Refined Murine Model of Idiopathic Pulmonary Fibrosis

Summary

This protocol delineates a method for inducing pulmonary fibrosis in mice through repeated oropharyngeal administration of bleomycin, yielding pathological features closely resembling those of idiopathic pulmonary fibrosis.

Abstract

Animal models play a crucial role in understanding disease mechanisms and testing potential therapeutic approaches. However, traditional murine models of idiopathic pulmonary fibrosis (IPF) that rely on a single-dose bleomycin injection fail to accurately replicate the complex pathological features observed in IPF patients. This study introduces an improved method using two successive oropharyngeal administrations of bleomycin in mice, which more effectively mimics the progression of IPF. This novel protocol induces key pathological changes, such as the formation of honeycomb-like cysts, fibroblastic foci, and bronchiolization of the alveolar epithelium, closely resembling the histopathology seen in human IPF. By utilizing this model, we are able to capture the progressive fibrotic changes over a longer time frame, which provides more reliable insights into the pathogenesis of IPF. Furthermore, this two-dose approach offers several advantages over conventional single-dose models, including better reproducibility and a more robust representation of the disease's development. This method enhances our ability to study the mechanisms underlying IPF and evaluate potential therapeutic interventions. The proposed protocol offers significant advancement in preclinical research and provides a valuable tool for investigating both the biology of pulmonary fibrosis and the efficacy of novel treatments.

Introduction

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease characterized by irreversible lung parenchyma damage, leading to debilitating symptoms such as dyspnea and a severe decline in quality of life^1,2. Histologically, IPF manifests as usual interstitial pneumonia (UIP), featuring spatially heterogeneous fibrosis, bronchiolization of the alveolar epithelium, and fibroblastic foci³. Despite its clinical significance, the complex pathogenesis of IPF and limited therapeutic options highlight the urgent need for robust experimental models to advance mechanistic understanding and treatment development.

Existing single-dose bleomycin models, while widely used⁴, fail to recapitulate key pathological hallmarks of human IPF, such as KRT5+ basal cell pods^5,6,7 and persistent fibroblastic foci, and exhibit self-resolving fibrosis⁸. Repetitive bleomycin administration improves pathological fidelity^9,10 but is hindered by technical complexity, high mortality, and prolonged modeling cycles^11,12. To address these limitations, we developed an optimized murine model featuring twice-repeated oropharyngeal bleomycin administration¹³. This approach combines the pathological completeness of repetitive injury models^9,10 with the technical advantages of non-invasive delivery, achieving sustained fibrotic progression beyond 60 days while maintaining low mortality. By closely mimicking human IPF pathology, including honeycomb-like cyst formation and persistent fibrosis, our method provides a robust platform for investigating disease mechanisms and evaluating therapeutic interventions.

The protocol is particularly suitable for researchers requiring chronic fibrotic models without surgical complications. It offers significant advantages over existing approaches in terms of reproducibility, clinical relevance, and compliance with animal welfare guidelines. Building upon established oropharyngeal techniques, these refinements advance preclinical IPF research by bridging critical gaps between experimental models and human disease pathology.

Protocol

C57BL/6 male mice aged 4-6 months were used here. All experiments involving mice were conducted in accordance with the animal protocol approved by the Laboratory Animal Research Center of Sun Yat-sen University (approval number SYSU-IACUC-MED-2021-B0076). The study adhered to the animal care guidelines outlined by the Sun Yat-sen University Institutional Animal Care and Use Committee.

1. Preparation for experiment

1. Reagents: Acquire bleomycin powder, phosphate-buffered saline (PBS), and isoflurane.
2. Equipment: Acquire pipette tips (200 µL), pipette, tracheal injection stand, 1 mL pipette tip box, and alcohol cotton balls.

2. Experimental procedure

1. Prepare a bleomycin stock solution at a concentration of 1.5 mg/mL using PBS as the solvent. Aliquot 600 µL of the stock solution into 2 mL microcentrifuge tubes and dilute it with 1200 µL of PBS to achieve a 3-fold dilution (final concentration: 0.5 mg/mL).
   NOTE: The bleomycin stock solution was stored at −20 °C and remained stable for up to 3 months.
2. Weigh each mouse prior to oropharyngeal administration. Calculate the required volume of bleomycin solution based on the mouse's body weight. The dosage is 1.5 mg/kg, and the injection volume (in µL) is equal to the mouse's weight (in grams) multiplied by 3 (due to the 3-fold dilution).
3. Place the mouse in an isoflurane anesthesia induction chamber. Monitor the mouse until its breathing slows, indicating adequate anesthesia, and then immediately remove it.
   NOTE: The transition from tachypnea to bradypnea signifies successful anesthesia. Immediate removal upon observed bradypnea is critical to prevent anesthetic overdose. Premature removal during tachypnea may lead to animal arousal during subsequent procedures.
4. Secure the mouse on the tracheal injection stand by hooking its teeth. Gently pull out the tongue using forceps and hold it with the thumb and index finger (avoid using forceps to minimize discomfort). Cover the mouse's nose with the middle finger to ensure proper delivery.
5. Using a 200 µL pipette tip, aspirate the calculated volume of diluted bleomycin solution, then slowly administer the bleomycin solution into the mouse's oropharynx without inserting the pipette tip deeply. This approach minimizes tissue damage and ensures effective delivery.
6. Listen for a distinct gurgling sound, indicating successful entry of the solution into the lungs. Once the sound ceases, remove the mouse from the stand and place it on a heating pad for recovery.
   NOTE: A characteristic gurgling sound serves as a reliable indicator of successful bleomycin delivery into the oropharynx. The absence of this acoustic cue suggests esophageal deposition, at which point the subject is excluded from subsequent experimental procedures due to failed administration.
7. Repeat the oropharyngeal administration procedure 7 days after the initial dose.
   NOTE: During the 60-day post-bleomycin period, monitor mice daily for: weight loss (>20% baseline weight), respiratory distress (labored breathing, tachypnea >160 bpm), activity decline (hunching, isolation).

3. Tissue collection

1. Euthanize the mice by cervical dislocation at the predetermined time points (14 and 60 days post-final bleomycin administration).
2. Perform a midline sternotomy to expose the thoracic cavity. The left atrial appendage was surgically incised. Subsequently, perfuse the lungs through the right ventricle with 10 mL of ice-cold PBS to remove circulating blood cells.
3. Cannulate the trachea and inflate the lungs with 1% paraformaldehyde (PFA) to ensure complete tissue fixation.
4. Excise the lungs and immerse them in 1% PFA for 1 h at room temperature to allow for proper fixation.
5. For tissue processing, rinse the fixed tissues in PBS 3x for 5 min each. Dehydrate the tissues sequentially using a graded ethanol series: 70% ethanol, 95% ethanol, and 100% ethanol, ensuring complete dehydration at each step. After dehydration, clear the tissues in xylene for 1 h at room temperature. Subsequently, embed the tissues in molten paraffin wax at 60 °C for at least 2 h, or until fully infiltrated. Finally, section the embedded tissues at a thickness of 7 µm using a rotary microtome.

4. Sample assessment

1. Perform hematoxylin and eosin (H&E) and Masson's trichrome staining to evaluate fibrosis severity.
   1. For H&E staining, perform sequential xylene deparaffinization for 5 min, 2x, and graded ethanol rehydration with 100% ethanol for 5 min followed by 100% ethanol for 1 min, then 95% ethanol for 1 min, and finally 80% ethanol for 1 min. Rinse with distilled water. Stain with hematoxylin for 5 min, followed by rinsing with tap water 3x and soaking for 5 min. Then, perform 80% ethanol dip for 1 min and counterstaining with eosin for 45 s. Perform dehydration with graded ethanol using 95% ethanol for 5 dips, followed by 100% ethanol for 5 dips, and finally, immerse in 100% ethanol for 2 min. Clear with xylene by dipping for 5 min, 2x and then perform neutral balsam mounting.
   2. For Masson's staining, after dewaxing as done in step 4.1.1, rinse slides in ddH₂O and stain with Ponceau S-fuchsin for 5 s, followed by 0.2% weak acid solution (acetic acid/water) rinse for 1 min. Perform differentiation in phosphomolybdic acid solution for 5 min with subsequent weak acid rinse for 1 min, followed by aniline blue counterstaining for 1 min and final weak acid wash for 1 min. Dehydrate sections through graded ethanol starting with 95% ethanol for 2-3 s followed by absolute ethanol for 5-10 s, 2x. Clear in xylene and mount with resin.
2. Conduct immunohistochemical (IHC) and immunofluorescence (IF) staining for markers such as α-SMA, KRT5, and ΔN-p63^14,15 to assess fibrotic and epithelial remodeling.
   1. For immunohistochemical staining, carry out dewaxing and rehydration of paraffin-embedded tissue sections prior to antigen retrieval in boiling 0.1 M sodium citrate buffer (pH 6.0) containing 0.5% Tween-20 for 90 s. Quench endogenous peroxidase activity with 3% H₂O₂, followed by membrane permeabilization with 0.5% Triton X-100/PBS and non-specific blocking. Incubate in primary antibody at 4 °C overnight, followed by 1 h room-temperature incubation with species-matched secondary antibodies and 30 min streptavidin-HRP conjugation. For chromogenic development, use Vector SK-4100 DAB substrate with hematoxylin counterstaining, ethanol dehydration, and neutral balsam mounting to complete the protocol.
   2. For immunofluorescence staining, following standard immunohistochemical processing (dewaxing, antigen retrieval, blocking, primary and secondary antibody incubation), label biotinylated targets with Streptavidin-conjugated Alexa Fluor 594 or 647 (1:500 dilution, 30 min incubation at room temperature). Perform nuclear counterstaining using DAPI (20 µg/mL final concentration) for 5 min, followed by mounting with antifade medium to preserve fluorescence signals.

Results

Compared to the traditional single-dose oropharyngeal bleomycin administration, this method successfully recapitulates hallmark pathological features of IPF, including fibroblastic foci (Figure 1B), honeycombing (Figure 1G) and bronchiolization of the alveolar epithelium (Figure 1H). Importantly, the model demonstrates the persistence of these features over time (Figure 1B), closely mimicking the chroni...

Discussion

In this study, we present a refined murine model of IPF utilizing twice-repeated oropharyngeal administration of bleomycin. This approach addresses the critical limitations of traditional single-dose models, which fail to recapitulate key pathological hallmarks of human IPF, such as KRT5+ and p63+ basal cell pods^7,14,15, persistent fibroblastic foci³, and irreversible fibrosis⁸. By...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagent used
bleomycin	MCE	HY-17565
H&E staining kit	Solarbio	G1120
isoflurane	RWD	R510-22-10
Masson's staining kit	Solarbio	G1340
PBS	Biosharp	BL302A
Antibodies used			Dilution
α-SMA	Abcam	ab5694	1:100
Cytokeratin 5	Immunoway	YT1271	1:100
DeltaN p63	CST	67825	1:100
Goat Anti-Rabbit IgG H&L(biotin)	Abcam	ab6720	1:800
Hydroxyproline	CST	73812	1:100
Peroxidase-conjugated Streptavidin	Jackson	016-030-084	1:100
Streptavidin / AF594	Bioss	bs-0437P-AF594	1:500