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^3,4.

The first historically developed model of pulmonary fibrosis was the bleomycin-induced model, which is often used in academia, with the best characterization and most widely used animal model currently⁵, recapitulating many of the features of IPF and other fibrotic ILDs, including lung inflammation, epithelial injury, fibroblast proliferation, and excessive extracellular matrix (ECM) deposition⁶. In addition, both acute and chronic pulmonary fibrosis can be modeled depending on the dose and frequency of bleomycin administration. It has been reported that the lungs enter an acute inflammatory phase within 5-7 days after bleomycin exposure, followed by a fibrotic phase beginning around day 7. By day 21, pronounced fibrotic remodeling is typically observed, allowing the model to recapitulate both the early inflammatory response and the late-stage fibrotic pathology in a temporally defined manner⁷ (Figure 1A). This allows the study of the entire disease process from the initial injury to the fibrotic stage, which is very important for drug development and mechanism studies at all stages^8,9.

Bleomycin modeling routes are diverse and can be delivered locally or systemically, including endotracheal, nasal, intravenous, and intraperitoneal routes. The most commonly used is an endotracheal injection. The disadvantage is that it creates incisions in the mice, which affects the survival rate of the modeled mice¹⁰. Improvements were later made to allow the use of an indwelling needle for tracheal intubation and drip injection in mice¹¹. However, the drug pushed by the syringe can only be distributed in the lungs as a block, which can lead to uneven distribution of bleomycin in each lung lobe, producing local overdensity and affecting the stability of the mouse model. In addition, chamber nebulizer cartridges have been used for bleomycin delivery, but the amount of inhaled bleomycin aerosol in each mouse cannot be precisely quantified, and the single induction time is long¹². Therefore, a refined aerosol-based intratracheal bleomycin delivery method is developed for reproducible and minimally invasive mouse models of pulmonary fibrosis. Bleomycin is aerosolized, enabling uniform dispersion and deposition in the lungs, thereby better recapitulating pulmonary disease pathology. It is hoped that the emergence of this method will accelerate research into drugs that can completely cure pulmonary fibrosis.

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.
2. Acclimate mice to the facility for 5-7 days before initiating the aerosol-based model.

2. Preparation of bleomycin solution

CAUTION: Bleomycin is a cytotoxic antitumor agent. Perform all steps in a certified biosafety cabinet while wearing appropriate PPE.

1. Using a sterile 1 mL syringe, withdraw 3 mL of sterile 0.9% sodium chloride and inject it into a vial containing 15 U of bleomycin hydrochloride.
2. Gently swirl the vial until the powder is fully dissolved to obtain a 5 U/mL stock solution.
3. Aliquot the stock solution into sterile 1.5 mL microcentrifuge tubes. Prepare working solutions of 3.75 U/mL and 2.5 U/mL by diluting with sterile saline.
   NOTE: For a 25 g mouse, these concentrations correspond to doses of 7.5 U/kg and 5 U/kg, respectively. Adjust concentrations if the mouse's weights vary.
4. Store working solutions at 4 °C for up to 1 week or at -80 °C for up to 1 month. Thaw and bring to room temperature before use.

3. Aerosol-based intratracheal bleomycin delivery

1. Anesthesia preparation and administration
   1. Prepare the anesthetic working solution by dissolving 0.1 g of sodium pentobarbital powder in 10 mL of sterile physiological saline. Filter using a 0.22 μm PES membrane filter. Store the solution at 4 °C in a light-protected environment and use within 3 days of preparation.
   2. Administer sodium pentobarbital solution via intraperitoneal injection using a 1-mL syringe with a 26-gauge needle at a dose of 60 mg/kg body weight. If anesthesia depth or duration is insufficient, adjust the dose based on animal response under the guidance of the attending veterinarian.
   3. Assess anesthesia depth several minutes after injection by firmly pinching the toe and confirming the absence of withdrawal reflex before proceeding with subsequent procedures.
2. Preparation of aerosol delivery apparatus
   1. Prepare the aerosol delivery apparatus, ensuring that the nebulizing needle, nebulizing syringe, and dosing pillar are all present and sterile (Figure 1B).
   2. Submerge the aerosol delivery syringe in sterile saline. Slowly aspirate the fluid and pause for 8 s. Expel the fluid rapidly. Repeat this three times to fill and flush the system.
   3. Refill the syringe with saline after the final expulsion. Attach the aerosolizing needle, ensuring the junction is filled with saline.
   4. Submerge the assembled needle in saline and repeat the aspirate-pause-expel cycle three more times to eliminate air bubbles.
      NOTE: To confirm readiness, expel saline against a dark background. Ensure that the rapidly ejected saline forms a fan-shaped mist against a dark background.
3. Intratracheal aerosol delivery procedure
   1. Prime the aerosol needle with bleomycin working solution by aspirating and expelling it thrice with 8 s pauses.
   2. Insert a 25 µL dosing pillar on top of the 50 µL pillar to calibrate volume. Aspirate the required volume (50 µL). Expel any excess liquid.
      NOTE: This dosing configuration ensures consistent delivery by minimizing human error.
   3. Place the anesthetized mouse on the intubation platform, hooking the upper incisors and taping the limbs to stabilize (Figure 1C).
      NOTE: Confirm rhythmic chest movements. If the animal appears active, administer an additional anesthetic dose.
   4. Use curved forceps to pull out the tongue gently. Insert a small animal laryngoscope to expose the glottis (Figure 1D).
   5. Insert the aerosolizing needle vertically through the glottis. Deliver the bleomycin aerosol rapidly (Figure 1E).
      NOTE: Angle the needle tip toward the desired lung for targeted delivery. Avoid inserting too deeply to prevent trauma. The tip of the nebulizer needle should be positioned close to but not touching the bifurcation point (first bifurcation) of the mouse's trachea to achieve the best modeling effect. If the tip is inserted only into the middle of the trachea, the aerosol may only strike the tracheal wall, forming large droplets that are coughed up or swallowed. If the tip is too close to the tracheal bifurcation, there is a risk of tracheal damage.

4. Post-procedure recovery

1. Place the mouse on a warming pad to maintain body temperature. Observe the mouse continuously until it recovers from anesthesia (typically 1-2 h).
2. Perform a toe pinch to check reflexes and confirm the mouse is alive.
3. Perform daily clinical monitoring for 21 days, including body weight, activity level, grooming, and respiratory signs.
4. Euthanize animals when any of the following criteria are met: (i) >20% loss of body weight compared with the pre-treatment baseline, or ≥15% loss accompanied by other clinical signs of distress; (ii) persistent severe respiratory distress at rest (labored or abdominal breathing, marked tachypnea or bradypnea, nasal flaring, or open-mouth breathing), with or without cyanosis of the extremities; (iii) inability to obtain food or water, marked lethargy, or a moribund appearance (no response to handling and/or loss of the righting reflex); or (iv) severe dehydration, hypothermia, or other signs of irreversible deterioration as judged by the attending veterinarian. When these humane endpoints are reached, euthanize the animals with an overdose of sodium pentobarbital (≥150 mg/kg, i.p.), followed by a secondary physical method (e.g., cervical dislocation), to ensure death and minimize pain and distress.

5. Terminal lung function measurement and tissue collection

1. On day 21, anesthetize the mouse with 90 mg/kg i.p. of the same anesthesia solution to induce deep anesthesia and suppress breathing.
2. Confirm complete anesthesia via toe pinch. Expose the trachea by cutting the skin and separating the muscles.
3. Make a small beveled incision in the trachea and insert the intubation needle. Secure with a surgical ligature.
4. Transfer the mouse to the computer-controlled small animal lung mechanics ventilator. Initiate mechanical ventilation and ensure no spontaneous breathing. Use the ventilator system to measure pulmonary function parameters.
5. Perform right ventricular perfusion with 5-10 mL of sterile saline until the lungs appear blanched.
6. Dissect out the entire lung. Fix the left lung in neutral buffered formalin for 24 h.
7. Embed the fixed lung tissue in paraffin, section it, and perform H&E staining, Masson's trichrome staining, and immunohistochemical staining for COL1A1 and α-SMA.
8. Place the right lung in a 2 mL homogenization tube with beads. Add water per the hydroxyproline kit's protocol. Homogenize and proceed with the assay.

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