A Refined Aerosol-Based Intratracheal Bleomycin Delivery Method for Reproducible and Minimally Invasive Mouse Models of Pulmonary Fibrosis

Summary

Here, we present a protocol to model pulmonary fibrosis in mice using aerosolized bleomycin delivered via the trachea. This optimized method enables uniform, quantitative, and precise delivery without surgical incision, enhancing the reproducibility and reliability of bleomycin-induced fibrosis.

Abstract

Pulmonary fibrosis is characterized by progressive deposition of fibrotic scar tissue within the lung parenchyma, leading to severely impaired gas exchange. It underlies a spectrum of chronic interstitial lung diseases, notably idiopathic pulmonary fibrosis, a condition associated with an exceedingly poor prognosis. Given the lack of effective therapies, robust mouse models are critical for elucidating underlying pathological mechanisms and evaluating novel antifibrotic interventions. Bleomycin-induced pulmonary fibrosis remains the most extensively utilized experimental model. Common routes of administration in mice include intravenous and intraperitoneal injections, invasive open-tracheal instillation, and noninvasive tracheal dripping. However, invasive surgical methods often cause secondary tissue injury, potentially compromising model reproducibility and stability. Conversely, noninvasive tracheal dripping usually results in uneven bleomycin distribution across lung lobes and poses a risk of asphyxiation, thus reducing reproducibility and increasing technical challenges. To address these limitations, a refined aerosol-based intratracheal delivery method is developed that is operationally simpler, minimally invasive, highly reproducible, and ethically superior by significantly reducing animal distress. Using a small-animal laryngoscope to visualize the rima glottidis directly, a specialized aerosolizing needle is inserted into the trachea, markedly narrower than the mouse tracheal diameter. Bleomycin solution is delivered under precisely controlled pressure, generating a fine aerosol. This ensures uniform and efficient distribution of the agent throughout the lung parenchyma. Moreover, one can selectively target the left or right lung by directing the needle into the appropriate bronchus. This optimized model's dose-response relationship is extensively characterized by systematically monitoring changes in lung function, histopathological manifestations, and lung hydroxyproline content. This refined experimental protocol is anticipated to facilitate laboratory standardization, ultimately accelerating the development and preclinical validation of novel antifibrotic therapeutics.

Introduction

Pulmonary Fibrosis (PF) is a group of lethal diseases characterized by progressive lung parenchymal damage and abnormal repair, of which Idiopathic Pulmonary Fibrosis (IPF) accounts for 30%-40%, with the median age of diagnosis of patients being 65 years old, and more than 80% of them being 60 years old older than 60 years old^1,2. As global aging intensifies, the incidence of IPF is increasing year by year, with a worldwide IPF incidence of 3-9/100,000/year, and according to a study in the United States of America in people over 65 years of age, the incidence in this population surged to 93.7/100,000/year

Protocol

All animal procedures were conducted under institutional guidelines approved by the Institutional Animal Care and Use Committee (IACUC). Proper animal welfare measures were implemented throughout the experiment. Male C57BL/6 mice aged 6-8 weeks, weighing approximately 25 g, were selected for the experiment. The Table of Materials lists all animals, reagents, and equipment used in this study and their commercial sources.

1. Animal selection and acclimation

1. House all mice under specific pathogen-free conditions with a 12 h light/dark cycle and ad libitum access to food and water.

Results

In this study, we proposed an aerosol-based intrabronchial delivery method to induce pulmonary fibrosis in mice using bleomycin. In preliminary experiments, a green dye was aerosolized and administered to the lungs of different mice. The dye showed a speckled distribution pattern throughout the lungs, indicating that the aerosol-based intrabronchial administration allowed for uniform dispersion of the agent across all lung lobes (Figure 1F). The green ink delivery shown in

Discussion

This study proposed an optimized noninvasive tracheal nebulization model of bleomycin-induced pulmonary fibrosis in mice. A small animal laryngoscope was used to locate the glottis, and a tiny nebulization needle was inserted into the mouse's airway. Pressure was manually applied to nebulize the medication into the lungs, thereby reducing the discomfort and suffocation associated with the retention needle used in noninvasive injection^13,14. Additionally, these ae.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 μm polyethersulfone syringe filter	Cytosci	CSTPE3320
1 mL disposable syringe	BD	300481
1.5 mL microcentrifuge tube	Servicebio	EP-150-M
2.0 mL reinforced lysis tube	Servicebio	HT-200-M
4 °C laboratory refrigerator	Meiling	BCD-402WPCX
50 mL conical centrifuge tube	Servicebio	EP-5001-J
-80 °C ultra-low temperature freezer	Haier Biomedical	DW-86L828J
Absolute ethanol	Sinopharm Chemical Reagent Co., Ltd	100092683
Acidic differentiation solution	Servicebio	G1039
Aerosol microsprayer for small animal intubation	Shanghai Yuyan Instruments Co., Ltd	Yan-30012
Basic surgical instrument set	Shanghai Yuyan Instruments Co., Ltd	YAN-21A3
Bleomycin hydrochloride solution	Hanhui Pharmaceutical Co., Ltd	H20055883
Bluing reagent	Servicebio	G1040
Computer-controlled small animal lung mechanics ventilator	EMKA Biotech Beijing Co.,Ltd.	FX-2
Electrical insulation tape	3M	1500
Eosin Y staining solution	Zhuhai Baso Biotech Co., Ltd	BA4022
Green marking ink	Pilot	Chiku-rin
Hematoxylin staining solution	Servicebio	G1004
Hydroxyproline quantification assay kit	Abcam	ab222941
Laminar flow hood	HDL	DL-CJ-1N
Laryngoscope for small animal procedures	Shenzhen Bilead Biology Technology CO., LTD	DXSHJ0
Light microscope	Nikon	E100
Low-toxicity dewaxing solution	Servicebio	G1128
Masson's trichrome staining kit	Zhuhai Baso Biotech Co., Ltd	BA4079B
Microplate reader with absorbance detection	Agilent	Synergy H1
Neutral buffered formalin	Servicebio	G1101-3ML
Neutral mounting medium	Sinopharm Chemical Reagent Co., Ltd	10004160
Recombinant Rabbit Monoclonal Anti-COL1A1 Antibody	Huabio	HA722517
Refrigerated centrifuge	Eppendorf	5424R
RNase-free water	Generalbiol	TYJC002
Small animal tracheal intubation platform	Shanghai Yuyan Instruments Co., Ltd	CG-02M
Sterile 0.9% sodium chloride solution	China Otsuka Pharmaceutical Co., Ltd	H12020024
Tissue homogenizer	Servicebio	KZ-III
Xylene	Sinopharm Chemical Reagent Co., Ltd	10023418
Zirconia grinding beads	Servicebio	G0203