A Heterotopic Mouse Model for Studying Laryngeal Transplantation

Laryngeal transplant models were described in mice and rats. Senior alter's previous work focused on devising a more efficient method using the donor carotid arteries for anastomosis, obviating the need to dissect the superior thyroid arteries. In this video, we described this procedure in mice.

This mouse laryngeal transplant model allows immunologic studies to be conducted with ease and at low cost. Although technically more challenging due to the smaller size of the vessels compared to a rat model, this procedure can be performed in approximately three hours if familiarity with the surgical steps is obtained. After the animal is fully anesthetized, transfer the mouse to the shaving area where 1.5%isoflurane with one liter per minute oxygen flow will be delivered via a face mask for maintenance of anesthesia.

Confirm the depth of anesthesia with a toe pinch. Shave the chest and neck of the mouse up to the jawline and apply hair removal cream. And after 30 seconds, wipe the cream off and transfer the mouse to the surgical area.

Immobilize the mouse and prep the surgical area thrice with povidone-iodine and alcohol, then drape the mouse. Make a small horizontal incision just superior to the suprasternal notch. Using fine scissors, elevate the skin bilaterally through that incision up to the mandible and excise a trapezoid shaped skin segment.

Excise the bilateral salivary glands using cautery at the superior part where a small vein traveling through the gland is visualized. Dissect the bilateral sternocleidomastoid muscles from the surrounding tissues and retract them laterally. Make a midline incision on the strap muscles and bilaterally excise them taking care not to damage the underlying thyroid gland and leaving it on the laryngotracheal esophageal or LTE complex.

After this step, bilateral carotid arteries are visible. Circumferentially dissect the common carotid arteries to the level of the clavicle inferiorly and to the level of the carotid bifurcation superiorly. The vagus nerve and internal jugular vein are dissected free from the carotid arteries and not included in the procured graft.

Dissect the internal and external carotid arteries far enough superiorly to be able to ligate and divide. Using a 10-0 nylon sutures, ligate the internal carotid arteries two to three millimeters superior to the carotid bifurcation. Ligate the external carotid arteries at least three millimeters superior to the branching point of the superior thyroid artery and ligate the common carotid arteries at the level of the sternum.

Cut all ligate vessels bilaterally. Keep the vascular pedicles on top of the LTE complex to avoid accidental damage during further dissection. To prevent any gas leakage or inadvertent loss of anesthesia, make sure the animal has expired before making any airway cuts.

Transect the trachea below the fifth tracheal ring and carry the incision through the esophagus down to the prevertebral fascia to free the LTE complex inferiorly. Free the trachea and esophagus from the underlying prevertebral fascia from inferior to superior. Create an anterior pharyngectomy just inferior to the hyoid.

Carry the incision down to the prevertebral fascia to free the LTE complex superiorly. Divide any remaining lymphoid or connective tissue attachments between the LTE complex and the surrounding tissue. And finally, remove the graft.

Place the procured graft in a sterile Petri dish and wash with normal saline to get rid of any blood clots. Dilate the bilateral carotid arteries using one millimeter micro dilators. Using a 30-gauge blunt tipped needle, inject approximately two milliliters of heparinized saline in each carotid artery to flush the graft.

Blood and saline will be seen flushing out of the contralateral carotid artery and small free vessel ends which confirms intact superior thyroid arteries. The graft can be left in heparinized saline and a short break can be taken for up to three hours before transplanting it into the recipient. Prepare the recipient mouse in the exact same manner as described for the donor following anesthesia induction, shaving, and surgical preparation steps.

Inject the recipient mouse with extended release analgesic subcutaneously 30 minutes prior to the start of surgery. Using a scalpel, make a midline neck incision extending from the jawline superiorly to the sternum inferiorly. Elevate the skin on the left side and retract it laterally.

Excise the left salivary gland cauterizing the superior vessels as previously described. Excise the adipose and lymphoid tissue by dividing any visible vessels with low temp cautery. Taking care not to damage the underlying external jugular vein.

Dissect the external jugular vein circumferentially. Use at least five millimeters of clear length of the vessel for anastomosis. Dissect the sternomastoid muscle and retract it laterally.

Circumferentially dissect the common carotid artery from the clavicle inferiorly up to the carotid bifurcation superiorly. Pass the background material under the external jugular vein and apply the double approximating B-3 vessel clamps. Place a 10-0 nylon suture through the anterior wall of the external jugular vein at the location of the desired monotomy, and use the suture to pull anteriorly intent the vessel.

Cut just deep to the suture with micro scissors to facilitate the creation of the proper size single slit venotomy, and to ensure the cut is completely through the venous wall. Using a 30-gauge blunt tipped needle, flush the inside of the vein with heparinized saline. Place the donor LTE complex between the recipient left carotid artery and left external jugular vein.

Align the free end of the donor left carotid artery toward the recipient left external jugular vein and bevel the end using sharp scissors. Using four 10-0 nylon interrupted sutures, anastomose the donor left carotid artery and recipient left external jugular vein in an end-to-side fashion. Slide the background material under the recipient common carotid artery and apply the double approximating A-3 vessel clamps.

Create an arteriotomy in the same fashion as the venotomy. Anastomose the donor right carotid artery to the recipient left carotid artery in an end-to-side fashion. Using six interrupted sutures with 10-0 nylon is ideal for the arterial anastomosis.

Remove the clamps on the venous side. If bleeding is encountered, applied gentle pressure with cotton tips. Remove the clamps on the artery and immediately apply gentle pressure with cotton tips.

Some bleeding is expected at the step which usually stops after one minute with gentle pressure. Check the integrity of blood flow in the artery in the vein. With intact arterial flow, pulsation of the donor carotid artery is usually seen and the donor thyroid gland will change from its flushed transparent color back to its original reddish color.

Red coloration of the small vessels on the LTE complex can also be observed. Close the skin incision with 5-0 monofilament suture in a running fashion. Apply a skin adhesive on the incision.

Inject one milliliter of warm saline subcutaneously to account for fluid loss during the surgery. Stop the anesthesia and transfer the mouse to a recovery cage. Observe the mouse on a heating pad until it is fully awake to avoid hypothermia.

Surgical success can be assessed immediately after removing the vessel clamps. Figure A is representative of a successful surgery because blood flow can be visualized in the carotid arteries of the donor larynx and the thyroid glands turned red. Figure B is representative of an unsuccessful surgery because blood flow cannot be visualized in the carotid arteries of the donor larynx and the thyroid glands remained pale.

After 15 days in the absence of immunosuppression, most recipients do not have blood flow to the transplant. The patency of the artery can be assessed macroscopically by dissecting the laryngotracheal complex free from the surrounding recipient tissue and visualizing the end-to-side anastomosis.Thanks. After watching this video, you should understand how to heterotopically transplant a laryngeal tracheoesophageal complex from one mouse to another in approximately three hours.

This feasible and relatively low cost model offers significant potential in studying the immune system's role in rejection of the laryngotracheal complex, thereby offering the potential for new therapies in organ transplantation.