(D-140) Compound Action Potential and Microvasculature Analysis During Uniaxial Stretching: Implications for Vocal Fold Paralysis

The recurrent laryngeal nerve (RLN) is the primary motor supply to the muscles of the vocal folds in the larynx.  Paralysis or paresis of one of the vocal folds, such as with unilateral vocal fold paralysis/paresis (UVP), often results in impaired speaking, breathing, or swallowing.[1-9] In general, the left RLN is impaired more often than the right in humans. Interestingly, 12-42% of laryngeal paralyses are diagnosed as idiopathic or unknown etiology.[11-14] Increased occurrence of pathology in the distal RLN segments, however, (i.e. closer to the larynx or trachea) suggested that chronic nerve compression occurred near the aortic arch or inferior trachea.

The Soft Tissue Biomechanics Laboratory (STBL) showed that those with idiopathic UVP have a higher aortic compliance compared to those of similar age and gender. The overall working hypothesis of this project is that the hypercompliance of the aorta itself contributes to the loss of function of the recurrent laryngeal nerve (which innervates the vocal fold) due to the dynamic motion that the aortic arch undergoes in a cardiac cycle. The mode and mechanism of mechanical insult that causes such damage, however, (e.g., levels of compression and/or stretch) have not yet been identified or quantified. When analyzing a nerve under stretch, it is necessary to know how the microvasculature and compound action potential (CAP) signal are altered. The STBL has a two-photon microscope to 3D image changes in microvasculature and a Uniax testing apparatus to stretch and apply load to nerves while CAPs are recorded.

The sciatic nerves of a rat were excised to simulate the RLN for experimental trials. The sciatic nerve was stimulated indirectly in a 3D printed chamber, which is composed of three solution wells: two outer KCl wells and an inner Krebs solution well. The wells were enclosed by Valap sealant, which is a mixture of Vaseline, paraffin wax, and lanolin oil. The nerve was extended across all the chamber wells with sutures tied to its ends and clamped to the Uniax device (Figure 1). The nerve was stimulated at 4.5V, 100us duration, and 0.33 Hz repetition. The signal was recorded using Ag/Cl electrodes, a diff amp, and a NI MAX chassis. After a CAP recording was obtained, the nerve was stretched to 1.05, 1.10, and 1.15 of its excision length and the applied force was quantified.

In another subset of trials, the rats were injected with 0.2mL heparin (an anticoagulant) via the tail vein. The rat was then euthanized, and its chest cavity cut open to expose the heart. The rat was perfused through the left ventricle with 250ml PBS, 100 ml 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI), and 500ml PFA; the right atrium served as an outlet for perfusion. Some nerves were unstretched and others were stretched to 1.05, 1.10, and 1.15 their physiological length. Both nerves were fixed overnight. These nerves were then cleared using an ethanol and BABB clearing process. An excitation of 910nm and 20X objective were used for two photon imaging of microvasculature.

Overall, there was no significant difference between CAP reductions of individual stretch trials; however, there was an evident reduction in CAP for the solution control and cut nerve when compared to the stretch trials. Also, the imaging trials showed that it is possible to map the microvasculature of the sciatic nerve.

With a sample size of n = 5 sciatic nerves, an average percent reduction in CAP was quantified for 1.05, 1.10, and 1.15 stretches. Negative controls were run where the nerve was cut and just solution was in the chamber. All the percent reductions were in reference to the ex-vivo length. The percent reductions were 25.22%, 40.54%, and 54.56% for 1.05, 1.10, and 1.15 stretches, respectively. There were no significant differences between any of the stretch trials. The cut and solution controls showed a 93.68% and 91.90% reduction in CAP, respectively (Table 1). These reductions were significantly different than the stretch trials.

Moreover, load was applied to a sample of n = 2 sciatic nerves to quantify the stress-strain relationship. The nerve was stretched to 1.05, 1.10, 1.15, and 1.20 and of its original length, applying a stress of 5.46 kPa, 13.62 kPa, 15.18 kPa, and 18.91 kPa, respectively (Table 2). The nerve was assumed to have a circular surface area with a diameter of 1.25 mm.[15] There was a significant difference between 1.05 stretch and all the other stretch values.

Currently, the nerve’s microvasculature without stretch is being mapped with two-photon excitation (Figure 2). The nerve will be introduced to 1.05, 1.10, 1.15, and 1.20 stretch, and the overall layout of the microvasculature and the potential diameter changes will be compared to the unstretched nerves.

There may have been minimal significant differences observed for CAP stretches or applied load trials because the sample size was not large enough. Also, when the suture is placed in the Uniax stretching apparatus, it often slips. This means that there will be a smaller load applied, which then decreases mechanical insult and potential decrease in CAP. In the future, the grip on the clamps will be improved to prevent this confounding variable.

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