Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

The overall goal of this procedure is to evaluate T-cell function in the absence of the thymus. This is accomplished by first intubating the rat and then opening the chest to expose the thymus. Next, the thymus is dissected from the surrounding tissues and hemostasis is obtained in the final step.

The incision is closed in layers. Ultimately, the peripheral blood mononuclear cell population can be evaluated by flow cytometric analysis, but the presence of naive T cells in the absence of thymus function. The main advantages of this technique over the existing methods of thymectomy are that with this technique, the airway is controlled by a simple intubation tube.

The size of the sternal incision is minimized. The positive pressure ventilation with peep prevents pneumothorax and the mortality rates are minimized. Demonstrating the procedure will be myself and Toria Reell, a fourth year medical student at Duke University for my laboratory.

To prepare the tracheal intubation cannula first use cutting pliers to remove the needle end of a two inch forcing gauge angio catheter needle. Pinch the lumen of the needle closed with needle nosed pliers, and then use the cutting pliers to remove the pinch section down to the edge of the open lumen portion. Next, cut the edges on both sides of the remaining end at a 30 to 45 degree angle to the needle edge and place the bottom three to four millimeters of the cannula into the open area of a pair of straight slip joint pliers.

Pull up until a slight upward curve has been formed, and use fine sandpaper to smooth down the edges at the end of the cannula. Then return the blunted instrument into the angio catheter tubing, ensuring that the tubing is slightly shorter than the blunted needle. Before beginning the procedure, rub a small amount of lubricant onto the end of the intubation cannula, and then turn on the ventilator to begin the flow of isof fluorine gas through the connector tubing.

After confirming sedation by toe pinch, transfer the animal to the incubation apparatus. Suspend the rat on the metal bar by its upper incisor teeth, and position a flexible high intensity light source one to two centimeters from the ventral surface of the neck. Next, use a pair of student standard pattern forceps to pull the tongue gently upwards and to the side of the bottom teeth.

Then grip the tongue between the thumb and forefinger of one hand while using the flat inside surface of one prong of the forceps. To press the lower end of the tongue ventrally, exposing the epi gloss and the aperture of the larynx. Visualize the vocal cords and then guide the upturned blunted end of the intubation cannula anteriorly through the open gloss into the trachea until the hub of the angio catheter touches the incisors.

Now remove the metal stylet and attach the anesthesia tubing to the angio catheter opening. To begin the isof fluorine ventilation, set the ventilator to pressure control settings with a pressure of 12 to 14 millimeters of mercury at a rate of 60 respirations per minute, and use a positive end expiratory pressure of three centimeters of water. Apply eye to a sedated adult rat After observing the bilateral chest wall expansion, to ensure the proper placement of the catheter, secure the endotracheal tube with a strip of cloth tape.

Then attach the blood oxygenation and heart rate monitor to the rat's foot, and begin the monitoring to remove the thymus. First sterilize the skin by applying povidone iodine to the entire chest of the animal. When the iodine is dry, wipe the surface of the skin with a 70%ethanol soaked gauze.

Next, cover the rat with clear plastic wrap, cutting a hole to expose the sterile operating field, and then identify the supra sternal notch in the upper thoracic region. Make a two centimeter midline incision through the skin with a scalpel starting distally and extending proximally along the sternum to two to three millimeters above the notch. Then perform a 1.5 centimeter partial median sternotomy, starting from the supra sternal notch using blunt tipped shea scissors.

Next, use blunt graft forceps to separate the pretracheal strap muscles. At this point, the trachea can be seen and the endotracheal catheter should be visualized inside. Place the prongs of a small ALM retractor underneath the separated strap muscles and sternum, and then open the retractor to expose the superior aspect of the thymus.

Use fine dumont forceps to release the lateral edges of the thymus tissue and expose the lower thymic lobes. Then pull the thymus gently and superiorly into the open incision site, taking care to avoid the surrounding tissues. If necessary, obtain hemostasis by holding pressure on any areas of bleeding with a sterile Q-tip.

Now deliver the lower thymic lobes into the incision and sharply lyce the posterior attachments using forceps and micro scissors. Then remove the intact thymus and carefully inspect it for any missing sections. Now, remove the ALM retractor and oppose the strap muscles with two interrupted five zero maxon sutures.

Then apply two drops of surgical grade cyanoacrylate tissue adhesive across the sutures. Oppose the sternum using 2 4 0 silk sutures placed through the gaps between the ribs just under the sternum to avoid the underlying muscle layer. Next, use a running four zero nylon suture to close the skin layer, and then discontinue the isof fluorine.

To shorten the postoperative anesthesia recovery period, clean the incision site and surrounding skin with saline, moist and gauze, followed by several drops of bupivacaine for local anesthesia, and one to two drops of cyanoacrylate glue to seal. The incision continue to ventilate. After the isof fluorine is turned off until the rat shows signs of independent respiration, then extubate the animal and allow it to fully recover under close observation in a cage placed on a warming pad.

In this experiment, the thoracic tissue was examined by immunohistochemistry for cytokeratin and hematin eosin stain revealing a denser nuclear staining of the excise thymic tissue by h and d, and a characteristic lacy pattern by cytokeratin staining, allowing the differentiation of the thymic tissue from, for example, the thoracic lymph nodes. Peripheral blood samples obtained from the euthanized rats at four weeks were then analyzed for the persistent depletion of naive T-cells. As expected ThermaCo rats maintain their overall T-cell counts compared to control rats, but demonstrated a loss of both their naive CD four and CD eight T-cell populations.

After watching this video, you should have a good understanding of how to safely and rapidly intubate a rat, dissect, and remove the thymus and close the mediastinum and incision in a way that minimizes any postoperative complications.