Chinese medicine, atomized inhalation preparations, stem cell repair preparations and pulmonary targeted nano delivery systems with functions of alleviating oxidative damage of alveolar epithelium, inhibiting pulmonary inflammatory cascade reaction, blocking the transformation of fibroblasts into myofibroblasts, down-regulating abnormal collagen secretion and deposition, delaying pathological alveolar remodeling and improving pulmonary ventilation & diffusion function. It is widely used for excavation of pathogenic molecular targets of pulmonary fibrosis, elucidation of interactive regulatory network among inflammation, apoptosis and fibrosis, as well as preclinical in-vivo efficacy verification of various anti-fibrotic medical products in large animals. It acts as an essential and scarce standardized large-animal gold-standard model in the fields of respiratory pharmacology, pathophysiology of interstitial lung disease and pulmonary targeted drug delivery.

beagle bleomycin-induced pulmonary fibrosis model, intratracheal instillation, diffuse pulmonary interstitial fibrosis, lung remodeling, respiratory dysfunction, anti-fibrotic drugs & pulmonary protective agents preclinical in-vivo evaluation