(C-113) Assessing Functional Compound Action Potential Measurements in Ex-Vivo Peripheral Nerves

Our goal is to find a potential cause for idiopathic unilateral vocal fold paralysis (iUVP). The current cause of the disease is unknown. The vocal folds are muscles that can be found on both sides of the larynx that expand and contract during speech. Paralysis can cause the vocal folds to remain open, leaving the airway unprotected. This can lead to issues in swallowing, talking, and breathing. iUVP most commonly presents on the left side.

               The recurrent laryngeal nerve (RLN) bifurcates off the vagus nerve, wraps around the aortic arch, and innervates the vocal folds. Previous research has shown that there is greater onset of iUVP when aortic arch compliance increases. Compliance refers to the ability for the aorta to expand under pressure. Therefore, the hyper-compliance of the aorta stretches the RLN causing damage to the nerve and conductance, which impacts functionality of the vocal folds potentially leading to iUVP.

The rat sciatic nerve (Figure 1) served as our preliminary animal model which later transitioned to the RLN in a juvenile porcine model then an adolescent porcine model. Once the nerve is harvested the process to stimulate and stretch the nerve is consistent in all three models. The nerve is then incubated in oxygenated krebs solution for one hour. This supplies the nerves with the proper nutrients and environment to recover from surgery. The pH is kept between 7.2 and 7.4 to mimic its in-vivo environment. After the hour incubation period, the nerve is individually tested in a chamber with three wells (Figure 2). The outer two are filled with a potassium chloride solution and the inner well is filled with krebs. Electrodes are then placed through the chamber into the wells. The nerve is stimulated and recorded both indirectly through the solution. The nerve is stimulated at 4.5 volts (for 100 microseconds every 3.33 seconds) via a train delay generator and a constant voltage isolated stimulator connected to the stimulating electrodes. The recording electrodes are connected to a differential amplifier that is used to filter out noise at 100 high-pass and 50k low-pass. The file is then processed in a MATLAB script to visualize and quantify the CAP.

The preliminary rat sciatic trials were tested in two groups the right sciatic nerve and the left sciatic nerve. For the right sciatic, three animals were tested with an average decrease in compound action potential of 91.25% when the nerve was cut. For the left sciatic, the same three animals were used, and an average decrease in compound action potential of 87.70% when the nerve was cut. For the RLN, quantifiable CAP was obtained in three pigs with an average decrease of 83.19% when the nerve was cut. A paired t-test was performed to determine that there is a statistical difference between the no stretch and cut amplitude for the rats’ right sciatic nerve values which yielded a p-value of 0.0293 (Figure 3).  A paired t-test was also run on the other two groups and the results were consistent. A one-way ANOVA statistical test was also performed between the three groups which yielded an F-value of 0.65 and a P-value of 0.547 meaning there is no statistical difference in the CAP amplitude reduction between the rat sciatic and pig RLN groups.

Porcine CAP success rates were greatly less than that of the rat model. According to other literature we found that the amount of epineurium on the exterior of the nerve could be inhibiting our ability to stimulate the nerve. Literature suggested the amount of epineurium on the RLN is much greater than that of the rat sciatic nerve. We also found that the amount of epineurium is less on a juvenile pig (2-3 months) than compared to an adolescent piglet (2-3 weeks). However, after five piglet tests no quantifiable CAP was found. More testing and research needs to be conducted to increase the success rate of quantifiable CAP. If the success rate can be increased and the results hold with an increased sample size of quantifiable CAP, we will confidently be able to report the average decrease in compound action potential when the left recurrent laryngeal nerve is cut in a porcine model.