Oropharyngeal Administration of Bleomycin in the Murine Model of Pulmonary Fibrosis

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 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.

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 patients^3,4. 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 fibrosis^7,8. 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 14-21^9,10,11 (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'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-β1. 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-β1, IL-β1, and TNF-α, have been shown to induce pulmonary fibrosis in rodents, typically by day 14^17,18,19. 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 delivery^{14,21,22,23,24,25,26}. 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.

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'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

1. 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.
   1. Dissolve bleomycin powder in sterile, pharmaceutical-grade PBS to a stock concentration of 10 U/mL. Bleomycin should be prepared under a chemical hood using proper chemotherapeutic precautions. Store the aliquots at -20 °C for up to 6 months.
   2. 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's purpose (e.g., survival or lethal dosing).
   3. Adjust the final volume of administration as needed. For these studies, dilute bleomycin to 0.375 U/mL, which equates to 50 μL for a 25 g mouse, resulting in a final working concentration of 0.75 U/kg.

2. Induction of anesthesia

1. 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.
2. Administer 10 µL 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.
   1. 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 µL 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.

3. Oropharyngeal administration

1. Once properly sedated, suspend the mouse on the procedural platform at a 60°-80° angle by hanging it by its front incisors to effectively open the oropharynx (Figure 2A).
2. Occlude the nasal passage with a smooth microvascular clamp, forcing the mouse to respire through its oropharynx.
3. Retract the tongue out of the oropharynx using forceps.
4. 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).
5. 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.
6. After aspiration is confirmed, carefully remove the nose clip.
7. 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 µL 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.

4. Animal recovery

1. After treatment, place the animal on its side in its cage with a heating pad underneath to maintain thermoneutrality.
2. 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.
3. 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's effectiveness.
4. Under ARC and IACUC protocols, euthanize animals if the weight loss exceeds 20% of the animal'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).

5. Tissue harvesting, processing, and end point analysis

1. Depending on the experimental question and the desired time point, euthanize the mice following IACUC protocols and harvest their lungs²⁸ at the appropriate time. The effects of bleomycin are often grossly visually apparent compared to control, indicating successful administration. In these studies, the mice were sacrificed on days 7, 14, and 21.
2. 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&E), and/or Masson's trichrome staining as previously described^28,29,30.
3. For collagen measurement, homogenize the right lung and use a commercially available kit (see Table of Materials) to measure hydroxyproline content as previously described³¹.
4. 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 described^32,33,34.

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&E) histologic staining. By day 7, fibrotic change of the alveolar septa can be seen, along with small inflammatory/fibrotic changes, compared to PBS control (Figure 3A,B). By day 14, larger, more confluent areas of fibrosis are seen, with significant destruction of the normal alveolar architecture (Figure 3C). At day 21, these significant fibrotic changes remain (Figure 3D). These histopathologic changes were quantified using the modified Ashcroft scoring system²⁹, which demonstrated a similar degree of fibrosis between days 14 and 21 (Figure 3E).

While histopathology remains the gold standard, additional assays can be performed to objectively quantify the fibrotic and inflammatory changes induced by bleomycin. To confirm the H&E findings, hydroxyproline assays were performed on day 14³¹, which demonstrated an increase in the total amount of collagen content with bleomycin (Figure 4A). Additionally, RNA was isolated from whole lung homogenates on day 14, and a quantitative polymerase chain reaction (qPCR) demonstrated the upregulation of profibrotic genes Col3a1 and Tgfb with bleomycin (Figure 4B). To characterize the immune changes induced by oropharyngeal bleomycin, flow cytometry was performed as previously described^32,33,34 (Figure 4C). In response to OA bleomycin, a robust infiltration of myeloid cells into the murine lung is observed, including interstitial macrophages (IM), monocyte-derived alveolar macrophages (MoAM), and neutrophils (Figure 4D). These data demonstrate that the oropharyngeal aspiration technique is a safe, convenient, and reproducible methodology for inducing pulmonary fibrosis with bleomycin in the murine animal model.

Figure 1: Timeline of experimental protocol. (A) Graphic representation of oropharyngeal administration of bleomycin into the murine lung. (B) Timeline of bleomycin disease course. The initial inflammatory phase lasts for 48-72 h and is followed by a fibrotic phase beginning approximately day 7, which resolves after 6 weeks for individual injections. Arrows represent common potential tissue harvest times (day 7, 14, or 21) depending on the experiment question and assays being utilized. Please click here to view a larger version of this figure.

Figure 2: Schematic of bleomycin oropharyngeal administration. (A) Once properly sedated, the mouse is strung by its front incisors at 60°-80° on a secure platform. (B) The nasal passage is occluded using a non-serrated microvascular clamp. The tongue is retracted from the oropharynx, and using a stepper pipette with a stub leur-stub tip, the bleomycin solution, or PBS control, is administered into the back of the oropharynx. A visible bubble of fluid should be visible until aspirated into the lower respiratory tract. Please click here to view a larger version of this figure.

Figure 3: Bleomycin administration via the oropharyngeal aspiration results in robust fibrotic histologic changes. Wildtype C57Bl/6 mice were treated with a one-time dose of either bleomycin (0.75U/kg) or PBS at day 0. (A) Sham controls demonstrate normal lung histology. (B-D) Bleomycin results in progressive fibrosis and architectural distortion of the murine lung. (E) Modified Ashcroft scoring of histologic samples. Error bars represent standard deviation. ****p < 0.0001 as determined by one-way ANOVA. Images are representative of three independent experiments performed with 4-8 animals per group. 20x images (right, scale bars: 200 µm) are from the designated insets on the 1x images (left, scale bars: 3 mm). Please click here to view a larger version of this figure.

Figure 4: Quantification of the profibrotic and proinflammatory changes induced by bleomycin. (A) Lungs were harvested at day 14 from wildtype C57Bl/6 mice treated with either bleomycin (0.75U/kg) or PBS. Hydroxyproline assays were performed, and collagen content was measured (mg per right lung). (B) RNA was isolated from homogenized lung tissue of bleomycin or PBS-treated animals at day 14. Quantitative PCR was performed to measure the level of Col1a1 and Tgfb transcripts. Fold change related to the PBS control group was calculated using the 2^CP method in reference to the internal housekeeping gene. (C) Flow gating strategy of myeloid populations in the murine lung. (D) Quantification of myeloid populations in the murine lung at day 14, in response to bleomycin. Data is representative of three independent experiments with 4-8 animals per group per experiment. Error bars represent standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as determined by unpaired student's t-test (hydroxyproline and qPCR) or two-way ANOVA. Please click here to view a larger version of this figure.

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 IPF³⁶. There are a multitude of ways to administer bleomycin in rodent models, including via intratracheal (IT), intranasal (IN), intravenous (IV), intraperitoneal, and subcutaneous injection⁶. While IV administration more closely mimics the mechanism of bleomycin-induced pneumonitis in humans, the IT route is more commonly used; fibrosis is achieved with a single dose, without systemic toxicities associated with higher doses, and is more cost-effective⁸. IT delivery requires either endotracheal intubation or surgical exploration into the neck and tracheostomy, both requiring deep levels of sedation and a high degree of procedural skill, while carrying significant associated periprocedural morbidity and mortality. IN administration is a reasonable alternative to IT, however, it has been shown to cause more variable patterns of distribution and lung injury^37,38. The OA approach has seen increased use and represents a seemingly more reproducible substitute for the delivery of bleomycin and other fibrosing agents into the murine lung^{14,21,22,23,24,25,26}. Recently, OA and IT routes of bleomycin administration were directly compared in mice, demonstrating equal efficacy and homogenous fibrotic injury, while minimizing periprocedural mortality²¹. Similarly, in this study, no mice died upon OA administration (data not shown), underscoring the favorable technical and ethical profile of this method.

The degree of fibrosis and timing of injury induced by bleomycin depends on several factors. Following bleomycin instillation into the murine lung, an initial inflammatory period of acute lung injury occurs, followed by a fibroproliferative phase that begins after 5-7 days¹⁰. Routinely, disease endpoints are assessed between days 14-21, when sustained fibrosis is achieved (Figure 3C). The present study focused on day 14, which has been shown to be the optimal time to measure lung fibrosis parameters, as the animals have developed extensive fibrosis, with less variability and lower mortality than is seen at day 21¹¹. Following a single dose, fibrosis has been noted to spontaneously resolve after approximately 6 weeks in mice. To capture the irreversibility seen with many forms of ILD in humans, investigators have developed repetitive dosing models to achieve permanent fibrotic changes in murine models^9,12.

Previous studies have demonstrated a strain variance in regards to the severity of fibrosis achieved with a single dose of bleomycin, with C57Bl/6 mice being "high responders", DBA/2 and Swiss mice being "intermediate responders", and BALB/c mice being "low responders"³⁹. Furthermore, the age and sex of the mice used also influence the degree of inflammation and fibrosis. Older mice (52-54 weeks) demonstrated increased fibrosis than younger mice (8-12 weeks), and male mice appear more susceptible to bleomycin, in general, than their female counterparts^9,40. These observations suggest underlying genetic factors influencing the inflammatory and wound healing response, and investigators should age- and sex-match mice when testing potential therapeutic avenues, as suggested by recent American Thoracic Society (ATS) official guidelines²⁵.

Bleomycin represents an inflammatory model of fibrosis. The initial epithelial damage induced by bleomycin results in transient, acute cytokine production and neutrophil recruitment in the lung^41,42. IPF itself is not clinically characterized by a strong immunologic phenotype, aside from acute exacerbations, though other types of ILD, such as those associated with connective tissue disease (CTD-ILD), are more clearly driven by immune dysregulation^43,44,45. Therefore, depending on the hypothesis being tested, investigators may choose to study interventions that intercept the disease axes at earlier or later time points. Furthermore, it is recommended that a second model be considered to validate key findings when screening preclinical therapies, such as silica, TGF-β1 adenoviral overexpression, diphtheria-induced epithelial damage, or humanized mouse models^{5,17,46,47,48}.

In conclusion, the oropharyngeal aspiration technique represents a robust, reproducible, translatable, and safe alternative to intratracheal delivery of bleomycin to induce fibrosis in the murine lung.

The authors have no conflicts of interest.

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.