The selective intra-bronchial method is significant because it offers investigators a reproducible and nonlethal model for studying acute lung injury, a major cause of acute respiratory distress syndrome in humans. The advantage of the selective intra-bronchial technique is the localization of the inflammatory response to the left lung offering the right lung as an uninjured control and minimizing animal distress. Demonstrating this procedure will be Alexander Tavares, a technician and pregraduate student in our lab.
To prepare 0.1 normal hydrochloric acid, add 11 milliliters of double distilled water to an amber glass bottle, followed by the slow addition of one milliliter of 37%12 normal acid. Next, slowly add four milliliters of one normal acid working stock into 35 milliliters double distilled water in a 50-milliliter conical tube. Then use an electronic pH probe to measure the pH of the 0.1 normal stock solution, titrating to a pH of 1.1 using sodium hydroxide or acid stock solution as needed to a final volume of 40 milliliters.
Before beginning the procedure, filter one to two milliliters of the acid solution through a sterile 0.22 microliter filter into a sterile microcentrifuge tube and confirm a lack of response to pain reflex in an anesthetized mouse. Using electric clippers in slow downward strokes gently shave the surgical area on the ventral surface of the mouse below the chin in the cervical region of the throat. Remove any loose hair, and swab the exposed skin with three consecutive 10%povidone-iodine solution and 70%isopropyl alcohol swabs.
When the skin has been prepped, place the mouse in the supine position on a clean surgical board, and cover the animal with a sterile surgical drape. Make a longitudinal 0.5 centimeter opening in the skin above the trachea and salivary glands, and use slightly curved serrated forceps to carefully retract the skin. Gently separate the salivary glands to expose the tracheal muscles and use slightly curved serrated forceps to gently push apart the paratracheal muscles.
Tease away the fascia surrounding the trachea until the cartilaginous rings of the trachea are completely exposed, and use fully curved serrated forceps to work away the connective tissue between the retrotrachea and the retrofascia. Slide fully curved serrated forceps behind the trachea and lift the trachea. Once the tip of the forceps is behind the trachea, grasp a 10 to 15 centimeter piece of 4-0 braided silk suture with the tips of the forceps and pull the suture behind the trachea so that there's an even length on either side.
The surgical board may be turned to bring the surgical area closer to the dominant hand for the next steps. Then gently pull the ends of the suture toward the anterior of the mouse, and hold the ends in place. For cannulation of the left mainstem bronchus, expose the needle of a 24-gauge by 3/4-inch angiocatheter and insert the needle bevel side up into the interior region of the trachea between the first and second tracheal rings.
When the needle tip can be visualized within the tracheal lumen, release the suture and advance the cannula over the needle into the trachea angling towards the left posterior side of the mouse. When resistances encountered, withdraw the needle. Once the cannula is in place, firmly grasp the injection port to prevent the catheter from shifting and secure a P-200 micropipette tip within the port.
Then instill 50 microliters of the filtered 0.1 normal acid solution into the catheter, followed by an equal volume of air. This step requires precise manipulation of the cannula, while the airway's obstructed, and should be completed quickly and smoothly to restore proper airflow and to minimize airway damage. Rapidly withdraw the catheter and tilt the surgical board at a 60 degree angle.
After 30 seconds, lay the surgical board flat, and remove the suture from behind the trachea. Then use a 4-0 wax-coated braided silk suture to close the skin incision with two to three stitches. The acute lung injury resulting from intra-bronchial acid instillation involves the entire left lung.
Indeed, following intravenous administration of Evans blue dye and lung perfusion, the dye is detected only within the left lung. Quantification of the dye extravasation into the left lung is significantly increased relative to sham selective instillation. Interstitial neutrophils accumulate in the left lung 24 hours after acid instillation in contrast to the right lung in which few interstitial neutrophils are observed.
Lung histology reveals tissue injury and inflammation at the organ and cellular levels with exudative inflammation 24 hours after injury as characterized by marked alveolar edema and neutrophil infiltration within the left lung. Note that lack of significant injury or leukocyte influx into the uninjured control right lung. 72 hours after injury the edema and cellular infiltrates are substantially decreased representing a resolving exudative phase.
As assessed by flow cytometry, alveolar neutrophils increase in the left lung 24 hours after the initial injury, and decrease substantially at 48 and 72 hours. It is important to visualize the catheter in the trachea before advancing. Angle the cannula towards the left mainstem bronchus during insertion and follow the acid instillation with 50 microliters of air.
Bronchoalveolar lavage fluid analysis, lung histology, immune cell phenotyping by flow cytometry, live mouse imaging, and pulmonary function testing can all be performed to assess the inflammation and resolution. The development of this self-limited model of acute lung injury has allowed researchers to make significant contributions to the field of resolution biology. Remember that concentrated hydrochloric acid is hazardous and that it should be handled in a chemical hood wearing the appropriate personal protective equipment.
