The overall goal of this procedure is to use intra tracheal intubation to study the effects of experimental treatments on the physiology and pathology of the mirroring lung. This is accomplished by first measuring a control mouse of a similar size to the experimental mouse from the mouth to the bronchial bifurcation point of the lung. Next, use the BioLite intubation illumination system to locate the trachea.
The fiber optic fiber is then quickly removed after insertion leaving the catheter in place. In the final step, a solution containing the experimental treatment is added to the lungs through the catheter. Ultimately, the even spread of the molecule of interest can be visualized throughout the lung through the use of an accompanying green dye.
The main advantage of this technique over other intubation methods is that with our technique, the fiber optic elimination system facilitates the location of the trike as well as the placement of the catheter, ensuring optimal delivery of the material of interest into the lung. Demonstrating the procedure will be on site, a post-OC from my laboratory Before beginning the procedure, measure the length between the mouth and bronchial point using a similar sized practice mouse to determine the proper length of catheter to be inserted during the intubation. Then apply vet ointment to both eyes of the anesthetized experimental animal.
After several minutes, pinch a foot to confirm sedation, and then place the animal on an intubation. Stand angled at about 60 degrees. Hook the upper incisors over a small rubber band located at the top of the stand to hold the animal in place, and then use a Q-tip to gently retract the mouse's tongue to one side.
The most difficult aspect of this procedure is to clearly locate the trachea, which is separate from the asso Fergus and into which the catheter should be inserted. In order to be successful, make sure that the larynx is well illuminated by the fiber optic fiber, and to be aware of the direction in which the catheter should be inserted. Carefully insert the intubation system until the larynx is visualized with the aid of the fiber optic like guide.
Once the epiglottis and the hyoid cartilages are visualized, insert the fiber over the epiglottis and between the hyoid cartilages advancing the system until the previously determined length of catheter has been inserted. Once the catheter is in place, quickly remove the fiber optic like guide to allow the animal to breathe Normally then administer the solution of interest into the catheter. A good intubation will result in the solution being immediately sucked down through the tube into the lungs.
As soon as the solution is sucked into the lungs, remove the mouse from the stand and put it back into the original cage. Observe the mouse until it starts moving. Once the animal's health is confirmed, return the cage to its rack.
In the initial experiments, green dye solution was used to intubate the mice for practice. The lungs were immediately resected after intubation to examine how evenly the color was distributed. The dye was observed in most parts of the lung indicating a successful intubation.
The technique was then applied to study bleomycin induced pulmonary fibrosis using C 57 BL six mice three weeks after intratracheal intubation with bleomycin or saline as a control. The lungs were examined by histological analysis to examine the development of bleomycin induced fibrosis. No fibrosis was visible at either low or high magnification in the lungs of saline treated animals.
In contrast, bleomycin intubated mice developed fibrosis throughout their lungs as was evident in both low and high magnification images. Hydroxyproline content was higher in bleomycin treated mouse lungs as compared to saline treated mouse lungs. Further demonstrating that bleomycin treated mice develop pulmonary fibrosis.
The damaged area was then overlaid with a 121 point lattice grid and the number of intersections or points falling over the fibrous areas were counted and expressed as the percentage of a total of 121 points, the percentage of the damaged areas thus counted was proportional to the amount of bleomycin administered. Be sure to obtain an idea in advance of how deep catheter should be inserted into the trachea and to confirm that the solution is sucked down through the catheter into the lung as soon as it is added.
