Direct Intrabronchial Administration to Improve the Selective Agent Deposition Within the Mouse Lung

To model lung disease in mice, experimental agents are often administered via intratracheal injection. This approach, however, cannot selectively target one lung, and the distribution may be asymmetric between lungs. By cannulating an individual lung in an anesthetized mouse, we can unilaterally administer experimental agents.

With modification, this approach may be used bilaterally, improving the symmetry of deposition across both lungs. Catheter preparation including polishing, slight angulation, and labeling is key element for success. Of course, practice this technique is also necessary.

Demonstrating the procedure will be Sumei Liao, a professional research assistant from my laboratory. After confirming a lack of response to toe pinch, suspend an anesthetized, eight-to 10-week-old, C57-Black/6 mouse from the incisors on a draped suture line in a supine orientation. Loosely secure the mouse with two to three pieces of hook, and loop the tape, avoiding restriction of ventilation, and turn on a light-emitting diode fiber-optic illuminator.

Position the operator behind the platform, dorsal to the mouse. Orient the gooseneck of the illuminator two to three centimeters away from the mouse so that it lights the larynx area through the skin. Using sterile forceps in the dominant hand, draw the tongue out of the oral cavity.

Use the nondominant hand and the sterile depressor to flatten the root of the tongue to widely expose the oropharynx. After releasing the forceps, use the dominant hand to intubate the extended catheter into the trachea via the oral cavity. Confirm placement by observing whether the bubble in the syringe moves up and down with each breath.

For selective lobar cannulation of the distal right lung, after intratracheal intubation, rotate the plastic board 30 degrees, and guide the hub of the catheter in parallel to the mouse midline to the appropriate weight-based depth, as indicated in the table. Using a gel-loading tip, deliver 20 microliters of 0.3%Evans blue dye into the catheter, followed by one to two 100-microliter aliquots of air by glass dropper. Then, withdraw the catheter, and maintain the mouse in position for 30 seconds before placing the animal on a warming blanket, with monitoring until full recovery.

For selective lobar cannulation of the distal left lung, after intratracheal intubation, rotate the plastic board minus 74 degrees, and gently advance the catheter into the left mainstem bronchus while placing modest pressure 90 degrees downward and toward the bookend. After cannulating the left lower lung segments, rotate the plastic board minus 30 degrees, and deliver 40 microliters of 0.3%Evans blue dye with a gel-loading tip. Then, deliver one to two 100-to 300-microliter aliquots of air via glass dropper, and withdraw the catheter to allow the mouse to recover, as demonstrated.

For right entire lung administration, after intratracheal intubation, rotate the plastic board 30 degrees, and guide the catheter in parallel to the mouse midline, reaching to depths necessary for right-sided distal lobar cannulation. Confirm the appearance of the tachypnea sign, and rotate the mouse minus 74 degrees to enable gravity assistance for agent delivery. Withdraw the catheter to a position that corresponds to the takeoff of the right mainstem bronchus, and ensure that the bevel of the catheter faces downward.

Deliver 30 microliters of 0.3%Evans blue dye to the right lung, followed by one to two 100-to 300-microliter aliquots of air. Then, withdraw the catheter, and allow the mouse to recover. For left entire lung administration, after intratracheal cannulation, rotate the plastic board minus 74 degrees, and gently advance the catheter into the left mainstem bronchus while placing modest pressure both 90 degrees downward and toward the bookend.

Confirm the no tachypnea sign, and rotate the mouse 86 degrees to allow gravity assistance with agent administration. Withdraw the catheter to the left mainstem bronchus, and rotate the bevel of the catheter to face downward. Deliver 30 microliters of 0.3%Evans blue dye with a gel-loading tip to the left lung, followed by one to two 100-to 300-microliter aliquots of air.

Then, withdraw the catheter, to allow the mouse to recover, as demonstrated. Bilateral delivery of FITC-dextran by both intratracheal and symmetric intrabronchial administration leads to asymmetric lung parenchymal FITC fluorescence with greater relative concentrations noted in the left lung. This suggests that asymmetric lung delivery of experimental agents after intratracheal administration is not a consequence of asymmetric presentation of these agents to each mainstem bronchus but that equal mainstem delivery is diluted by differences in lung weight and mass.

Dose-adjusted intrabronchial administration improves the symmetry of lung parenchymal agent delivery. Despite this correction, however, persistent heterogeneity is still observed within the different lobes of the right lung. 21 days after intratracheal bleomycin-induced fibrotic lung injury, inflammation is observed predominantly in the left lung, with less injury notable in the right lung.

Conversely, dose-adjusted intrabronchial bleomycin administration results in a slightly decreased injury to the left lung and an increased injury to the right lung, with the greatest injury evident in the middle and superior lobes. During entire lung administration, don't forget to withdraw the catheter and tend the bevel of the catheter face down.