Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

All animal care and experimental procedures were approved by the Italian Ministry of Health (authorization n. 456/2016-PR) and performed according to the Declaration of Helsinki conventions.

1. Mice

1. After purchasing them, allow the mice to acclimate for at least 7 days before the injection.
   NOTE: Mice were housed in the animal facility under pathogen-free conditions, were maintained under constant temperature and humidity on a 12 h light/dark cycle, and were given free access to water and standard pellet food.
2. Use female C57BL/6 mice and inject them at 12 to 16 weeks of age.

2. Endotracheal injection of bleomycin

1. Bleomycin preparation
   CAUTION: Based on the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), bleomycin is classified as a GHS08 health hazard.
   1. Prepare bleomycin under a chemical hood.
   2. To obtain the desired working concentration (0.05 U/100 µL), resuspend 15 U of lyophilized bleomycin sulfate in 30 mL of sterile saline.
   3. Carefully mix the sample by inverting the tube to avoid clot formation.
   4. Properly label the tube with the date of resuspension, store it at 4 °C, and use its content within 24 h.
   5. Prior to the instillation, equilibrate the bleomycin solution to room temperature.
      NOTE: In this experiment, a single dose of 1.5 U/kg body weight of bleomycin was used to induce lung injury in C57BL/6 mice. Nevertheless, each mouse strain has a different sensitivity to bleomycin^6,7. Titration of bleomycin should be performed to determine the optimal dose in the mouse strain used for the experiments.
2. Anesthesia
   1. Prepare anesthesia by dissolving 0.2 g of 2,2,2-tribromoethanol in 9 mL of sterile saline and 1 mL of absolute ethanol (at a working concentration of 20 mg/mL).
   2. Mix thoroughly by inverting the tube to avoid clot formation.
   3. Properly label the tube with the date of preparation, store it at 4 °C in darkness, and use it within 3 days.
   4. Anesthetize the mice with an intraperitoneal injection of 250 µL of tribromoethanol solution (at a final dose of 200 mg/kg body weight) per mouse, using a 1 mL syringe and a 26 G needle.
      NOTE: With this dose, the mice are unconscious for at least 20 min. When necessary, adjust the dosage according to the mouse response, in consultation with the veterinarian.
   5. Monitor the mouse breathing. The respiration rate will slightly slow down. After a few minutes, pinch one of the mouse feet to check the lack of pedal reflex.
3. Endotracheal injection
   1. Maintain aseptic conditions during all the procedure. Use sterile surgical instruments and materials, wear sterile gloves and avoid contact with any non-sterile surface.
   2. Lie down the anesthetized mouse on its back on a surgical platform and hold it in place by delicately fixing its legs with surgical tape strips.
      NOTE: Gentle taping of mouse legs is recommended to avoid mouse sliding away from the surgical platform during its rotation (step 2.3.10).
   3. Place the mouse on heated mat to keep the rectal temperature stable at 37 °C throughout the intervention. Measure the rectal temperature by a rectal probe.
   4. Gently hyperextend the mouse neck by placing a “pillow”, for example, a dental cotton roll, beneath its cervical region.
   5. Gently shave the throat with a razor blade.
   6. Remove hair from the surgical area with alcohol and disinfect the mouse skin several times with 1% povidone-iodine solution.
   7. Pinch the skin with a pair of anatomical forceps and make a short incision in correspondence of the mouse sternohyoid muscle, using a pair of ring-handled, curved blunt scissors.
      NOTE: The incision of the skin is about 0.5 cm in length. The corresponding skin piece which is removed is so small that it will create no tension in the mouse neck.
   8. Stop the bleeding with cotton wool sticks.
   9. Exteriorize the trachea by blunt dissection, gently cleaning it from fat and other tissues.
   10. Rotate the surgical platform to orient the mouse with its head toward the operator.
       NOTE: This position allows the operator, during the injection, to angle the syringe so that it follows the natural path of the trachea straight to the lungs.
   11. Place the mouse under an operating microscope to help with the visualization of the trachea. Adjust the illumination and set the magnification (between 1 and 1.2), focus, and sharpness. The trachea can be easily distinguished as a white translucent tube, and the tracheal rings are clearly visible.
   12. Mix the bleomycin solution by gently pipetting, and aspirate 100 µL into a 0.5 mL syringe with a 25 G needle, avoiding bubble formation.
   13. Once the trachea in clearly visualized, carefully puncture it with the needle tip at an angle of 30° (Figure 1A).
   14. Slowly instill 100 µL of bleomycin or sterile saline (vehicle control) directly into the lumen of the trachea. Wait a few seconds until the entire volume travels down the needle, and then remove it from the trachea.
   15. Observe a few seconds of apnea, which occurs when the needle is correctly inserted into the trachea so that the mouse will immediately inhale the entire volume of the liquid.
   16. If the mouse is not inhaling the liquid, carefully monitor its breathing and adjust the needle position. If the mouse stops breathing, immediately remove the needle and allow the mouse to resume breathing normally before reinserting it.
   17. Safely discard the syringe and needle after the injection.
   18. Close the subcutaneous fascia and the skin wound with a 5-0 absorbable suture.
       NOTE: When not completely reabsorbed, remove sutures 7-10 days post-surgery.
4. Animal recovery
   1. Place the injected mouse on its side on a heating pad for recovery.
   2. Monitor the mouse breathing and observe the mouse until it starts moving and regains sternal recumbency and full consciousness.
   3. Once it is confirmed that the mouse is in good condition, return it to the original cage. Do not return it to the company of other animals until it has fully recovered.
   4. To ensure prolonged analgesia and avoid any residual post-interventional pain, administer subcutaneously buprenorphine (at a final dose of 0.05 mg/kg body weight) to mouse every 12 h post endotracheal injection.
   5. Examine the mice for 24 h after the endotracheal injection of bleomycin and do so twice a day. Monitor the mice for respiratory distress, weight loss, behavior abnormalities, and for any sign of morbidity.

3. Tail vein infusion of human umbilical cord mesenchymal stromal cells

1. Cell preparation
   NOTE: The isolation, characterization, and cultivation of mesenchymal stromal cells from human umbilical cord has previously been described^8,9,10. hUC-MSC should be aseptically manipulated and infused; therefore, perform all steps under a sterile hood.
   1. Expand the hUC-MSC in 75 cm² culture flasks to early passages (1–3 maximum).
      NOTE: The hUC-MSC should be 70% confluent at the day of their infusion into mice.
   2. Wash the cells with 10 mL of sterile phosphate-buffered saline (PBS) at room temperature.
   3. Add 2 mL of trypsin and incubate the cells at 37 °C for about 1 min, until they start detaching.
   4. Neutralize the trypsin by adding 8 mL of hUC-MSC complete medium containing 10% fetal bovine serum (FBS).
   5. Collect the cells by centrifugation at 350 x g for 10 min.
   6. Resuspend the pellet in sterile saline and count the cells using a Bürker chamber. Prepare the cell suspension for infusion by diluting the cells to a final concentration of 2.5 x 10⁵ in 200 µL of sterile saline per mouse. Prepare an excess cell suspension to ensure there is enough volume for infusing all mice.
   7. Keep the cells on ice prior to the infusion. Infuse within a few hours, as described in section 3.3.
2. Anesthesia
   NOTE: In order to minimize the risk of damaging the mouse tail vein during injection the mouse must not move. Therefore anesthesia has been preferred over simple mouse restraining.
   1. Anesthetize the mice by 4% isoflurane inhalation in an induction chamber.
   2. Monitor the mouse breathing. The respiration rate will slightly slow down. After a few minutes, pinch one of the mouse feet to check for proper anesthetization.
3. Tail vein infusion
   1. Once unconsciousness has been confirmed, place the mouse under a sterile hood for the aseptic hUC-MSC intravenous infusion.
   2. Maintain general anesthesia throughout the experiment via a facial mask with a continuous flow of 1.5% isoflurane.
   3. To promote vasodilation and allow for an easier injection, soak the mouse tail in warm water for 2 min.
   4. Mix the cell suspension by gently pipetting to make sure that the cells do not form clumps. Aspirate 200 µL into a 1 mL syringe with a 26 G needle, avoiding bubble formation.
   5. Hold the mouse tail by the tip and gently straighten it.
   6. Locate the lateral vein of the mouse tail; gently scrape it with a scalpel and wipe it with 70% ethanol.
      NOTE: Gently scraping of the tail is performed to remove hair, making the site of injection smoother and cleaner.
   7. Starting from the distal portion of the tail, insert the needle into the vein at a 15° angle and slowly infuse 200 µL of hUC-MSC or sterile saline (vehicle control) (Figure 1B).
   8. Monitor the successful intravenous infusion by the liquid entering the vein without resistance and by a lack of extravasation. Wait a few seconds until the entire volume travels down the needle, and then remove it from the vein.
   9. To prevent bleeding, briefly apply pressure to the entry wound with a sterile gauze.
   10. Safely discard the syringe and the needle after the infusion.
4. Animal recovery
   1. Place the infused mouse on its side on a heating pad for recovery.
   2. Monitor the mouse breathing and observe the mouse until it starts moving and regains sternal recumbency and full consciousness.
   3. Once it is confirmed that the mouse is in good condition, return it to the original cage. Do not return it to the company of other animals until it has fully recovered.
   4. Examine the mice for 24 h after the tail vein infusion and every other day, to monitor their health status and detect any suffering or pathological sign early.

4. Organ explant and tissue processing

1. Sacrifice the mice at days 8, 14, or 21 after the bleomycin administration (Figure 1C) by overdose of injectable anesthetic.
2. Excise the trachea and the lungs and immediately wash them in ice-cold PBS.
3. Snap-freeze the right lungs in liquid nitrogen and store them at -80 °C for a subsequent molecular analysis¹⁰.
4. Inflate the left lungs with 4% paraformaldehyde and fix them in 10% neutral-buffered formalin solution for 24 h; then, dehydrate them in graded alcohol series, clear them in xylene, and embed them in paraffin¹⁰.