Name: Oropharyngeal Administration of Bleomycin in the Murine Model of Pulmonary Fibrosis (Video) | JoVE Journal
Uploaded: 2025-05-09T14:00:00.000+00:00
Duration: 6 min 3 s
Description: Discover a minimally invasive oropharyngeal aspiration technique for administering bleomycin in murine pulmonary fibrosis models. This standardized method enables reproducible study of immune mechanisms and fibrotic disease progression.

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This work was supported by the NIH Ruth L. Kirchstein National Research Service Award (NRSA) Institutional Research Training Grant (T32) awarded to RW (2T32HL072752-16). The authors would also like to acknowledge the support of the Saul and Joyce Brandman Foundation Lung Health Center.

Animal studies described in these experiments were conducted under protocols (ARC-2021-025, ARC-2010-039) approved by the UCLA Animal Research Committee (ARC) and the Institutional Animal Care and Use Committee (IACUC). Full compliance with all state and federal regulations and policies regarding laboratory animal use was maintained. Animals were housed in UCLA&#39;s Animal Care Facility and cared for by the skilled staff of the UCLA Division of Laboratory and Animal Medicine (DLAM) under pathogen-free conditions. Wildtype C57BL/6 mice were commercially obtained and allowed to acclimate for at least 14 days. Male mice aged 8-12 weeks were used for these studies, with an average body weight of 20-25 g. Female mice may also be used, though it is important to sex- and age-match animals across experimental groups and conditions27. The commercial details of the animals, reagents, and equipment used in this study are listed in the Table of Materials.
1. Oropharyngeal administration of bleomycin
<ol>
	<li>Preparation of bleomycin 
	NOTE: Use pharmaceutical-grade bleomycin to ensure consistency and reproducibility between animals and experiments. Dose bleomycin in units of drug per kg animal (U/kg), not milligrams (mg) per kg.
	<ol>
		<li>Dissolve bleomycin powder in sterile, pharmaceutical-grade PBS to a stock concentration of 10 U/mL. Bleomycin should be prepared&#160;under a chemical hood using proper chemotherapeutic precautions. Store the aliquots at -20 &#176;C for up to 6 months.</li>
		<li>Prepare the final working concentration. The dose is based on weight (0.5-3 U/kg), and adjustments are made as necessary depending on the bleomycin used and the experiment&#39;s purpose (e.g., survival or lethal dosing).</li>
		<li>Adjust the final volume of administration as needed. For these studies, dilute bleomycin to 0.375 U/mL, which equates to 50 &#956;L for a 25 g mouse, resulting in a final working concentration of 0.75 U/kg.</li>
	</ol>
	</li>
</ol>
2. Induction of anesthesia
<ol>
	<li>Prepare the anesthesia cocktail by diluting ketamine and xylazine in PBS to working concentrations of 10 mg/mL and 1 mg/mL, respectively. Perform this step under sterile conditions using pharmaceutical-grade reagents and in accordance with institutionally approved protocols.</li>
	<li>Administer 10 &#181;L of the cocktail per gram body weight of the animal (working concentration: ketamine 100 mg/kg, xylazine 10 mg/kg) via intraperitoneal injection using a 27.5 G needle and a 1 mL syringe.
	<ol>
		<li>Ensure the mouse is properly anesthetized and unresponsive to noxious stimuli, such as toe pinch. Effects should be seen within 5 min. If still responsive, administer additional ketamine/xylazine in 20 &#181;L increments until the desired level of anesthesia is achieved. Apply ophthalmic ointment to prevent eye dryness while under anesthesia. 
		NOTE: Ketamine is preferred over other injected anesthetics and sedatives due to its favorable side effect profile. It has minimal effects on hemodynamics, including heart rate and respiratory rate. Inhaled isoflurane can be used as an alternative anesthetic agent. In these studies, ketamine/xylazine is preferred because it results in prolonged sedation and minimal coughing or reflux of bleomycin after administration.</li>
	</ol>
	</li>
</ol>
3. Oropharyngeal administration
<ol>
	<li>Once properly sedated, suspend the mouse on the procedural platform at a 60&#176;-80&#176; angle by hanging it by its front incisors to effectively open the oropharynx (Figure 2A).</li>
	<li>Occlude the nasal passage with a smooth microvascular clamp, forcing the mouse to respire through its oropharynx.</li>
	<li>Retract the tongue out of the oropharynx using forceps.</li>
	<li>Using a stepper pipette with a stub leur-stub tip, gently place the desired volume of bleomycin, or saline control, into the back of the oropharynx. Ensure a bubble of liquid is grossly visible (Figure 2B).</li>
	<li>Continue holding the tongue in place until the animal aspirates the solution. This should be visibly and often audibly apparent. 
	NOTE: If the animal quickly aspirates the solution, visualization in the back of the oropharynx may be transient, within a few seconds. Regardless, if successful, the animal will demonstrate an abrupt and transient change in its breathing pattern, taking rapid, shallow breaths. Bubbling of the fluid may occur, further indicating that the fluid has successfully entered the lower respiratory tract. Occasional coughing may also occur. This is usually a negligible volume of the bleomycin solution and should not affect the experimental results, allowing the animal to remain included in the study. Avoid repetitive dosing, as it increases the risk of asphyxiation and alters the final weight-based dosing.</li>
	<li>After aspiration is confirmed, carefully remove the nose clip.</li>
	<li>Observe the animal in the hanging position for 15-30 s to ensure no reflux of the bleomycin solution, then return it to its cage. 
	NOTE: A maximum volume of 50 &#181;L is recommended to minimize the risk of asphyxiation. Depending on the desired dose of bleomycin and the weight of the mouse, adjust the concentration of the bleomycin solution as needed. When practicing this technique, use a water-based dye such as Evans blue to confirm that the solution is administered into the lower respiratory tract, rather than into the stomach14.</li>
</ol>
4. Animal recovery
<ol>
	<li>After treatment, place the animal on its side in its cage with a heating pad underneath to maintain thermoneutrality.</li>
	<li>Monitor the mice until they are fully conscious. This typically takes 1-2 h, depending on the dose of ketamine used and the metabolism of the animal. Gently pinch the toes and keep the animals euthermic to facilitate awakening.</li>
	<li>Clinically monitor the mice on a daily basis for changes in body weight, grooming, activity level, and respiratory status. Similar to other delivery methods of bleomycin, animals may experience significant weight loss over the 14-21 day course, which is a key marker of the model&#39;s effectiveness.</li>
	<li>Under ARC and IACUC protocols, euthanize animals if the weight loss exceeds 20% of the animal&#39;s starting weight. The prevalence and severity of weight loss depend on the dose of bleomycin used and the demographics of the mice (see above).</li>
</ol>
5. Tissue harvesting, processing, and end point analysis
<ol>
	<li>Depending on the experimental question and the desired time point, euthanize the mice following IACUC protocols and harvest their lungs28 at the appropriate time.&#160;The effects of bleomycin are often grossly visually apparent compared to control, indicating successful administration.&#160;In these studies, the mice were sacrificed on days 7, 14, and 21.</li>
	<li>For histology, dissect the lungs en bloc and fix them in 4% PFA for 24 h. Proceed with paraffin embedding, sectioning, hematoxylin and eosin (H&#38;E), and/or Masson&#39;s trichrome staining as previously described28,29,30.</li>
	<li>For&#160;collagen measurement, homogenize the right lung and use a commercially available kit (see Table of Materials) to measure hydroxyproline content as previously described31.</li>
	<li>For flow cytometry, digest the right lung using the tissue dissociator and an enzymatic solution to obtain a single-cell suspension. Perform flow cytometric staining and analysis as previously described32,33,34.</li>
</ol>

The authors have no conflicts of interest.

A detailed video protocol is provided on the oropharyngeal aspiration technique for administering bleomycin for use in the murine model of pulmonary fibrosis. Additionally, we highlight potential downstream applications to quantify fibrotic and inflammation changes induced by OA bleomycin.
While no one animal fully recapitulates the complexity of human disease, the bleomycin mouse model has been used for the past five decades and remains the most widely implemented to study the pathogenesis of...

Interstitial lung disease (ILD) refers to a heterogeneous group of disorders characterized by progressive and irreversible scarring of the alveoli space, interstitium, and distal airways1. Idiopathic pulmonary fibrosis (IPF) is the most common form of ILD and carries a median survival of approximately three years2. IPF is an ultimately terminal condition, with orthotopic lung transplantation being a salvage therapy for select patients. There are currently two FDA-approved therapies for IPF, both of which merely slow the rate of progression rather than stabilize or improve lung function for patients3,4. Significant research efforts are underway to elucidate the underpinnings of IPF and identify new therapeutic targets. Myriad animal models exist to study IPF pathogenesis, each with its own advantages and disadvantages5. While no one model is able to fully recapitulate the complexity of human disease, these approaches do offer significant insight into the molecular mechanisms of IPF and can complement translational studies.
The bleomycin murine model remains the most widely used and well-characterized in vivo model of IPF6. Bleomycin is a peptide agent that induces single- and double-stranded DNA breaks. Following its discovery in 1962, bleomycin was found to be effective in treating a number of cancers, including testicular tumors and lymphoma, however its use has been limited by dose-dependent pneumonitis and resultant pulmonary fibrosis7,8. This pulmonary toxicity is recapitulated in mice. When administered in a single dose, following an initial inflammatory phase, fibrosis can be seen beginning near day 5, peaking on days 14-219,10,11 (Figure 1). Spontaneous resolution occurs after roughly 6 weeks, though permanent fibrotic changes can be achieved with repetitive dosing12. Given the transient and inflammatory nature, there are some inherent drawbacks with the bleomycin model13, however it offers a rapid, robust, and reproducible system to begin to answer some of the major gaps in our field&#39;s understanding of ILD and allows investigators to compare results over the past five decades. Other installation approaches include the asbestosis and silica murine models, which offer similar time courses (days 14-28)6,14,15,16. However, these models generate a histologic pattern more consistent with pneumoconiosis than IPF and require the use of airborne particulates, necessitating careful handling. Alternatively, animal models exist that utilize epithelial-driven transgene expression, such as diphtheria-toxin and TGF-&#946;1. These recapitulate the non-inflammatory alveolar type 2 epithelial cell injury seen in IPF, however take slightly longer (21-30d) and require the use of specialized animals that must be backcrossed into any existing transgenic models of interest. Lastly, adenoviral-mediated overexpression of cytokines, including TGF-&#946;1, IL-&#946;1, and TNF-&#945;, have been shown to induce pulmonary fibrosis in rodents, typically by day 1417,18,19. These cytokine overexpression models allow for convenient intranasal delivery, though require the careful purification and handling.
Multiple approaches exist for the delivery of bleomycin, including intratracheal (IT), intranasal, intraperitoneal, subcutaneous, and intravenous routes6. IT delivery is the most common method, traditionally involving either endotracheal intubation or surgical tracheostomy20, both of which require deep sedation, technical finesse, and are associated with perioperative morbidity and mortality. Recently, the oropharyngeal aspiration (OA) technique has been shown to be equally efficacious as IT, with considerably fewer side effects and an easier route of delivery14,21,22,23,24,25,26. Here, we present a detailed visual protocol for the OA method of bleomycin delivery into the murine lung and highlight various potential downstream applications for data quantification.

<table><tbody><tr><td>anti-mouse CD45, Brlliant Violet 605</td><td>BioLegend</td><td>103155</td><td></td></tr><tr><td>anti-mouse CD64, AlexaFluor 647</td><td>BioLegend</td><td>139322</td><td></td></tr><tr><td>anti-mouse Ly6G, AlexaFluor 700</td><td>BioLegend</td><td>127622</td><td></td></tr><tr><td>anti-mouse MerTK, PE/Cy7</td><td>BioLegend</td><td>151522</td><td></td></tr><tr><td>anti-mouse SiglecF, PE</td><td>BD Biosciences</td><td>552126</td><td></td></tr><tr><td>BD Luer-Stub Adaptors</td><td>Fisher Scientific</td><td>13-681-21</td><td></td></tr><tr><td>Bleomycin</td><td>McKesson</td><td>1129996</td><td>From NorthStar Rx 16714088601</td></tr><tr><td>Endotracheal Mouse Intubation Kit</td><td>Kent Scientific</td><td>ETI-MSE</td><td></td></tr><tr><td>Fixable Live/Dead Violet</td><td>Thermo</td><td>L34955</td><td></td></tr><tr><td>FlowJo v10 Software</td><td>FlowJo</td><td></td><td></td></tr><tr><td>gentleMACS Dissociator</td><td>Miltenyi</td><td>130-093-235</td><td></td></tr><tr><td>Hydroxyproline Assay Kit</td><td>Sigma</td><td>MAK463</td><td></td></tr><tr><td>Liberase TM</td><td>Roche</td><td>5401127001</td><td></td></tr><tr><td>Moria Vessel Clamp</td><td>Fine Science Tools</td><td>18350-11</td><td></td></tr><tr><td>Mouse Endotracheal Intubation Kit</td><td>Kent</td><td>ETI-MSE</td><td></td></tr><tr><td>Stepper Pipette</td><td>Dymax</td><td>TI15469</td><td></td></tr><tr><td>Wildtype C57BL/6 mice&amp;nbsp;</td><td>Jackson Laboratories</td><td>JAX, stain #000664</td><td></td></tr></tbody></table>

oropharyngeal administration of bleomycin in the murine model of pulmonary fibrosis

Oropharyngeal Administration of Bleomycin in the Murine Model of Pulmonary Fibrosis

Interstitial lung disease (ILD) represents a broad spectrum of disorders characterized by the progressive and often irreversible scarring of the lung parenchyma, the most common being idiopathic pulmonary fibrosis (IPF). Several animal models of IPF have been developed, with the bleomycin murine model being the most widely used. Bleomycin is a chemotherapeutic known to induce DNA damage in the alveolar epithelium, resulting in acute lung injury and pulmonary fibrosis in humans. Rodent models of IPF use bleomycin administration via various methods, the most common being intratracheal (IT). Recently, the oropharyngeal aspiration (OA) technique has been shown to be equally efficacious as IT for multiple fibrosing agents, with considerably fewer side effects and an easier route of delivery. This protocol details the OA method of bleomycin delivery into the murine lung and highlights examples of potential downstream applications for data quantification. This methodology offers a simple, quick, and safe way to utilize this widely used animal model for studying the molecular mechanisms underlying IPF.

The protocol described here summarizes the oropharyngeal aspiration route of administration in the bleomycin murine model. In these experiments, animals were treated with either bleomycin (0.75U/kg body weight) or PBS for sham control. On days 7, 14, and 21, mice were euthanized, their lungs explanted, and tissue fixed, as previously described35. Fibrosis was assessed using hematoxylin and eosin (H&amp;E) histologic staining. By day 7, fibrotic change of the alveolar septa can be seen, along with ...

This protocol demonstrates the oropharyngeal aspiration technique for use in the bleomycin murine model of pulmonary fibrosis.

Interstitial lung disease (ILD) represents a broad spectrum of disorders characterized by the progressive and often irreversible scarring of the lung ...

oropharyngeal-administration-bleomycin-murine-model-pulmonary

Research

JoVE Journal

Biology

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

An Improved Method for Rapid Intubation of the Trachea in Mice

We describe a non-invasive multimodal imaging approach based on Micro-CT and fluorescence molecular tomography for longitudinal assessment of the mouse lung fibrosis model induced by double intratracheal instillation of bleomycin.

A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice

We describe a method to significantly enhance orthotopic engraftment of lung cancer cells into the murine lungs by pre-conditioning the airways with injury. This approach may also be applied to study stromal interactions within the lung microenvironment, metastatic dissemination, lung cancer co-morbidities, and to more efficiently generate patient derived xenografts.

Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Intratracheal (IT) administration of experimental agents in mice often results in asymmetric delivery to the distal lungs.  In this report, we describe a direct intrabronchial (IB) approach to cannulate each lung in living mice non-operatively.  This approach can be used to selectively administer agents to one lung or may be adapted to improve the symmetric agent delivery to both lungs.

Direct Intrabronchial Administration to Improve the Selective Agent Deposition Within the Mouse Lung

Various animal models of pulmonary fibrosis have been established using bleomycin to clarify the pathogenesis of pulmonary fibrosis and find new drug targets. However, most pulmonary fibrosis models targeting lung tissue have uneven drug administration. Here, we propose a model of uniform pulmonary fibrosis induced by nasal bleomycin nebulization.

A Mouse Model of Pulmonary Fibrosis Induced by Nasal Bleomycin Nebulization

This protocol describes the isolation of pulmonary interstitial macrophages (IMs) and their adoptive transfer after IL-33 stimulation of the lung alveoli in a mouse model, which can facilitate the in vivo study of idiopathic pulmonary fibrosis (IPF).

Adoptive Transfer of IL-33-Stimulated Macrophages into Bleomycin-Induced Mouse Models to Study Their Effect on Idiopathic Pulmonary Fibrosis In Vivo

Here, we present a protocol to utilize micro-computed tomography (micro-CT) to quantify ventilated regions in a unilateral bleomycin-induced pulmonary fibrosis model via intratracheal instillation on the lesional side.

Unilateral Lung Volume Analysis Using Micro-CT for Enhanced Assessment of Pulmonary Fibrosis in Preclinical Models

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.

Refined Murine Model of Idiopathic Pulmonary Fibrosis

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.

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

Discover a minimally invasive oropharyngeal aspiration technique for administering bleomycin in murine pulmonary fibrosis models. This standardized method enables reproducible study of immune mechanisms and fibrotic disease progression.

Oropharyngeal Administration of Bleomycin in the Murine Model of Pulmonary Fibrosis

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