Co-transplantation of Schwann cells and neural stem cells in the laminin-chitosan-PLGA nerve conduit to repair the injured recurrent laryngeal nerve in SD rats.

The objective of this paper is to investigate the possibility and efficacy of recurrent laryngeal nerve repair by transplantation of co-cultured Schwann cells and neural stem cells (NSCs) in laminin-chitosan-poly-lactic-co-glycolic acid (laminin-chitosan-PLGA) nerve conduits in rats. A laminin-chitosan-PLGA conduit was used in a rat recurrent laryngeal nerve transection model. The rat recurrent laryngeal nerve was dissected to generate a 5 mm defect. Then, a laminin-chitosan-PLGA nerve conduit with or without Schwann cells and NSCs in the lumen was transplanted into the defect. A total of 96 female rats were randomised into six groups: co-culture of NSCs and Schwann cells in the nerve conduit group (CO), Schwann cells only in the nerve conduit group (SC), neural stem cells only in the nerve conduit group (NSC-only), nerve conduit group (null), autologous nerve graft group (autograft) and sham operation group (sham). Regenerated nerves were evaluated by histological and functional assessment at 8 and 12 weeks after surgery. The diameter and area of the regenerated myelin sheath, as well as the secretion of brain-derived neurotrophic factor and glial cell-derived neurotrophic factor in laryngeal muscle or regenerated nerve tissue in the CO group, were significantly better than they were in the SC, NSC-only and null groups (all P values < 0.05). Immunofluorescence showed that the CO group had significantly more neurofilament-200 immunoreactive and S-100 immunoreactive fibres than the SC, NSC-only and null groups (all P values < 0.05). The performance of the CO groups and autograft groups was found to be similar by laryngoscopy. Arytenoid cartilage motion recovery in these two groups was significantly better than it was in the other groups (all P values < 0.05). Our results indicated that co-culture of Schwann cells and NSCs in laminin-chitosan-PLGA conduits might promote injured nerve regeneration. This method might be a promising alternative for defective nerve repair.

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.

Recurrent laryngeal nerve regeneration using a self-assembling peptide hydrogel.

OBJECTIVES/HYPOTHESIS: To regenerate defected recurrent laryngeal nerves (RLNs), various methods have been developed. However, no consistently effective treatments are currently available because of their insufficient functional recovery. RADA16-I, a self-assembling peptide used clinically as a hemostat, reportedly supports neurite outgrowth and functional synapse formation in vitro. The purpose of this study was to investigate the effect of RADA16-I hydrogels on transected RLNs in rats. STUDY DESIGN: Animal experiments with controls. METHODS: Fifteen adult rats were divided into the following three groups: RADA16-I (+), RADA16-I (-), and neurectomy. A 6-mm gap of the left RLN was bridged using an 8-mm silicone tube in the RADA16-I (-) and RADA16-I (+) groups. Subsequently, RADA16-I hydrogel was injected into the tube in the RADA16-I (+) group. The surgical incisions were closed without any further treatment in the neurectomy group. After 8 weeks, laryngoscopy and electrophysiological and histological examinations were performed to evaluate the effect of RADA16-I on nerve regeneration and thyroarytenoid muscle atrophy. RESULTS: Although most rats in the three groups exhibited no improvements of their vocal fold movement, partial recovery was observed in one rat in the RADA16-I (+) group. The neurofilament-positive areas and the number of myelinated nerves in the RADA16-I (+) group were significantly higher than in the RADA16-I (-) group. The area of the left thyroarytenoid muscle in the RADA16-I (+) group was significantly larger than that of the neurectomy group. CONCLUSIONS: Our results suggested that RADA16-I hydrogel was effective for RLN regeneration. LEVEL OF EVIDENCE: NA Laryngoscope, 130:2420-2427, 2020.

Regenerative Effects of Basic Fibroblast Growth Factor on Restoration of Thyroarytenoid Muscle Atrophy Caused by Recurrent Laryngeal Nerve Transection.

OBJECTIVES: Vocal fold atrophy following unilateral vocal fold paralysis is caused by atrophy of the thyroarytenoid (TA) muscle and remains a challenge. Medialization procedures are popular treatment options; however, hoarseness often remains due to the reduction in mass or tension of the TA muscle. Therefore, in addition to medialization procedures, TA muscle reinnervation is desirable. In vivo studies have shown the potential for basic fibroblast growth factor (bFGF) to affect muscular and nerve regeneration. The present study aimed to examine the regenerative effects of bFGF on restoration of TA muscle atrophy caused by recurrent laryngeal nerve transection. STUDY DESIGN: Prospective animal experiments with controls. METHODS: TA muscle atrophy was induced by unilateral transection of the recurrent laryngeal nerve. One month after transection, different doses (200 ng, 100 ng, 10 ng) of bFGF in 50 µL were repeatedly injected into the TA muscle four times with an interval of 1 week between injections. Saline only was injected in the sham group. Larynges were harvested for histologic and immunohistochemical examination 4 weeks after the final injection. RESULTS: The cross-sectional TA muscle area was significantly larger in the bFGF-treated groups compared with the sham-treated groups. Immunohistochemistry indicated that bFGF significantly increases the number of neuromuscular junctions and satellite cells in the TA muscle. CONCLUSIONS: These results suggest that local application of bFGF to the TA muscle may improve TA muscle atrophy caused by recurrent laryngeal nerve paralysis. Furthermore, bFGF may have regenerative effects on both nerves and muscles.

Failed Vocalis Muscle Monitoring During Thyroid Surgery Resulting From Residual Muscle Relaxation.

Postoperative paralysis of the vocal cords resulting from intraoperative surgical injury of the recurrent laryngeal nerve is one of the most serious complications of elective thyroid surgery. The chance of injury is reduced by using intraoperative neuromonitoring (IONM). This educational report describes a case of IONM failure resulting from residual muscle relaxation that was restored by administration of sugammadex. It discusses possible pharmacology explanations and provides recommendations for how to prevent this situation in daily practice.

Effect of laryngotracheal topical anesthesia on recurrent laryngeal nerve monitoring during thyroid Surgery.

STUDY OBJECTIVE: Intraoperative neuromonitoring of the recurrent laryngeal nerve (RLN) is often used as an adjunct for RLN identification and preservation during thyroidectomies. Laryngotracheal anesthesia (LTA) with topical lidocaine reduces coughing upon emergence from anesthesia and in the immediate postoperative period; however, its use is prohibited with concerns that it could decrease the sensitivity of the intraoperative neuromonitoring. We hypothesize that there is no difference in measurements of nerve conduction made before and after LTA administration. DESIGN: An observational study in which all patients were subjected to LTA administration was conducted. Recurrent laryngeal nerve threshold currents were measured before and after the intervention. SETTING: Tertiary medical center operating room. PATIENTS: Eighteen patients (total of 25 nerves at risk) with American Society of Anesthesiologists classes 1 to 3 undergoing thyroid surgery. INTERVENTIONS: After the thyroid was removed and threshold currents at the RLN were obtained, LTA with endotracheal lidocaine was applied on the left and right side of the in situ endotracheal tube (2 cc of 4% lidocaine per side). Threshold currents were reassessed at 5 and 10 minutes after LTA administration. MEASUREMENTS: Threshold currents (minimum stimulus current applied to the RLN required to generate a discernible electromyographic response at the vocal cords) were recorded along the RLN for a baseline at 5 and 10 mm from the insertion point of the RLN into the larynx. Threshold currents were reassessed at the same 2 positions on the RLN at 5 and 10 minutes after LTA administration. Differences in mean values, between threshold currents recorded at the 3 different times, at 2 positions on the RLN, were used to compare effects of LTA on nerve conduction. MAIN RESULTS: There were no statistically significant differences when comparing threshold currents before and after LTA administration. CONCLUSIONS: Laryngotracheal anesthesia had no significant effect on RLN nerve conduction in the period assessed.

Cricothyroid Muscle Botulinum Toxin Injection to Improve Airway for Bilateral Recurrent Laryngeal Nerve Paralysis, A Case Series.

Bilateral vocal fold paralysis most commonly results from iatrogenic trauma to the recurrent laryngeal nerve during surgical procedures in the anterior neck. Patients may require tracheostomy because of acute or gradual onset of dyspnea and airway compromise. The intralaryngeal injection of Botox has been considered as a possible therapy for these airway symptoms of bilateral vocal fold paralysis. Chronic unopposed activity of intact cricothyroid muscles could potentially result in gradual medialization of the vocal folds in patients with bilateral recurrent laryngeal nerve paralysis. This case series describes three patients who successfully underwent injections of botulinum toxin into the bilateral cricothyroid muscles to offer sustained relief of dyspnea resulting from bilateral vocal fold paralysis.

The effect of routine reversal of neuromuscular blockade on adequacy of recurrent laryngeal nerve stimulation during thyroid surgery.

Testing of the integrity of the recurrent laryngeal nerve during thyroid surgery has become routine practice for many surgeons to aid dissection and minimise the chance of inadvertent nerve injury. We hypothesised that routine reversal of an intermediate-acting, non-depolarising neuromuscular blocking agent would improve conditions for stimulation of the recurrent laryngeal nerve. We conducted a single-centre, randomised, double-blind placebo-controlled trial of patients undergoing thyroid surgery by the same surgeon. After randomisation, the participants received either neostigmine 2.5 mg with glycopyrrolate 0.4 mg or placebo, at 30 minutes after induction of anaesthesia and administration of 0.4 mg/kg of atracurium. The primary outcome was the subjective assessment by the surgeon as to whether the neuromuscular function was adequate for stimulation of the recurrent laryngeal nerve using a neuromuscular integrity monitor (NIM). Time to NIM stimulation was 44.6 minutes in the placebo group and 41.4 minutes in the intervention group (P=0.268). Of the 21 patients who received the neuromuscular blockade reversal, 20 (95.2%) had adequate surgical conditions for NIM stimulation, compared to 9 out of 18 patients (50%) in the placebo group (P=0.002). Three of the ten patients (30%) with inadequate reversal showed no evidence of residual blockade assessed peripherally. The routine reversal of neuromuscular blockade at 30 minutes post induction appears to result in adequate surgical conditions for safe stimulation of the recurrent laryngeal nerve. Return of neuromuscular function at a peripheral site does not guarantee adequate laryngeal muscle function for use of the NIM.

Influence of intravenous anesthetics on neuromonitoring of the recurrent laryngeal nerve during thyroid surgery.

Limited reports are available in the literature on the impact of intravenous administration of anesthetics on laryngeal electromyographic (EMG) activity. The purpose of this study was to determine the influence of the two commonly used intravenous anesthetics (propofol and thiamylal) on EMG amplitude evoked from the recurrent laryngeal nerve (RLN) during thyroid surgery. A total of 40 patients were randomized to receive a bolus of propofol (0.5 mg/kg; n = 20) or thiamylal (1.5 mg/kg; n = 20) to increase anesthetic depth when the surgeon found patient movement intraoperatively. Evoked potentials were obtained before and every 1 minute after the administration of each agent for up to 5 minutes by stimulating the RLN. The magnitude of evoked potentials at each time point and hemodynamic response were compared within groups. The mean amplitude of evoked potentials did not change significantly after administration of either propofol or thiamylal (p > 0.05 within groups). Mean arterial pressure measured from 1 minute to 5 minutes was significantly lower in the propofol group than in the thiamylal group (p < 0.05). Heart rate measured within 5 minutes did not differ significantly within groups. Low dose of propofol (0.5 mg/kg) or thiamylal (1.5 mg/kg) did not affect EMG readings during neuromonitoring of the RLN in thyroid surgery. Our results show that thiamylal provides better hemodynamic stability than propofol, and is therefore a preferable agent to increase anesthesia depth and prevent further patient movement during intraoperative neuromonitoring.

Electromyographic study of differential sensitivity to succinylcholine of the diaphragm, laryngeal and somatic muscles: a swine model.

Neuromuscular blocking agents (NMBAs) might diminish the electromyography signal of the vocalis muscles during intraoperative neuromonitoring of the recurrent laryngeal nerve. The aim of this study was to compare differential sensitivity of different muscles to succinylcholine in a swine model, and to realize the influence of NMBAs on neuromonitoring. Six male Duroc-Landrace piglets were anesthetized with thiamylal and underwent tracheal intubation without the use of an NMBA. The left recurrent laryngeal nerve, the spinal accessory nerve, the right phrenic nerve and the brachial plexus were stimulated. Evoked potentials (electromyography signal) of four muscle groups were elicited from needle electrodes before and after intravenous succinylcholine bolus (1.0 mg/kg). Recorded muscles included the vocalis muscles, trapezius muscle, diaphragm and triceps brachii muscles. The onset time and 80% recovery of control response were recorded and analyzed. The testing was repeated after 30 minutes. The onset time of neuromuscular blocking for the vocalis muscles, trapezius muscle, diaphragm and triceps brachii muscle was 36.3 ± 6.3 seconds, 38.8 ± 14.9 seconds, 52.5 ± 9.7 seconds and 45.0 ± 8.2 seconds during the first test; and 49.3 ± 10.8 seconds, 40.0 ± 12.2 seconds, 47.5 ± 11.9 seconds and 41.3 ± 10.1 seconds during the second test. The 80% recovery of the control response for each muscle was 18.3 ± 2.7 minutes, 16.5±6.9 minutes, 8.1±2.5 minutes and 14.8±2.9 minutes during the first test; and 21.5±3.8 minutes, 12.5 ± 4.3 minutes, 10.5 ± 3.1 minutes and 16.4 ± 4.2 minutes during the second test. The sensitivity of the muscles to succinylcholine, ranked in order, was: the vocalis muscles, the triceps brachii muscle, the trapezius muscle and the diaphragm. We demonstrated a useful and reliable animal model to investigate the effects of NMBAs on intraoperative neuromonitoring. Extrapolation of these data to humans should be done with caution.

Electromyographic response persists after peripheral transection: endorsement of current concepts in recurrent laryngeal nerve monitoring in a porcine model.

BACKGROUND: Recurrent laryngeal nerve palsy is a serious complication of endocrine surgery to the neck. Permanent lesions are still occurring in about one in a hundred, despite standardized surgical approach to the nerve and the availability of recurrent laryngeal nerve monitoring. Intraoperative recurrent laryngeal nerve monitoring is based on the visual or acoustic registration of evoked electromyography of the laryngeal muscles. Primarily, it proves conductivity of the stimulated nerve segment towards the muscle, so that stimulation distal of the lesion should show persistent electromyographic response. METHODS: In a porcine model, an iatrogenic nerve lesion of the recurrent laryngeal nerve was set. Subsequently, the proximal and distal dissected nerve portion was stimulated and the evoked electromyographic response of the laryngeal muscles was recorded by needle and laryngeal surface electrodes. RESULTS: As expected, no signal was obtained from the proximal segment. Meanwhile, the distal segment showed unchanged amplitude of the electrophysiological response for the observation period of more than 1 h. CONCLUSION: This result demonstrated a remarkable pitfall for the neuromuscular monitoring at the recurrent laryngeal nerve: In the human surgical setting, this might have resulted in the false assumption of an anatomical intact nerve. The persistence of distal electromyographic conduction strengthens the proposal to stimulate the vagal nerve as the proximal portion of the nerve as a part of a systematic protocol.

Nimodipine improves reinnervation and neuromuscular function after injury to the recurrent laryngeal nerve in the rat.

OBJECTIVES: Injury of the recurrent laryngeal nerve (RLN) is associated with a high degree of neuronal survival, but leads to various levels of vocal fold motion impairment or laryngeal synkinesis, which has been attributed to misdirected reinnervation of the target muscles in the larynx or aberrant, competing reinnervation from adjacent nerve fibers. The aim of the present study was to evaluate the impact of the regeneration-promoting agent nimodipine on reinnervation and neuromuscular function following RLN crush injury. METHODS: Sixty adult rats were randomized into nimodipine-treated or untreated groups and then underwent RLN crush injury. Reinnervation of the posterior cricoarytenoid muscle (PCA) was assessed by electrophysiological examination, retrograde tracing of lower motor neurons before and after injury, and quantification of neuromuscular junctions in the PCA muscle. RESULTS: At 6 weeks after injury, the nimodipine-treated animals showed significantly enhanced neuromuscular function and also demonstrated a higher number of motor neurons in the brain stem that had reinnervated the PCA, compared to the untreated animals. The somatotopic organization of ambiguus motor neurons innervating the larynx was similar before injury and after reinnervation. CONCLUSIONS: Nimodipine improves regeneration and neuromuscular function following RLN injury in the adult rat, and could be of use in future strategies following RLN injury.

[Central projections of the rat recurrent laryngeal nerve]. / Proyecciones centrales del nervio laríngeo recurrente de la rata.

Laryngeal nerves contain the fibres that control the laryngeal function. The studies carried out on the rat with the purpose of having a better knowledge of the functional components and the real origin of the fibres conveyed by the recurrent laryngeal nerve (RLN) are few and in disagreement. No one of such papers were developed using biotinylated dextrane amines (BDA), a powerful tool for tracing neural pathways. The aim of our study was to identify in the rat using BDA, the nuclei of real origin of the fibres of the RLN, knowing in this way the functional components of this nerve. The study has been developed in 31 adult male Sprague-Dawley rats, applying the BDA into the lesioned RLN. The results obtained in all the animals show that the rat's RLN does not contain afferent fibres, whereas the efferent fibres were originated within the ipsilateral nucleus ambiguus (NA). So, in the rat, the RLN seems to contain exclusively efferent fibres, probably been the superior laryngeal nerve who conveyed the afferent fibres.

Capsaicin-induced activation of pulmonary vagal C fibers produces reflex laryngeal closure in the rat.

Our recent studies show that intravenous administration of capsaicin induces enhancement of the intralaryngeal thyroarytenoid (TA) branch but a reduction of the intralaryngeal abducent branch, suggesting that the glottis is likely closed by capsaicin. The aim of the present study was to examine whether the glottis is adducted by intravenous administration of capsaicin. Electromyographic (EMG) activity of the TA muscle, subglottal pressure (SGP), and glottal behavior were evaluated before and after intravenous administration of capsaicin in male Wistar rats that were anesthetized and tracheostomized. Catheters were placed in the femoral artery and vein, as well as in the right jugular vein. Low and high doses of capsaicin (0.625 and 1.25 microg/kg) produced apnea and increases in the amplitude of the TA EMG. This enhancement of the TA EMG was observed during apnea as well as during recovery from apnea. Moreover, the onset of the TA EMG was advanced such that it commenced earlier during inspiration. Concomitantly, the SGP substantially increased. Increases in both the TA EMG and SGP were abolished after bilateral sectioning of the recurrent laryngeal nerve. In some animals, movement of the vocal folds was recorded by taking a motion picture with a digital camera under a surgical microscope. With intravenous administration of capsaicin, a tight glottal closure, decreases in blood pressure, and bradycardia were observed. These results strongly suggest that glottal closure is reflexively induced by intravenous administration of capsaicin and that closure of the glottis is beneficial for the defense of the airway and lungs when an animal is exposed to environmental irritants.

Lidocaine block of the recurrent laryngeal nerve in adductor spasmodic dysphonia: a multidimensional assessment.

OBJECTIVES/HYPOTHESIS: Lidocaine block of the recurrent laryngeal nerve (RLN) has been reported as a procedure for surgical selection of patients with adductor spasmodic dysphonia (ADSD). However, its effects on phonation have not been rigorously assessed in a prospective fashion using strict entry criteria and multiple measures of phonatory function. This investigation assessed the phonatory effects of RLN lidocaine block in ADSD to explore its potential as a diagnostic tool. STUDY DESIGN: Single group, pre/postexperimental trial. METHODS: Twenty-one consecutive patients with suspected ADSD underwent unilateral RLN block, causing temporary ipsilateral vocal fold paralysis. Voices were recorded before and during the block. Patients completed self-ratings of overall level of dysphonia severity, vocal effort, and laryngeal tightness. Blinded listeners completed auditory-perceptual ratings, and the frequency of phonatory breaks was acoustically analyzed. RESULTS: During the block, patients reported significant reductions on overall severity (P = .045), vocal effort (P < .001), and laryngeal tightness (P = .002). Listeners rated the voices during the block as significantly more breathy (P < .001), less strained (P < .001), and less severe (P = .059). Acoustic analysis confirmed significantly fewer phonatory breaks during the block (P < .001). Patient-based ratings of improvement were more consistent than listener ratings, and reduction in overall severity correlated with perceived breathiness. CONCLUSIONS: Although individuals varied in their outcomes, group results suggest that response to RLN lidocaine block warrants further study as a possible diagnostic tool in ADSD.

Effects of SZ1677, a new non-depolarizing steroidal neuromuscular blocking drug, and rocuronium on two laryngeal muscles and the anterior tibial muscle in guinea pigs.

BACKGROUND: SZ1677 is a new neuromuscular blocking drug structurally related to rocuronium. We compared the effect of an ED(90) of SZ1677 (25 microg/kg) with that of rocuronium (100 microg/kg) in guinea pig laryngeal and peripheral muscles. METHODS: Electromyography was used to quantify neuromuscular blockade at the posterior cricoarytenoid muscle, the thyroarytenoid muscle and the anterior tibial muscle after SZ1677 (n = 10) and rocuronium (n = 9). RESULTS: Maximum neuromuscular blockade was similar after SZ1677 and rocuronium (83 +/- 11% vs. 89 +/- 11%; thyroarytenoid muscle: 91 +/- 8% vs. 97 +/- 3%; anterior tibial muscle: 91 +/- 15% vs. 96 +/- 3%, respectively). Onset time of neuromuscular blockade at the laryngeal muscles was similar for the two neuromuscular blocking drugs; it was shorter at the thyroarytenoid muscle (67 +/- 32 s vs. 42 +/- 40 s) than at the posterior cricoarytenoid muscle (101 +/- 26 s vs. 102 +/- 108 s). Onset time at the anterior tibial muscle was longer after SZ1677 (114 +/- 34 s) than after rocuronium (68 +/- 46 s); P < 0.05. Neuromuscular recovery was faster after SZ1677 (interval 25%-75%: posterior cricoarytenoid muscle: 222 +/- 66 s; thyroarytenoid muscle: 192 +/- 92 s; tibial muscle 149 +/- 55 s) than after rocuronium (450 +/- 148 and 464 +/- 183 s, 292 +/- 86 s, respectively); P < 0.05. CONCLUSIONS: In guinea pigs, SZ1677 offers a rapid onset of neuromuscular blockade at a laryngeal adductor muscle with a shorter duration than rocuronium. Regardless of the drug used, the course of neuromuscular blockade differs not only between peripheral muscles and the larynx but also between antagonistic laryngeal muscles. The differences seem to be species specific.

Tracing axons of peripheral nerves in rats: a potential technique to study the equine recurrent laryngeal nerve.

To study the fascicular anatomy of peripheral nerves, three different groups of retrograde axonal tracers were evaluated: fluorophores, horseradish peroxidase conjugated to subunit B of cholera toxin (CT-HRP), and adeno-associated virus (AAV). The hindlimb nerves in rats served as a model to identify the most efficient tracer in regard to labeling axons within peripheral nerves. The rat's tibial and common peroneal nerves were injected with the different tracers and the sciatic nerve was subsequently examined for evidence of labeled axons. The CT-HRP clearly provided the best results in this rat model. Subsequently, CT-HRP was injected into the recurrent laryngeal nerve (RLN) of two horses in order to identify the location and distribution pattern of the RLN axons within the course of the cervical vagus nerve trunk. No labeling could be observed in either of the two horses.