Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

This protocol can be successfully applied both to induce important inflammatory and fibrotic response in both lungs by endotracheal injection of Bleomycin and to validate new anti-inflammatory and anti-fibrotic systemic therapies by tail vein infusion. The main advantage of this technique is that direct injection of Bleomycin into the mouse trachea ensures rapid targeting of the lungs without any spills in the upper airway allowing safe, robust and reproducible induction of lung inflammatory and fibrosis. Demonstrating the procedure will be Dr.Fiorenza Orlando, a veterinary doctor from our collaborating laboratory.

To begin, position the anesthetized mouse lying on its back on a surgical platform. Delicately fix its legs with surgical tape strips to hold it in place. To hyper extend the neck of the mouse, place a support such as dental cotton roll under its cervical region.

Then use a razor blade to gently shave the throat. Use a pair of anatomical forceps to pinch the skin. Use a pair of ring handled curved blunt scissors to make a short incision about half a centimeter in length in correspondence to the mouse sternohyoid muscle.

Use cotton wool sticks to stop any bleeding. Then exteriorize the trachea by blunt dissection and gently clean it from fat and other tissues. Rotate the surgical platform to orient the mouse with its head towards the operator which allows the operator during the injection to angle the syringe properly.

To visualize the trachea, place the mouse under an operating microscope. Adjust illumination and set magnification between 1 and 1.2 then adjust the focus and sharpness. The trachea can be easily distinguished as a white translucent tube with clearly visible tracheal rings.

Gently pipette to mix the Bleomycin solution and aspirate 100 microliters into a 0.5 milliliter syringe with a 25 gauge needle avoiding bubble formation. Once the trachea is clearly visualized, carefully puncture it with the needle tip at a 30 degree angle. Slowly inject 100 microliters of Bleomycin directly into the lumen of the trachea.

After waiting a few seconds for the entire volume to travel down the needle, remove it from the trachea. Observe a few seconds of apnea occurring when the needle is correctly inserted into the trachea indicating that the entire volume of liquid has been inhaled. Safely discard the syringe and needle.

Close the subcutaneous fascia and the skin wound with a 5-0 absorbable suture and allow the animal to recover. After anesthetizing the mouse again and once unconsciousness has been confirmed, place the mouse under a sterile hood. Use a facial mask with a continuous flow of 1.5%isoflurane to maintain gentle anesthesia throughout the experiment.

To promote vasodilation and allow for easier injection, soak the tail in warm water for two minutes. Mix a suspension of human umbilical cord mesenchymal stromal cells by gently pipetting to avoid cell clump formation. Aspirate 200 microliters into a one milliliter syringe with a 26 gauge needle avoiding bubble formation.

Hold the tail by the tip and gently straighten it. Then locate the lateral vein of the tail. Gently scrape the tail with a scalpel and wipe it with 70%ethanol.

Starting from the distal portion of the tail, insert the needle into the vein at a 15 degree angle. Slowly infuse 200 microliters of human umbilical cord mesenchymal stromal cells. To monitor successful intravenous infusion, observe the liquid entering the vein without resistance and a lack of extravasation.

After waiting a few seconds until the entire volume travels down the needle, remove it from the vein. Then briefly apply pressure to the entry wound with a sterile gauze to prevent bleeding and allow the animal to recover. Safely discard the syringe and the needle after the infusion.

After Bleomycin injection, lung histopathological changes were assessed by Hematoxylin and Eosin and Picrosirius Red staining confirming extensive inflammatory infiltration and fibrosis compared to normal lung architecture after saline injection. Bleomycin-induced lung inflammation and fibrosis were largely attenuated by human umbilical cord mesenchymal stromal cell infusion but not by saline. Collagen deposition was measured by hydroxyproline assay.

Following Bleomycin injection, hydroxyproline levels progressively increased from day eight to day 14 and 21. Whereas the addition of human umbilical cord mesenchymal stromal cells significantly reduced the effects of Bleomycin. Expression levels of Col1A1 type one collagen gene in whole lung mRNA also showed a progressive increase at days eight, 14 and 21 which was partially reduced by human umbilical cord mesenchymal stromal cell treatment.

Immunostaining with specific antibodies showed that infused human umbilical cord mesenchymal stromal cells rapidly and effectively reached mouse lungs, although only a few cells were detected. These data suggest a rapid dislocation of the cells from the site injury despite the prolonged protective effect. The use of an operating microscope ensures high confidence of success by allowing the operator to accurately monitor correct placement of the needle into the mouse trachea prior to installation, thus minimizing the risk of damaging it.