Temporal expression of Laminin-111 in the developing rat larynx.

Laminin-111 is a basement membrane protein that participates in motor innervation and reinnervation. During axonal pathfinding, laminin-111 interacts with netrin-1 (NTN1) and changes its attractant growth cone properties into repulsion. While previous models of recurrent laryngeal nerve (RLN) transection show increased Laminin-111 and NTN1 production after injury, developmental expression in the larynx has not been defined. This study investigates the expression of laminin-111 in laryngeal muscles during primary laryngeal innervation of Sprague Dawley rats. Adult larynges and embryos were sectioned for immunohistochemistry with ßIII-Tubulin, laminin subunit α-1 (LAMA1), NTN1, and α-bungarotoxin. Sections were processed for single-molecule inexpensive RNA fluorescence in situ hybridization analysis of LAMA1 mRNA. LAMA1 expression increased in all intrinsic laryngeal muscles, except the medial thyroarytenoid (MTA), at E20.5. At E20.5 there was increased expression in the lateral thyroarytenoid (LTA) and posterior cricoarytenoid (PCA) compared to the MTA. NTN1 upregulation was limited to the LTA and lateral cricoarytenoid (LCA) at E16.5 without any increase in the MTA or PCA. LAMA1 and NTN1 expression did not strictly follow expected patterns relative to the known timing of innervation and does not appear to be acting similarly to its role following RLN injury. These differences between developmental and post-injury innervation provide targets for investigations of therapeutics after nerve injury.

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

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.

The injury of recurrent laryngeal nerve and thyroid regeneration after irreversible electroporation ablation of most part of thyroid gland-an experimental study on swine model.

To study the thyroid regeneration and injury of recurrent laryngeal nerve after irreversible electroporation (IRE). 12 pigs were divided into three groups: six pigs underwent IRE, other pigs were used as controls. IRE was performed near tracheoesophageal groove, to ablate most part of thyroid gland. Parathyroid and thyroid function, recurrent laryngeal nerve injury and thyroid computed tomography (CT) imaging were regularly investigated. The histopathology results were analyzed to detect thyroid regeneration. Masson's trichrome method for collagen and immunohistochemistry were performed for Soluble protein-100 (S100) and neurofilaments on nerve section. In IRE group, there were no symptoms of recurrent laryngeal nerve-related injury. No abnormalities of recurrent laryngeal nerve were shown on hematoxylin-eosin (HE) staining, Masson's trichrome staining, Neurofilament (NF) staining and S100 staining. There were no significant changes for thyroid and parathyroid function in all pigs. Immediately after IRE, CT showed hypoattenuation in the ablated thyroid gland and it became swelling. 14 days after IRE, thyroid CT showed hetergenous attenuation in the electroporation zone, and the size and attenuation of thyroid gland were normal after two months. There was cell apoptosis in the thyroid gland after IRE. Seven and 14 days after IRE, there was fragmentation of nucleus within the follicle, and some follicles were empty. Two months later, complete regeneration of thyroid tissue was shown. IRE was shown to be both effective and safe with complete regeneration of thyroid tissue and preservation of the function and structure of the recurrent laryngeal nerve.

Expression of trophic factors receptors during reinnervation after recurrent laryngeal nerve injury.

OBJECTIVE: An injury of the recurrent laryngeal nerve (RLN) triggers axonal regeneration but results in a poor functional recovery. Netrin-1 and glial cell-derived neurotrophic factor (GDNF) expression are up-regulated in laryngeal muscles during RLN regeneration, but the role of their receptors produced in the nucleus ambiguus is unknown. The aim of this work was to determine the timing of the production of Netrin-1 and GDNF receptors during RLN regeneration and correlate this with the previously identified timing of up-regulation of their trophic factors in the laryngeal muscles. STUDY DESIGN: Laboratory experiment with rat model. METHODS: The right RLN was transected and dextran amine tracer applied. At 7, 14, and 21 days postinjury (DPI), brainstems were removed and harvested. Immunostaining was performed for Netrin-1 (deleted in colorectal carcinoma [DCC], UNC5A) and GDNF receptors (rearranged during transfection [Ret], glycosylphosphatidylinositol-linked cell surface receptors [GFRα1, GFRα2, GFRα3]). The timing and type of receptor production relative to injury as well as their position in the nucleus ambiguus was analyzed. RESULTS: Netrin-1 UNC5A receptors were minimal in the nucleus ambiguus during RLN regeneration. DCC, the receptor that plays an attract role, was immunopositive from 7 to 21 DPI. All GDNF receptors, except GFRα2, were clearly positive from 7 to 14 DPI. No differences of production were observed according to the position of the motor neurons in the nucleus ambiguus. CONCLUSION: An injury of the RLN leads to a higher production of Netrin-1 DCC and GDNF receptors in the nucleus ambiguus. The timing of receptor production is similar to up-regulation of their trophic factors in the laryngeal muscles. LEVEL OF EVIDENCE: NA. Laryngoscope, 129:2537-2542, 2019.

Polysialylated Neural Cell Adhesion Molecule Supports Regeneration of Neurons in the Nucleus Ambiguus After Unilateral Recurrent Laryngeal Nerve Avulsion in Adult Rats.

OBJECTIVES: A correlation appears to exist between the expression of the polysialic acid neural cell adhesion molecule (PSA-NCAM) and repair in central nervous system (CNS) diseases. However, the expression of PSA-NCAM in the CNS after peripheral nerve injury remains unclear. This study aimed to evaluate the expression of PSA-NCAM in the ipsilateral nucleus ambiguus (NA) after unilateral recurrent laryngeal nerve (RLN) avulsion. MATERIALS AND METHODS: The left RLN of adult Sprague Dawley rats were avulsed. The expression of PSA-NCAM, PSA-NCAM/NeuN, and PSA-NCAM/Tuj1 in the brain stem was investigated using immunohistochemistry. The results were subjected to one-way analysis of variance followed by the Tukey post hoc test for statistical analyses. RESULTS: PSA-NCAM-positive and PSA-NCAM/NeuN and PSA-NCAM/Tuj1 double-labeled positive cells were observed 7 days post injury in the ipsilateral NA. PSA-NCAM/NeuN and PSA-NCAM/Tuj1 double-labeled cells were observed at 21 and 7 days post injury, respectively. PSA-NCAM/NeuN double-labeled cells were also found in the contralateral NA. CONCLUSIONS: After unilateral avulsion of the RLN, the expression of PSA-NCAM in the ipsilateral NA was correlated with the proliferation and the differentiation of neural cells. PSA-NCAM expression may be used as a predictor of the initiation of repair in neural cells.

Transition of myosin heavy chain isoforms in human laryngeal abductors following denervation.

The objective of this study was to investigate the myofiber subtype transition of human posterior cricoarytenoid (PCA) muscle after the injury to recurrent laryngeal nerve (RLN). PCA muscle specimens were obtained from 38 bilateral vocal fold paralysis patients underwent arytenoidectomy. According to the duration of RLN injury, all the cases were divided into five denervation groups: 6-12 months, 1-2, 2-3, 3-6, and >6 years. The normal PCA muscles from total laryngectomy patients were chosen as controls. Immunofluorescence was adopted to detect the expression level of myosin heavy chain (MHC)-I and MHC-II in PCA muscle. Quantitative real-time PCR was also used to assess the transcriptional level of MHC subtypes (MHC-I, MHC-IIa, MHC-IIb, MHC-IIx, embryonic-MHC, and peri-natal-MHC). Immunofluorescence showed that MHC-I-positive myofibers in denervation groups were much lower than control group, respectively, while MHC-II-positive myofibers were significantly higher than control group (P < 0.05). With the extension of denervation, the number of MHC-I-positive myofibers gradually decreased, while MHC-II gradually increased and peaked in 1- to 2-year group. Transcriptional level of MHC-I, MHC-IIa, and MHC-IIb in denervation groups significantly down-regulated compared with the control (P < 0.05), respectively. However, MHC-IIx, embryonic-MHC, and peri-natal-MHC significantly up-regulated in all denervation groups, and the highest level was in 1- to 2-year denervation group. Data from the present study demonstrated that the maximum transition of MHC subtypes in human PCA muscles occurred in 1-2 years after denervation, suggesting that laryngeal reinnervation before the occurrence of irreversible transition of MHC subtypes could maintain the structural integrity of laryngeal PCA muscles.

GFAP immunoreactivity within the rat nucleus ambiguus after laryngeal nerve injury.

Changes that occur in astroglial populations of the nucleus ambiguus after recurrent (RLN) or superior (SLN) laryngeal nerve injury have hitherto not been fully characterised. In the present study, rat RLN and SLN were lesioned. After 3, 7, 14, 28 or 56 days of survival, the nucleus ambiguus was investigated by means of glial fibrillary acidic protein (GFAP) immunofluorescence or a combination of GFAP immunofluorescence and the application of retrograde tracers. GFAP immunoreactivity was significantly increased 3 days after RLN resection and it remained significantly elevated until after 28 days post injury (dpi). By 56 dpi it had returned to basal levels. In contrast, following RLN transection with repair, GFAP immunoreactivity was significantly elevated at 7 dpi and remained significantly elevated until 14 dpi. It had returned to basal levels by 28 dpi. Topographical analysis of the distribution of GFAP immunoreactivity revealed that after RLN injury, GFAP immunoreactivity was increased beyond the area of the nucleus ambiguus within which RLN motor neuron somata were located. GFAP immunoreactivity was also observed in the vicinity of neuronal somata that project into the uninjured SLN. Similarly, lesion of the SLN resulted in increased GFAP immunoreactivity around the neuronal somata projecting into it and also in the vicinity of the motor neuron somata projecting into the RLN. The increase in GFAP immunoreactivity outside of the region containing the motor neurons projecting into the injured nerve, may reflect the onset of a regenerative process attempting to compensate for impairment of one of the laryngeal nerves and may occur because of the dual innervation of the posterior cricoarytenoid muscle. This dual innervation of a very specialised muscle could provide a useful model system for studying the molecular mechanisms underlying axonal regeneration process and the results of the current study could provide the basis for studies into functional regeneration following laryngeal nerve injury, with subsequent application to humans.