Our research seeks to understand the immune mechanisms underlying the pathogenesis of pulmonary fibrosis. One of the biggest challenges in the field remains translating in vitro and in vivo models to human pulmonary fibrosis. A number of animal models exist to study pulmonary fibrosis.
We seek to standardize one of the most commonly used models, the bleomycin murine model, so that it's readily adaptable for other labs to use. We present a minimally invasive, highly reproducible, and safe method of administering bleomycin in the murine model of pulmonary fibrosis. This animal model compliments in vitro in human studies.
It involves running the cell types critical for pathogenesis and physiologically replicates changes in ventilation, perfusion, and gas exchange that occur during injury. We predict that interventions that severely reduce histopathological severity in vivo may indicate better clinical outcomes. Proceed to prepare bleomycin solutions in a chemical hood while following proper chemotherapeutic precautions.
First dissolve bleomycin powder in sterile PBS to prepare a stock concentration of 10 units per milliliter. Depending on the specific bleomycin used and the experimental objective, prepare the final working concentration as per the required dose based on body weight. To adjust the final volume of administration, dilute bleomycin to 0.375 units per milliliter, ensuring a total volume of 50 milliliters for a 25 gram mouse.
Suspend the properly anesthetized mouse on the procedural platform at a 60 to 80-degree angle by hanging it by its front incisors to effectively open the oropharynx. Occlude the nasal passage using a smooth microvascular clamp to force respiration through the oropharynx. Using forceps, retract the tongue out of the oropharynx.
Using a stepper pipette with a stub lure stub tip, gently place the desired volume of bleomycin or saline control into the back of the oropharynx. Ensure a visible bubble of liquid is grossly apparent. Continue holding the tongue in place until the mouse aspirates the solution, which is visibly and often audibly apparent.
Once aspiration is confirmed carefully remove the nose clip. Observe the mouse in the hanging position for 15 seconds to ensure no reflux of the bleomycin solution before returning it to its cage. Then place the animal on its side in its cage with a heating pad underneath to maintain thermo neutrality.
Gently pinch the toes and ensure the animals remain euthermic to facilitate awakening. Monitor the mice until they regain full consciousness, which typically takes one to two hours depending on the ketamine dose and the animal size and metabolism. Clinically monitor the mice daily for changes in body weight, grooming, activity level, and respiratory status.
Track weight as a key marker of the model's effectiveness. At the appropriate time, harvest the lungs from the properly euthanized mice. For histology, dissect the lungs on block and fix them in 4%paraformaldehyde for 24 hours.
Proceed with paraffin embedding, sectioning, and staining using hematoxylin and eosin or Masson's trichrome. For soluble collagen measurement, homogenize the right lung. For flow cytometry, digest the right lung using a tissue dissociator and an enzymatic solution to obtain a single cell suspension.
Perform flow cytometric staining and analysis following standard protocols. Compared to PBS control, fibrotic changes in the alveolar septa and small inflammatory or fibrotic areas were observed in bleomycin-treated lung samples by day seven. By day 14, larger and more confluent fibrotic regions appeared with significant destruction of normal alveolar architecture.
By day 21, the fibrotic changes persisted with no more significant increase. The modified Ashcroft scoring system confirmed that fibrosis levels were similar between days 14 and 21. Hydroxyproline assays performed on day 14 demonstrated an increase in total soluble collagen content in bleomycin-treated lungs compared to control.
Quantitative polymerase chain reaction analysis showed an upregulation of profibrotic genes Col1A1 and TGFB in response to bleomycin. Flow cytometry analysis revealed a robust infiltration of myeloid cells including interstitial macrophages, monocyte-derived alveolar macrophages, and neutrophils in the lungs.
