The overall goal of this procedure is to present a model of cellular transplantation based on the use of olfactory and sheathing cells in a laryngeal nerve injury model. This is accomplished by first sectioning and performing anastomosis of the recurrent laryngeal nerve. Next, the olfactory and sheathing cells obtained from olfactory mucosa or olfactory bulbs are transplanted.
Then two to three months after surgery, the animals are evaluated by laryngoscopy and electromyography. Finally, the recurrent laryngeal nerve is isolated for histological studies. Ultimately, results can be obtained that show olfactory and sheathing cells improving axonal regrowth of the recurrent laryngeal nerve and functional recovery of the vocal cords through laryngoscopy, electromyography, and histological measurements.
This method can answer key questions in the field of peripheral nerve injury that involve the measure of axonal growth and the ability to quantify functionality of the recovery. Generally, individuals new to this method will struggle because recurrent lal nerves anastomosis require microsurgical skills After generating GFP labeled primary cultures of olfactory bulbs, or OB and olfactory mucosa or OM as outlined in the written protocol. Prepare for surgery by autoclaving all instruments.
Once the rat has been anesthetized by intraperitoneal injection of ketamine and chlorpromazine, verify its level of sedation by performing a toe. Pinch, shave the surgical site and place the rat in dorsal decubitus, disinfect the skin with alternating washes of alcohol and Betadine solution. After that, assemble a sterile surgical environment and maintain sterile procedures throughout surgery.
Use a scalpel to make a two centimeter medial cervical cutaneous incision. Next, following the medial white line, perform a vertical incision of the infra hyoid muscles using an orthostatic retractor between the infra hyoid muscles. Expose the larynx and then the recurrent laryngeal nerve or RLN.
Then under a surgical microscope, use micro scissors to cut the nerve fully at the level of the seventh tracheal ring with one point of 11 T suture. Perform anastomosis to de innervate the larynx just before transplantation. Use 0.05%trypsin EDTA to remove the OB and OM cells from the dishes.
Then to stop the digestion, add five milliliters of warm medium after centrifuging the cells at 432 G for five minutes. Use a hemo cytometer to count the cells and adjust the cellular concentration in this model between 1.2 and six times 10 to the six cells in 30 microliters of D-M-E-M-F 12 were used in a micro fuge tube. Prepare a one to two ratio of cells to growth factor, reduced matrigel and place on ice just prior to the transplantation.
Remove the tube from the ice and hold it to warm the mixture. Using a pipette, depose the cell matrigel solution on the anastomosis site and allow the mixture to self-assemble on the injured nerve when it is set. Use four aught sutures to close the muscles and skin.
Place the rat in an individual cage and keep it under a heating lamp for 24 hours. Administer analgesics for the next five days, approximately two months after surgery after anesthetizing. Rats as described earlier in this video, use a toe pinch to confirm sedation.
Place the rat in dorsal decubitus before inserting a 30 degree video. Laryngoscope adjusted to provide the best view of the larynx to reproduce the same view For each recording. Determine anatomical landmarks such as the posterior commissure, the right vocal cord where it inserts into the vocal apophysis of the OID and the left focal cord where it inserts into the vocal hypothesis of the oid.
Record vocal cord movements by selecting three sequences of successive maximal abduction and maximal abduction. To analyze laryngeal function, determine absolute angular movement by measuring the difference between the movement of maximal and maximal abduction of the vocal chords. Determine the dynamic score by measuring the movement amplitude and the functional score by measuring the synkinesis and paradoxical movements to carry out electromyography.
Place an electrode on the hamstring muscle of the rat after exposing the larynx and trachea as demonstrated for recurrent nerve anastomosis. Expose the cricothyroid cartilage. Next, using micro scissors, open the cricothyroid cartilage to expose the posterior cricothyroid or PCA muscle.
Insert a monopolar needle electrode into the muscle with the animal under spontaneous ventilation. Use an acquisition system to record the electrical muscular activity of the PCA muscle. Analyze the muscular activity in terms of richness and synchronization with the respiration by assigning a qualitative score from zero to three using the following scale.
Zero equals an untried tracing without increase during inspiration. One equals a rhymed tracing with inspiration increasing, but poor tracing two equals a rhymed tracing with richer activity and three equals a rhymed tracing. That's very rich constituting an interference pattern similar to a maximal intentional activity.
Next, to avoid damaging the RLN because of its small size, expose the vagus nerve, then attach an electrode and stimulate at this site. Record the latency and potential duration in the PCA muscle. Finally, after euthanizing the animal using a pentobarbital overdose under a microscope, locate the anastomosis, suture and remove the distal stump of the RLN between the anastomosis and larynx.
Perform histological analysis according to the text protocol shown here after transplantation of cells from primary cultures of olfactory, mucosa and olfactory bulbs are examples of EMG traces obtained in the PCA muscle during respiration. Animals of the control group presented a rich electrical muscular activity with an increase during inspiration that was not present in the Rerated group. In this figure, histological analyses of the RLN by toluidine blue staining show typical fiber profiles from normal re innervated and transplanted animals.
This measurement allows quantification of the number of myelinated fibers, the mean size of the fibers, and the mean thickness of the myelin as shown here. To complete the histological analyses. Tracking of GFP positive cells was performed after intra nervous transplantation in the sciatic nerve.
This site was chosen rather than the RLN because the R lns small size prevents intra nervous transplantation. Hans master anastomosis of the recurrent perial nerve can be done in 15 minutes if it's performed properly After its development. These techniques paved the way for researcher in the field of peripheral nerve injury to explore the use of cellular transplantation in a rat model.
After watching this video, you should have a good understanding how to transplant cells in the recurrent nerve injury injury model and evaluate functional recovery of the vocal cords through electromyography a lab endoscopy and histological measurements.
