This protocol lets us investigate drug-eluting stents in a small animal model of laryngotracheal stenosis letting us leverage genetically modified animals in a cost effective manner. Transoral placement of the drug-eluting airway stent spares the animals from an open incision into the trachea representing a more clinically relevant model of laryngotracheal stenosis. This technique facilitates preclinical investigation of local drug delivery strategies to treat laryngotracheal stenosis.
This technique provides a feasible and effective platform for the translational investigation of drug-eluting airway stents in a small animal model that can be applied for future investigation in larger animal models as well as clinical application. technique particularly the transoral intubation, induction of trachea injury, and the airway stent placement step. Visual demonstration of this technically challenging surgery is critical as many of the fine details cannot be fully described in words.
To make a 1%rapamycin containing polymer solution, add six milligrams of rapamycin to 600 milligrams of 70-30 PLLA-PCL in a glass vial. Under a fume hood, add six milliliters of dichloromethane to the rapamycin supplemented and control vial and add 120 microliters of glycerol to each vial. Then cap the vials and allow the PLLA-PCL solution to homogenize for six to 12 hours at room temperature.
To create a murine airway stent, using sterile materials and technique, apply one milliliter of rapamycin supplemented PLLA-PCL solution onto the venous cannula of a 22 gauge fluorinated ethylene propylene based angiocatheter while slowing rotating the catheter. When all of the solution has been applied, prop the molded angiocatheter with the tip of the angiocatheter facing downward and the hilt of the catheter on the edge of a glass Petri dish in a vacuum hood. After 24 hours under vacuum at room temperature, gently twist and slide the catheter from the dried stent construct and circumferentially check the stent for any defects that may have resulted during the casting process.
To aid in visualization, it is helpful to draw a thin black line down the length of the stent. Trim each end of the casted stents with fine straight scissors so that the edges are axial and use the scissors to cut each stent into three millimeter axial segments. Then load each three millimeter stent onto a new 22 gauge venous catheter.
For laryngotracheal stenosis induction, after confirming a lack of response to toe pinch, place the first mouse onto a surgical platform in the supine position and loop a small piece of thread affixed to the top of the platform around the central incisors. Secure the limbs to the table with tape and make a 1.5 centimeter midline vertical incision in the neck of the mouse. Divide the overlying thymus and lateralize the two resulting lobes to visualize the trachea.
Divide the overlying sternohyoid and sternothyroid muscles at the superior attachment bilaterally and pass a 22 gauge angiocatheter transorally through the larynx into the trachea. Using small forceps, apply pressure to the anterior larynx of the mouse to assist in the correct placement of the catheter. A correctly placed angiocatheter can be visualized by its white color through the trachea.
To induce the injury, pass a Bleomycin-coated wire brush through the inserted angiocatheter and slowly withdraw the catheter so that only the wire brush remains within the trachea. Using fine forceps, apply counterpressure on the trachea to mechanically disrupt the tracheal lumen with the brush before reinserting the catheter into the trachea over the brush. Remove the wire brush and reapply the Bleomycin before inserting and removing the brush four more times as just demonstrated.
After the last Bleomycin application, remove the catheter from the trachea. For transoral placement of a PLLA-PCL, ensure that a thin black vertical line has been incorporated into each stent and transorally intubate the mouse with the pre-loaded angiocatheter. The stent should be visualized within the trachea through the transcervical incision.
When the stent is in place, use fine forceps to firmly but gently grasp the trachea through the incision to hold the stent in place and remove the catheter. It is critical to place the stent as carefully as possible. Too much pressure on the trachea will result in collapse of the stent causing airway obstruction and death to the animal.
Close the incision with tissue glue or absorbable 4-0 chromic sutures used here and allow the mouse to recover with monitoring before returning the animal to its home cage. At the appropriate experimental endpoint, place the mouse of interest in the supine position on the surgical platform and use fine curved iris scissors to reopen the incision. Expose the trachea as demonstrated and use the scissors to divide the distal trachea below the level of the stent.
Next, divide the proximal trachea below the larynx and above the stent and separate the divided trachea from the esophagus posteriorly. Then remove the stent from the trachea and fix the trachea in 10%formalin for 24 hours. The biodegradable rapamycin loaded PLLA-PCL stent construct used in this study is capable of eluting rapamycin in a consistent and predictable fashion under physiological conditions.
In this image, the miniaturized biocompatible stent can be observed in situ within the trachea as indicated by the black marker on the stent visualized through the translucent mouse trachea. After 21 days, the black dye marker can be used to confirm the maintenance of the stent position within the trachea. Biocompatibility testing using immunofluorescent staining for markers of acute and chronic inflammation demonstrate that the PLLA-PCL stent construct is not immunoreactive as determined by the minimal number of immune cells present after placement in the absence of injury.
Manipulating the airway in a delicate and atraumatic fashion is critical to the success of the procedure. Excessive force, multiple intubations, and stent collapse can lead to animal death. After treatment with rapamycin-eluting stent, assessment of survival, gene expression via quantitative real-time PCR, immunoassay including flow cytometry, ELISA, and immunohistochemistry can be performed.
As it provides a successful platform for drug elution in a mouse model, this technique paves the way for future investigations of locally applied therapeutics in laryngotracheal stenosis. Dichloromethane is a corrosive liquid. Be sure to always use a fume hood, personal protective equipment, and non-corrosive materials
