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.

Animal Study to Evaluate the Effect of Carbon Dioxide Insufflation on Recurrent Laryngeal Nerve Function in Transoral Endoscopic Thyroidectomy.

Data with regard to potential recurrent laryngeal nerve (RLN) compromise caused by intra-neck CO2 insufflation during transoral endoscopic thyroidectomy vestibular approach (TOETVA) are missing. RLN electromyographic (EMG) profiles, metabolic and hemodynamic parameters (oxygen saturation, heart rate, blood pressure, experimental time, CO2 partial pressure, pH, O2 partial pressure), central venous pressure (CVP), airpocket temperature and pressure were recorded in a TOETVA animal model. Twelve pigs were randomly divided into different groups according to increasing CO2 insufflation pressures. Nerves segments were then collected for histopathology. Significant variation of metabolic and hemodynamic parameters were registered when CO2 insufflation pressures increased x3 and x5 the baseline parameters. Combined EMG amplitude drop and latency increase also were documented. There was no significant change in the intraluminal temperature. RLNs structure were preserved with normal axons, no fibrosis, and no vacuolization and without loss of myelinated fibers during the experiment. RLN EMG profiles (but not histology) were altered when CO2 insufflation pressures increased.

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.

A new strategy for the surgical management of RLN infiltrated by well-differentiated thyroid carcinoma.

Well-differentiated thyroid carcinoma (WDTC) represents the most common endocrine malignancy. Despite excellent prognoses exceeding 90% in 10-year follow-up, there are clinically controversial issues. One of these is extrathyroidal tumour extension invading recurrent laryngeal nerve (RLN). The spread outside of the thyroid parenchyma and invasion to the surrounding structures, classified as always T4a, are the most important negative prognostic factor for the WDTC. Conversely, resection of the RLN leads to vocal cord paralysis with hoarseness, possible swallowing problems, and finally decreased quality of life. We propose a new algorithm for intraoperative management based on the MACIS classification, which would allow swift status evaluation pre/intraoperatively and consider a possibility to preserve the infiltrated RLN without compromising an oncological radicality. In the case of a preoperative vocal cord paralysis (VCP) and confirmation of the invasive carcinoma, a resection of the RLN and the nerve graft reconstruction are indicated. Preoperatively, unaffected vocal cord movement and intraoperatively detected RLN infiltration by the invasive WDTC require an individual assessment of the oncological risk by the proposed algorithm. Preservation of the infiltrated RLN is oncologically acceptable only in specific groups of patients of a younger age with a minor size of primary tumour.

Modulation of nerve fibers in the rat thyroarytenoid muscle following recurrent laryngeal nerve injury.

CONCLUSION: Regeneration of nerve fibers in the thyroarytenoid (TA) muscle occurred actively after damage on the recurrent laryngeal nerve (RLN) compared with the vagus nerve (VN). However, remyelination did not occur after damage on the RLN. OBJECTIVES: To determine the regeneration process of nerve fibers in the TA muscle following transection and immediate anastomosis of the RLN or VN. METHODS: Three types of animal model were prepared: an RLN anastomosis model (RLNa), a VN anastomosis model (VNa), and a peroneal nerve anastomosis model (PNa). Animals were sacrificed at five time points following the procedure. The modulation of axons, myelin sheaths, Schwann cells (SCs), nerve terminals (NTs), and acetylcholine receptors (AchRs) in the TA or tibialis anterior muscles was examined by immunohistochemical analysis. The ratios of the expression areas in axons, myelin sheaths, and SCs, and the number of NTs and AchRs in the treated (T) and untreated (U) sides (T/U) were evaluated. RESULTS: At 18 weeks, the T/U ratios of expression in RLNa, VNa, and PNa were 68.5, 0, and 100.4%, respectively, in axons; 0, 0, and 97.6% in myelin sheaths; 53.7, 0, and 93.6% in SCs; 61.0, 0, and 96.4% in NTs; and 99.4, 67.0, and 101.2% in AchRs.

[The short duration functional electrical stimuli do not cause mRNA expression change of K(ATP) channel and acute inflammation cytokine in recurrent laryngeal nerve].

OBJECTIVE: To investigate whether the inflammation response and K(ATP) channel damage happened because of acute injury after functional electrical stimuli (FES) act on the recurrent laryngeal nerve (RLN). METHOD: Thirty rabbits were divided into 3 groups by 10 rabbits for each group. The unilateral RNL were stimulated by FES with cuff electrode in first group. There was electrode on the RNL without FES in second group. The third group was normal control. The electrical generator and isolation unit provide electrical stimulation to the RLN at 50 Hz, with 4 mA current, for 2ms pulse duration. The total train duration was 5 seconds (2 seconds on; 3 seconds off). The RLN were collected in 1 hour after 3 hours electrical stimuli. We measured mRNA expression of injury acute response inflammation cytokine IL-10, TGF-beta1, TNF-alpha and K(ATP) Subunit protein Kir6.1, Kir6.2, SUR2AB, using real-time reverse transcribed-polymerase chain reaction. PCR products were verified by electrophoresis in agarose gels. We investigated the vocal synchronic adductive movement with RLN FES by endoscopy. We investigated the nerve local RNL morphological burn. Data were analyzed with the One-way ANOVA analysis of SPSS version 13.0 for Windows. RESULT: There were no significance difference among three groups in mRNA expression of inflammation cytokines IL-10, TGF-beta1, TNF-alpha and K(ATP) channel protein Kir6.1, Kir6., SUR2AB. We did not find the nerve morphological burn in all rabbits RLN. The vocal synchronic adductive movement were normal during stimuli time. CONCLUSION: The short duration functional electrical stimuli do not cause mRNA expression change of K(ATP) channel and acute inflammation cytokine in recurrent laryngeal nerve.

Dysfunction of the recurrent laryngeal nerve and the potential of gene therapy.

Injury to the recurrent laryngeal nerve causes vocal fold paresis or paralysis resulting in poor voice quality, and possibly swallowing dysfunction and/or airway compromise. Injury can occur as part of a neurodegenerative disease process or can be due to direct nerve trauma or tumor invasion. Management depends upon symptoms, the cause and severity of injury, and the prognosis for recovery of nerve function. Surgical treatment techniques can improve symptoms, but do not restore physiologic motion. Gene therapy may be a useful adjunct to enhance nerve regeneration in the setting of neurodegenerative disease or trauma. Remote injection of viral vectors into the recurrent laryngeal nerve is the least invasive way to deliver neurotrophic factors to the nerve's cell bodies within the nucleus ambiguus, and in turn to promote nerve regeneration and enhance both nuclear and nerve survival. The purpose of this review is to discuss the potential role for gene therapy in treatment of the unsolved problem of vocal fold paralysis.

Electrophysiological characterization of vagal afferents relevant to mucosal nociception in the rat upper oesophagus.

Emerging evidence indicates a nociceptive role of vagal afferents. A distinct oesophageal innervation in the rat, with muscular and mucosal afferents travelling predominantly in the recurrent (RLN) and superior laryngeal nerve (SLN), respectively, enabled characterization of mucosal afferents with nociceptive properties, using novel isolated oesophagus-nerve preparations. SLN and RLN single-fibre recordings identified 55 and 14 units, respectively, with none conducting faster than 8.7 m s(-1). Mucosal response characteristics in the SLN distinguished mechanosensors (n = 13), mechanosensors with heat sensitivity (18) from those with cold sensitivity (19) and a mechanoinsensitive group (5). The mechanosensitive fibres, all slowly adapting, showed a unimodal distribution of mechanical thresholds (1.4-128 mN, peak approximately 5.7 mN). No difference in response characteristics of C and Adelta fibres was encountered. Mucosal proton stimulation (pH 5.4 for 3 min), mimicking gastro-oesophageal reflux disease (GORD), revealed in 31% of units a desensitizing response that peaked around 20 s and faded within 60 s. Cold stimulation (15 degrees C) was proportionally encoded but the response showed slow adaptation. In contrast, the noxious heat (48 degrees C) response showed no obvious adaptation with discharge rates reflecting the temperature's time course. Polymodal (69%) mucosal units, > 30% proton sensitive, were found in each fibre category and were considered nociceptors; they are tentatively attributed to vagal nerve endings type I, IV and V, previously morphologically described. All receptive fields were mapped and the distribution indicates that the posterior upper oesophagus may serve as a 'cutbank', detecting noxious matters, ingested or regurgitated, and triggering nocifensive reflexes such as bronchoconstriction in GORD.

Distribution of class I and II human leukocyte antigens in the larynx.

OBJECTIVE: To examine the antigenic distribution of human leukocyte antigens (HLA) of the human larynx. STUDY DESIGN AND SETTING: Twelve human larynges were examined for Class I (HLA-A, -B, -C) and Class II (HLA-DR) histocompatibility antigens using mouse monoclonal antibodies in an indirect immunoperoxidase assay. Structures of the larynx and surrounding tissues were examined and given a semiquantitative score based on HLA Class I and II expression. RESULTS: The mucosal surface epithelium of the larynx stains 2+ or stronger for HLA Class I antigens and 1+ for Class II antigens. The deeper submucosal glands stain 1+ for Class I antigens and 2+ or stronger for Class II antigens. Thyroid cartilage showed 2+ or stronger staining of the chondrocytes for Class I antigens only. Thyroid follicular cells also stain only for Class I antigens. Perichondrium and Schwann cells of nerves stain stronger for Class I antigens than Class II antigens. Cartilage matrix, muscle cells, and axons of nerves do not stain for either class of antigens. Endothelium stains 3+ for both classes of antigens. CONCLUSIONS: The detailed distribution of major transplantation antigens in the human larynx is elucidated. Class II antigens implicated as initiators of organ transplant rejection were primarily found in 6 areas: mucosal surface epithelium, submucosal glands, ducts, vascular endothelium, perichondrium, and Schwann cells of nerves. The relevance of these findings to the initiation and detection of laryngeal allograft graft rejection is discussed.

Adenoviral GDNF gene transfer enhances neurofunctional recovery after recurrent laryngeal nerve injury.

To assess the possibility of gene therapy for recurrent laryngeal nerve (RLN) injury, we examined functional and histological recovery after glial cell line-derived neurotrophic factor (GDNF) gene transfer in a rat RLN crush model. Adenoviral vector encoding beta-galactosidase gene (AxCALacZ) or human GDNF gene (AxCAhGDNF) was injected into the crush site of the RLN. Neurons in the nucleus ambiguus on the crushed side were labeled with X-gal or GDNF immnohistochemistry after AxCALacZ or AxCAhGDNF injection. Reverse transcription-polymerase chain reaction analysis revealed expression of human GDNF mRNA transcripts in brainstem tissue containing the nucleus ambiguus on the crushed side after AxCAhGDNF injection. Animals injected with AxCAhGDNF displayed significantly improved motor nerve conduction velocity of the RLN and recovery rate of vocal fold movement at 2 and 4 weeks after treatment as compared to controls. AxCAhGDNF-injected animals showed a significantly larger axonal diameter and improved remyelination in crushed RLN as compared to controls. Adenoviral GDNF gene transfer may thus promote laryngeal function recovery after RLN injury. Inoculation of adenoviral vector containing the GDNF gene at the site of damage soon after nerve injury may assist patients with laryngeal paralysis caused by nerve injury during head and neck surgery.

Enhanced expression of neuronal nitric oxide synthase and phospholipase C-gamma1 in regenerating murine neuronal cells by pulsed electromagnetic field.

Pulsed electromagnetic field (PEMF) has been shown to improve the rate of peripheral nerve regeneration. In the present study we investigated the expression of neuronal nitric oxide synthase (nNOS) and phospholipase C-gamma1 (PLC-gamma1) in regenerating rat laryngeal nerves during the exposure to PEMF after surgical transection and reanastomosis. Axons were found to regenerate into the distal stump nearly twice faster in PEMF-exposed animals than in the control. Consistently, motor function was better recovered in PEMF-treated rats. The expression of nNOS and PLC-gamma1 was highly enhanced in the regenerated nerves.

Thoracic vagus section distal to the recurrent laryngeal nerve reduces substance. P-immunoreactive innervation in the rat bronchial tree.

The vagal nerve trunk in the mediastinum of mammals divides into two main branches, the thoracic vagus nerve and the recurrent laryngeal nerve, in which the sensory nerve axons are largely involved in neurogenic inflammation in the tracheobronchial airways. A previous study demonstrated that cutting the right-side thoracic vagus nerve but not the recurrent laryngeal nerve inhibited capsaicin-induced neurogenic inflammation in the right bronchial tree of the rat. The effect of left thoracic vagus nerve section is still not known. The main purpose of the present study was to investigate the effect of sectioning the right or left thoracic vagus nerve on the innervation density of substance P-immunoreactive axons in bilateral bronchial trees. Following nerve degeneration, the whole mounts of airway tissues were processed with substance P immunohistochemistry. Denervation of either thoracic vagus nerve reduced the innervation density of axons by 38-71% in different parts of the ipsilateral bronchial tree. The effect of right recurrent laryngeal nerve section was less specific; the innervation density was reduced by 21-39% in the trachea and bronchi of both sides. Capsaicin-induced neurogenic plasma leakage was decreased in the left mainstem bronchus and lobar bronchi after left thoracic vagus nerve section. It is concluded that the thoracic vagus nerve largely contributed to the sensory innervation in the ipsilateral bronchial airways and modulated their functions.