Name: Refined Murine Model of Idiopathic Pulmonary Fibrosis (Video) | JoVE Journal
Uploaded: 2025-06-17T13:00:00.000+00:00
Duration: 7 min 51 s
Description: Learn a refined murine model of idiopathic pulmonary fibrosis using repeated oropharyngeal bleomycin administration in mice. This method reduces mortality while recapitulating key pathological features including fibroblastic foci and collagen deposition.

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5 minutes

This research was funded by Shenzhen Scienceand Technology Program (Grant #JCYJ20230807110800002 to C.L.), Natural Science Foundation of Guangdong (#2024A1515011087 to C.L.), and Science and Technology Program of Guangzhou (# 202201020299 to Z.L.).

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
<ol>
	<li>Reagents: Acquire bleomycin powder, phosphate-buffered saline (PBS), and isoflurane.</li>
	<li>Equipment: Acquire pipette tips (200 &#181;L), pipette, tracheal injection stand, 1 mL pipette tip box, and alcohol cotton balls.</li>
</ol>
2. Experimental procedure
<ol>
	<li>Prepare a bleomycin stock solution at a concentration of 1.5 mg/mL using PBS as the solvent. Aliquot 600 &#181;L of the stock solution into 2 mL microcentrifuge tubes and dilute it with 1200 &#181;L of PBS to achieve a 3-fold dilution (final concentration: 0.5 mg/mL). 
	NOTE: The bleomycin stock solution was stored at &#8722;20 &#176;C and remained stable for up to 3 months.</li>
	<li>Weigh each mouse prior to oropharyngeal administration. Calculate the required volume of bleomycin solution based on the mouse&#39;s body weight. The dosage is 1.5 mg/kg, and the injection volume (in &#181;L) is equal to the mouse&#39;s weight (in grams) multiplied by 3 (due to the 3-fold dilution).</li>
	<li>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.</li>
	<li>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&#39;s nose with the middle finger to ensure proper delivery.</li>
	<li>Using a 200 &#181;L pipette tip, aspirate the calculated volume of diluted bleomycin solution, then slowly administer the bleomycin solution into the mouse&#39;s oropharynx without inserting the pipette tip deeply. This approach minimizes tissue damage and ensures effective delivery.</li>
	<li>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.</li>
	<li>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 (&#62;20% baseline weight), respiratory distress (labored breathing, tachypnea &#62;160 bpm), activity decline (hunching, isolation).</li>
</ol>
3. Tissue collection
<ol>
	<li>Euthanize the mice by cervical dislocation at the predetermined time points (14 and 60 days post-final bleomycin administration).</li>
	<li>Perform a midline sternotomy to expose the thoracic cavity. The left atrial appendage was&#160;surgically incised. Subsequently, perfuse the lungs through the right ventricle with 10 mL of ice-cold PBS to remove circulating blood cells.</li>
	<li>Cannulate the trachea and inflate the lungs with 1% paraformaldehyde (PFA) to ensure complete tissue fixation.</li>
	<li>Excise the lungs and immerse them in 1% PFA for 1 h at room temperature to allow for proper fixation.</li>
	<li>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 &#176;C for at least 2 h, or until fully infiltrated. Finally, section the embedded tissues at a thickness of 7 &#181;m using a rotary microtome.</li>
</ol>
4. Sample assessment
<ol>
	<li>Perform hematoxylin and eosin (H&#38;E) and Masson&#39;s trichrome staining to evaluate fibrosis severity.
	<ol>
		<li>For H&#38;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.</li>
		<li>For Masson&#39;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.</li>
	</ol>
	</li>
	<li>Conduct immunohistochemical (IHC) and immunofluorescence (IF) staining for markers such as &#945;-SMA, KRT5, and &#916;N-p6314,15 to assess fibrotic and epithelial remodeling.
	<ol>
		<li>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 &#176;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.</li>
		<li>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 &#181;g/mL final concentration) for 5 min, followed by mounting with antifade medium to preserve fluorescence signals.</li>
	</ol>
	</li>
</ol>

<ol><li>Albera, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Challenges+in+idiopathic+pulmonary+fibrosis+trials%3A+the+point+on+end-points%5BTitle%5D)+AND+%22Eur+Respir+Rev%22%5BJournal%5D)">Challenges in idiopathic pulmonary fibrosis trials: the point on end-points.</a> Eur Respir Rev. 20 (121), 195-200 (2011).</li>
<li>du Bois, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Predictors+of+Mortality+in+Patients+with+Idiopathic+Pulmonary+Fibrosis+%28IPF%29%5BTitle%5D)+AND+%22Am+J+Respir+Crit+Care+Med%22%5BJournal%5D)">Predictors of Mortality in Patients with Idiopathic Pulmonary Fibrosis (IPF).</a> Am J Respir Crit Care Med. 179, A1114(2009).</li>
<li>Martinez, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Idiopathic+pulmonary+fibrosis%5BTitle%5D)+AND+%22Nat+Rev+Dis+Primers%22%5BJournal%5D)">Idiopathic pulmonary fibrosis.</a> Nat Rev Dis Primers. 3, 17074(2017).</li>
<li>Wollin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mode+of+action+of+nintedanib+in+the+treatment+of+idiopathic+pulmonary+fibrosis%5BTitle%5D)+AND+%22Eur+Respir+J%22%5BJournal%5D)">Mode of action of nintedanib in the treatment of idiopathic pulmonary fibrosis.</a> Eur Respir J. 45 (5), 1434-1445 (2015).</li>
<li>Moeller, A., Ask, K., Warburton, D., Gauldie, J., Kolb, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+bleomycin+animal+model%3A+A+useful+tool+to+investigate+treatment+options+for+idiopathic+pulmonary+fibrosis%5BTitle%5D)+AND+%22Int+J+Biochem+Cell+Biol%22%5BJournal%5D)">The bleomycin animal model: A useful tool to investigate treatment options for idiopathic pulmonary fibrosis.</a> Int J Biochem Cell Biol. 40 (3), 362-382 (2008).</li>
<li>Fernanda de Mello Costa, M., Weiner, A. I., Vaughan, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Basal-like+Progenitor+Cells%3A+A+Review+of+Dysplastic+Alveolar+Regeneration+and+Remodeling+in+Lung+Repair%5BTitle%5D)+AND+%22Stem+Cell+Rep%22%5BJournal%5D)">Basal-like Progenitor Cells: A Review of Dysplastic Alveolar Regeneration and Remodeling in Lung Repair.</a> Stem Cell Rep. 15 (5), 1015-1025 (2020).</li>
<li>Kathiriya, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+alveolar+type+2+epithelium+transdifferentiates+into+metaplastic+KRT5%28%2B%29+basal+cells%5BTitle%5D)+AND+%22Nat+Cell+Biol%22%5BJournal%5D)">Human alveolar type 2 epithelium transdifferentiates into metaplastic KRT5(+) basal cells.</a> Nat Cell Biol. 24 (1), 10-23 (2022).</li>
<li>Chung, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+repeated+lung+injury+and+genetic+background+in+bleomycin-induced+fibrosis%5BTitle%5D)+AND+%22Am+J+Respir+Cell+Mol+Biol%22%5BJournal%5D)">Role of repeated lung injury and genetic background in bleomycin-induced fibrosis.</a> Am J Respir Cell Mol Biol. 29 (3 Pt 1), 375-380 (2003).</li>
<li>Ley, B., Collard, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidemiology+of+idiopathic+pulmonary+fibrosis%5BTitle%5D)+AND+%22Clin+Epidemiol%22%5BJournal%5D)">Epidemiology of idiopathic pulmonary fibrosis.</a> Clin Epidemiol. 5, 483-492 (2013).</li>
<li>Costabel, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+changing+treatment+landscape+in+idiopathic+pulmonary+fibrosis%5BTitle%5D)+AND+%22Eur+Respir+Rev%22%5BJournal%5D)">The changing treatment landscape in idiopathic pulmonary fibrosis.</a> Eur Respir Rev. 24 (135), 65-68 (2015).</li>
<li>Degryse, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Repetitive+intratracheal+bleomycin+models+several+features+of+idiopathic+pulmonary+fibrosis%5BTitle%5D)+AND+%22Am+J+Physiol+Lung+Cell+Mol+Physiol%22%5BJournal%5D)">Repetitive intratracheal bleomycin models several features of idiopathic pulmonary fibrosis.</a> Am J Physiol Lung Cell Mol Physiol. 299 (4), L442-L452 (2010).</li>
<li>Rydell-Törmänen, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+matrix+alterations+and+acute+inflammation%3B+developing+in+parallel+during+early+induction+of+pulmonary+fibrosis%5BTitle%5D)+AND+%22Lab+Invest%22%5BJournal%5D)">Extracellular matrix alterations and acute inflammation; developing in parallel during early induction of pulmonary fibrosis.</a> Lab Invest. 92 (6), 917-925 (2012).</li>
<li>Wang, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+mouse+model+of+pulmonary+fibrosis%3A+twice-repeated+oropharyngeal+bleomycin+administration+mimicking+human+pathology%5BTitle%5D)+AND+%22Biochem+Cell+Biol%22%5BJournal%5D)">A novel mouse model of pulmonary fibrosis: twice-repeated oropharyngeal bleomycin administration mimicking human pathology.</a> Biochem Cell Biol. 103, 1-7 (2025).</li>
<li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((miR-184+targets+TP63+to+block+idiopathic+pulmonary+fibrosis+by+inhibiting+proliferation+and+epithelial-mesenchymal+transition+of+airway+epithelial+cells%5BTitle%5D)+AND+%22Lab+Invest%22%5BJournal%5D)">miR-184 targets TP63 to block idiopathic pulmonary fibrosis by inhibiting proliferation and epithelial-mesenchymal transition of airway epithelial cells.</a> Lab Invest. 101 (2), 142-154 (2021).</li>
<li>Zuo, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((p63%28%2B%29Krt5%28%2B%29+distal+airway+stem+cells+are+essential+for+lung+regeneration%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">p63(+)Krt5(+) distal airway stem cells are essential for lung regeneration.</a> Nature. 517 (7536), 616-620 (2015).</li>
<li>Parlar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+cannabinoid+receptor+2+synthetic+agonist%2C+AM1241%2C+on+bleomycin+induced+pulmonary+fibrosis%5BTitle%5D)+AND+%22Biotech+Histochem%22%5BJournal%5D)">Effects of cannabinoid receptor 2 synthetic agonist, AM1241, on bleomycin induced pulmonary fibrosis.</a> Biotech Histochem. 96 (1), 48-59 (2021).</li>
<li>Uysal, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Role+of+Cannabinoid-1+Receptor+Ligands+in+the+Ovalbumin-Induced+Mouse+Model+of+Allergic+Asthma%3A+Is+It+Related+to+Transient+Receptor+Potential+Vanilloid-1+Channels%5BTitle%5D)+AND+%22Cannabis+Cannabinoid+Res%22%5BJournal%5D)">The Role of Cannabinoid-1 Receptor Ligands in the Ovalbumin-Induced Mouse Model of Allergic Asthma: Is It Related to Transient Receptor Potential Vanilloid-1 Channels.</a> Cannabis Cannabinoid Res. 10 (1), e121-e133 (2025).</li>
</ol>

The authors have no conflicts of interest to declare.

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 pods7,14,15, persistent fibroblastic foci3, and irreversible fibrosis8. By...

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 life1,2. 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 pods5,6,7 and persistent fibroblastic foci, and exhibit self-resolving fibrosis8. Repetitive bleomycin administration improves pathological fidelity9,10 but is hindered by technical complexity, high mortality, and prolonged modeling cycles11,12. 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 models9,10 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.

<table><tbody><tr><td>Reagent used</td><td></td><td></td><td></td></tr><tr><td>bleomycin</td><td>MCE</td><td>HY-17565</td><td></td></tr><tr><td>H&amp;amp;E staining kit</td><td>Solarbio</td><td>G1120</td><td></td></tr><tr><td>isoflurane</td><td>RWD</td><td>R510-22-10</td><td></td></tr><tr><td>Masson&#039;s staining kit</td><td>Solarbio</td><td>G1340</td><td></td></tr><tr><td>PBS</td><td>Biosharp</td><td>BL302A</td><td></td></tr><tr><td>Antibodies used</td><td></td><td></td><td>Dilution</td></tr><tr><td>&amp;alpha;-SMA</td><td>Abcam</td><td>ab5694</td><td>1:100</td></tr><tr><td>Cytokeratin 5</td><td>Immunoway</td><td>YT1271</td><td>1:100</td></tr><tr><td>DeltaN p63</td><td>CST</td><td>67825</td><td>1:100</td></tr><tr><td>Goat Anti-Rabbit IgG H&amp;amp;L(biotin)</td><td>Abcam</td><td>ab6720</td><td>1:800</td></tr><tr><td>Hydroxyproline</td><td>CST</td><td>73812</td><td>1:100</td></tr><tr><td>Peroxidase-conjugated Streptavidin</td><td>Jackson</td><td>016-030-084</td><td>1:100</td></tr><tr><td>Streptavidin / AF594</td><td>Bioss</td><td>bs-0437P-AF594</td><td>1:500</td></tr></tbody></table>

refined murine model of idiopathic pulmonary fibrosis

Refined Murine Model of Idiopathic Pulmonary Fibrosis

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.

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...

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.

Animal models play a crucial role in understanding disease mechanisms and testing potential therapeutic approaches. However, traditional murine models of ...

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Biology

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Learn a refined murine model of idiopathic pulmonary fibrosis using repeated oropharyngeal bleomycin administration in mice. This method reduces mortality while recapitulating key pathological features including fibroblastic foci and collagen deposition.

Refined Murine Model of Idiopathic Pulmonary Fibrosis

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