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TrkA inhibitor promotes motor functional regeneration of recurrent laryngeal nerve by suppression of sensory nerve regeneration - PubMed

  • ️Wed Jan 01 2020

TrkA inhibitor promotes motor functional regeneration of recurrent laryngeal nerve by suppression of sensory nerve regeneration

Hiroshi Suzuki et al. Sci Rep. 2020.

Abstract

Recurrent laryngeal nerve (RLN) injury, in which hoarseness and dysphagia arise as a result of impaired vocal fold movement, is a serious complication. Misdirected regeneration is an issue for functional regeneration. In this study, we demonstrated the effect of TrkA inhibitors, which blocks the NGF-TrkA pathway that acts on the sensory/automatic nerves thus preventing misdirected regeneration among motor and sensory nerves, and thereby promoting the regeneration of motor neurons to achieve functional recovery. RLN axotomy rat models were used in this study, in which cut ends of the nerve were bridged with polyglycolic acid-collagen tube with and without TrkA inhibitor (TrkAi) infiltration. Our study revealed significant improvement in motor nerve fiber regeneration and function, in assessment of vocal fold movement, myelinated nerve regeneration, compound muscle action potential, and prevention of laryngeal muscle atrophy. Retrograde labeling demonstrated fewer labeled neurons in the vagus ganglion, which confirmed reduced misdirected regeneration among motor and sensory fibers, and a change in distribution of the labeled neurons in the nucleus ambiguus. Our study demonstrated that TrkAi have a strong potential for clinical application in the treatment of RLN injury.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1

Assessment of left arytenoid mobility at 14 weeks after surgery. (a) Images of arytenoid angles in the CLOSE phase (maximal adducted position) and the OPEN phase (maximal abducted position). The baseline was defined as a horizontal line through the intersection of the left and right arytenoid lines in the CLOSE phase. The difference between the maximal adducted (α) and abducted (β) angles was defined as the arytenoid motion angle. Blue line: maximal abducted arytenoid line, black line: baseline. (b) Representative findings of vocal fold mobility. The fixed case (left columns, arytenoid motion angle < 1°) (Video 1) and the recovered case (right columns, arytenoid motion angle > 5°) (Video 2) of the left arytenoid cartilage are shown. The upper columns are in the CLOSE phase and the lower columns are in the OPEN phase. (c) Degree of arytenoid motion angle in the PGA-C Control and the TrkAi/PGA-C. In left arytenoid motion angles, there was a significant difference between the two groups. *p < 0.05.

Figure 2
Figure 2

Electromyography (EMG) of the posterior cricoarytenoid (PCA) muscles 15 weeks after the procedure. (a,b) Representative EMG of the PCA muscles under stimulation of left RLNs of PGA-C Control (a) and TrkAi/PGA-C (b). Red waves were stimulated at the point 5 mm proximal from the surgical site. Blue waves were stimulated at the point 5 mm distal from the surgical site. (c) The motor nerve conduction velocities (MNCVs) of the right RLN (untreated-control side) and left RLN (treated side) were assessed in PGA-C Control and TrkAi/PGA-C. The normal control and treated sides were compared in both groups, but there was no significant difference. ns = p > 0.05 (Student’s t test). (d) The compound muscle action potentials (CMAPs) in PGA-C Control and TrkAi/PGA-C were compared by determining the treated/untreated CMAP ratio. There was a significant difference between the two groups. *p < 0.05. (e,f) The resting potentials of both right and left PCA muscles in PGA-C (e) and TrkAi/PGA-C (f). Upper red waves were the resting potential of the right PCA muscle and lower blue waves were the left PCA muscle. (e) In PGA-C Control, the resting potential waves of left PCA were not synchronized with right waves. (f) In PGA-C/TrkAi, only two cases showed resting potential wave synchronized with the right-side wave.

Figure 3
Figure 3

Electron microscopic findings of regenerated recurrent laryngeal nerves (RLNs). (a–f) Transverse sections of RLN distal to the treated site. (a,d) Normal control RLN (right RLN). (b,e) Left RLN in the PGA-C Control. (c,f) Left RLN in the TrkAi/PGA-C. A larger number of thicker myelinated fibers were observed in TrkAi/PGA-C (c,f) than in PGA-C Control (a,e). The scale bars are 20 µm for Panels a–c and 2 nm for d–f. (g) In the histogram of the diameters of myelinated axons, the distribution of axon diameters was compared in the untreated-control side (right RLN, white bar) and treated side (left RLN) of PGA-C Control (black bar) and TrkAi/PGA-C (gray bar). The distribution of axon diameters in TrkAi/PGA-C had shifted to the right more than in the PGA-C Control.

Figure 4
Figure 4

Assessment of thyroarytenoid (TA) muscle atrophy in PGA-C Control and TrkAi/PGA-C. (a,b) TA muscles in representative longitudinal sections stained with H&E. Scale bar is 500 µm. Arrowhead: arytenoid cartilage, *, TA muscle; #, lateral cricoarytenoid muscle. (a) PGA-C control. (b) TrkAi/PGA-C. (c–e) Images of TA muscles at a higher magnification. Scale bar is 50 µm. (c) Right TA muscle (untreated-control side). (d) Left TA (treated side) in PGA-C control. (e) Left TA (treated side) in TrkAi/PGA-C. Apparent atrophy of TA muscles was not observed between the treated and untreated sides in both groups. (f) The quantitative analysis did not show significant atrophic changes in treated/untreated ratio (%) of the area of the entire TA muscles. ns = p > 0.05.

Figure 5
Figure 5

Assessment of retrograde labeling in ganglions of the vagus and nucleus ambiguus. (a) Harvested left ganglion of the vagus. PB: pharyngeal branch, SL: superior laryngeal nerve, IX: glossopharyngeal nerve, X: main trunk of the vagus nerve, XI: spinal accessory nerve. (b) Neurons in the ganglion of the vagus (surrounded by two broken lines) labeled with Fast Blue (FB) (arrowhead) and diamidino yellow dihydrochloride (DY) (arrow). The scale bar is 50 µm. (Below B) FB dye labeled the cytoplasm blue. DY dye labeled the nucleus yellow. (c) Comparison of the number of labeled neurons in the ganglion of the vagus. Labeled cell number had significantly decreased in TrkAi/PGA-C. *p < 0.05. (d) Triple-labeled neurons with FB, DY (FB/DY), and for ChAT immunofluorescence in the nucleus ambiguus. The arrows indicate DY labeled nuclei. The arrowheads indicate an FB labeled cytoplasm. In the ChAT-positive neurons, FB and/or DY labeled neurons (Merge) were counted. The scale bar is 20 µm. (e) The rostrocaudal distribution of labeled neurons in the nucleus ambiguus. The distribution of FB (injected into PCA muscles) labeled motor neurons from the obex had significantly shifted to a rostral location in TrkAi/PGA-C. There was no significant difference in the distribution of DY and FB + DY labeled neurons. FB + DY: FB and DY double-labeled neurons, **p < 0.01.

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