The overall goal of this procedure, is to provide quick, easy and reliable administration of reagents into the murine lungs via intubation. The method also limits the risk of residual damage to the surrounding tissues and is inexpensive. This method can help answer key questions in the basic lung biology and mechanisms of disease fields.
Such as acute lung injury and fibrogenesis. The main advantages of this technique are that is highly re-produceable, easy to master, does not require specialized equipment or lengthy recovery times. The procedure must be performed using sterilized tools.
So autoclave both the blunt end forceps and the depressor. In a sterilized hood, prepare a working stock of re-agent to administer via the intubation. A bolus volume between 30 and 45 microliters is recommended.
Sonicate the solution for 10 minutes at 35 kilohertz to assure even mixing. Now, prepare the workspace. Designate at least a square meter of space for the workspace itself.
As well as distinct locations for the animals both before and after the procedure. Now, prepare a procedure board. This customized model is cut from an aluminium sheet and reinforced by a T bracket.
Similar commercial boards are also available. Using a few strips of tape, fix the base of the procedure board to the bench, so it will be directly in front of the experimenter. Then tie a length of 4.0 suture between the two positioning screws of the procedure board.
Next, build a spirometer. Remove the plungers from three one-milliliter syringes. Then load up to 60 microliters of PBS into the top of each syringe barrel.
Loosely secure one of these three syringes to the hub of a catheter. When in use, air emanating from the trachea, will cause the PBS in the barrel to rise and fall. Place this next to the procedure board.
Place the other two additional syringes within reach to serve as backups. Next, into a fourth syringe, aspirate 300 microliters of air, and place it to one side of the board. Also prepare a few six inch lengths of tape to secure the animal to the board.
Then set up an isoflurane chamber if needed. Attach the oxygen, isoflurane and vacuum lines to the appropriate ports on the chamber and the clearance vacuum. Begin with anesthetizing the mouse.
Importantly because factors such as the age, weight, and health of the animal can all modify the response the anesthetics, each lab should optimize the isoflurane exposure to meet their own needs. While waiting, load the bolus of reagent into a pipette tip, and set it aside. When the mouse is sedated, suspend it by it's upper incisors from the thread on the procedure platform, with the animals dorsum lying flat against the platform surface.
Now, being careful not to restrict ventilation, loosely tape the lower portion of the the thoracic cavity, just above the diaphragm, to the platform, to keep the animal aligned during the procedure. Next, set the gooseneck illuminator to 80 to 100%Position the light one to two centimeters from the surface of the skin near the solar plexus and regularly check that it isn't over heating the skin. From behind the platform use the blunt end forceps to locate the tongue.
Then being careful to avoid the lower incisors, gently grip and draw the tongue out of the oral cavity. While securing the tongue, flatten it with the depressor In the non-dominant hand, allowing the forceps to release. Now, guide the gooseneck approximately from the solar plexus to the main stem bronchi to illuminate the trachea.
The trachea can also be easily distinguished by the action of respiration, which will cause the light to fluctuate in intensity as it leaves the trachea. When correctly positioned, the trachea can be seen as a central pin of light, with minimal ambient light in the oral cavity itself. The catheter should not enter the oral cavity until the view of the trachea is ideal.
The trachea cannot be brought into view through manipulation of either the light source or the depressor. Researchers should release the tongue and re-attempt the procedure. Next, angle the syringe to follow the natural path of the trachea.
And lower the 22 gauge catheter tip, with the attached syringe containing the PBS, straight into the lumen. Now, feed the catheter in an additional five millimeters and remove the tongue depressor. If correctly placed the PBS bubble in the barrel will begin to rise and fall with each breath.
Be patient when looking for this indicator as both heavy and light sedation can modify the frequency of respiration. Then shift the syringe to the non dominant hand. While gripping the hub, gently remove the syringe leaving the catheter in place.
From the dominant hand, deposit the prepared bolus of reagent directly into the center of the catheter hub. Then attach the second syringe with 300 microliters of air and eject the air into the hub. This will help ensure that the bolus is well distributed in the lungs.
Now, remove the syringe and replace it with a syringe containing the bubble of PBS. The bubble will continue to rise and fall if the procedure has been performed successfully. Now, remove the catheter and tape.
And place the animal in a dry warm place until it regains consciousness. Typically within a couple of minutes. Intubated mice were monitored daily for weight loss.
Mice were collected at four, 10, and 17 days post intratracheal intubation with bleomycin. Bronchoalveolar lavage was collected from these lungs and analyzed. Elevated levels of IgM demonstrated a significant time dependent increase in lung permeability indicative of epithelial and/or endothelial barrier dysfunction.
The right lungs were fixed and stained with Masson's trichrome. The fibrogenic response, a well established marker of total collagen content, was quantified. The histology sections evidenced treatment dependent thickening of the pulmonary interstitium, and increases in fibrotic lesions.
Once mastered this technique can be done in under a minute if properly performed. While attempting this procedure it's important to remember that manipulation of the soft tissues surrounding the trachea is not necessary or recommended. And it can compromise both the health of the animal, and the integrity of the experiment.
