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

We aim to develop a reproducible, minimally invasive, aerosol-based, drug delivery model for inducing pulmonary fibrosis and improving preclinical drug evaluation. Our model enables consistent and reliable drug delivery while reducing surgical trauma and improving reproducibility across experiments. We plan to apply this model to test our unique therapy for pulmonary fibrosis and a clinical lung injury.

To begin, using a sterile, one-milliliter syringe, withdraw three milliliters of sterile 0.9%sodium chloride, then inject sodium chloride into a vial containing 15 units of bleomycin hydrochloride. Gently swirl the vial until the bleomycin powder is fully dissolved to obtain a five-units-per-milliliter stock solution. Aliquot the stock solution into sterile, 1.5-milliliter micro centrifuge tubes.

Then prepare working solutions of 3.75 units per milliliter and 2.5 units per milliliter by diluting the stock with sterile saline. Prepare the aerosol delivery apparatus ensuring the nebulizing needle, nebulizing syringe, and dosing pillar are all present. Submerge the aerosol delivery syringe in sterile saline.

Slowly aspirate the fluid, pause for eight seconds, then rapidly expel the fluid. Then refill the syringe with sterile saline after the final expulsion. Attach the aerosolizing needle and ensure that the junction is filled with saline.

Then submerge the fully assembled needle in sterile saline. To prime the aerosolizing needle with the bleomycin-working solution, aspirate and expel it three times with eight-second pauses between each cycle. Aspirate the bleomycin-working solution into the syringe.

Insert a 25-microliter-dosing pillar on top of the 50-microliter pillar to calibrate the total volume. Expel any excess solution. Then place the anesthetized mouse on the intubation platform.

Hook the upper incisors to the wire loop and tape the limbs to stabilize the mouse in position. Using curved forceps, gently pull out the tongue. Insert a small animal laryngoscope to visualize and expose the glottis.

Then insert the aerosolizing needle vertically through the exposed glottis and deliver the bleomycin aerosol rapidly into the trachea. After that, place the mouse on a warming pad to maintain body temperature. Observe the mouse continuously until it fully recovers from anesthesia.

Perform a toe pinch to check for reflexes and confirm that the mouse is alive. Hematoxylin and eosin staining revealed mild alveolar wall thickening and localized fibrosis in the five-units-per-kilogram bleomycin group. While the 7.5 units per kilogram group showed extensive alveolar collapse and severe fibrotic lesions.

The Modified Ashcroft Score quantification demonstrated a dose-dependent increase in fibrosis severity in bleomycin-treated mice compared to saline controls. Masson's trichrome staining revealed increased collagen deposition in the five-units-per-kilogram group and extensive interstitial fibrosis in the 7.5 units per kilogram group. Quantification of collagen-positive areas confirmed a significant, dose-dependent increase in collagen deposition following bleomycin treatment.

Immunohistochemical staining showed increased expression of Col1A1 protein in lungs of both bleomycin groups with stronger and broader staining observed in the 7.5-units-per-kilogram group. Hydroxyproline content was significantly higher in both bleomycin-treated groups confirming increased collagen accumulation in lung tissue. Static lung compliance was significantly reduced in a dose-dependent manner in the bleomycin-treated groups.

Inspiratory capacity was significantly lower only in the 7.5-units-per-kilogram bleomycin group compared to saline. Respiratory system elastins increased significantly in the 7.5-units-per-kilogram group compared to saline. Respiratory system compliance was significantly decreased only in the 7.5-units-per-kilogram group.