Regeneration Of Recurrent Laryngeal Nerve Research Articles

Early Changes in Porcine Larynges Following Injection of Motor-Endplate Expressing Muscle Cells for the Treatment of Unilateral Vocal Fold Paralysis.

No curative injectable therapy exists for unilateral vocal fold paralysis. Herein, we explore the early implications of muscle-derived motor-endplate expressing cells (MEEs) for injectable vocal fold medialization after recurrent laryngeal nerve (RLN) injury. Yucatan minipigs underwent right RLN transection (without repair) and muscle biopsies. Autologous muscle progenitor cells were isolated, cultured, differentiated, and induced to form MEEs. Three weeks after the injury, MEEs or saline were injected into the paralyzed right vocal fold. Outcomes including evoked laryngeal electromyography (LEMG), laryngeal adductor pressure, and acoustic vocalization data were analyzed up to 7 weeks post-injury. Harvested porcine larynges were examined for volume, gene expression, and histology. MEE injections were tolerated well, with all pigs demonstrating continued weight gain. Blinded analysis of videolaryngoscopy post-injection revealed infraglottic fullness, and no inflammatory changes. Four weeks after injection, LEMG revealed on average higher right distal RLN activity retention in MEE pigs. MEE-injected pigs on average had vocalization durations, frequencies, and intensities higher than saline pigs. Post-mortem, the MEE-injected larynges revealed statistically greater volume on quantitative 3D ultrasound, and statistically increased expression of neurotrophic factors (BDNF, NGF, NTF3, NTF4, NTN1) on quantitative PCR. Minimally invasive MEE injection appears to establish an early molecular and microenvironmental framework to encourage innate RLN regeneration. Longer follow-up is needed to determine if early findings will translate into functional contraction. NA Laryngoscope, 134:272-282, 2024.

Platelet-rich plasma loaded nerve guidance conduit as implantable biocompatible materials for recurrent laryngeal nerve regeneration

Vocal cord paralysis caused by recurrent laryngeal nerve (RLN) injury during thyroidectomy results in hoarseness, aspiration, and dyspnea. We evaluated the usefulness of nerve guidance conduits (NGCs) constructed from an asymmetric polycaprolactone (PCL)/Pluronic F127 porous membrane and filled with platelet-rich plasma (PRP) for functional RLN regeneration. We evaluated the proliferation and migration of Schwann cells (SCs) after PRP treatment in vitro. For the in vivo study, rabbits were divided into a non-loaded NGC group and a PRP-loaded NGC group. The left RLNs were resected and interposed with the NGCs. Functional and histological examinations of the vocal cords were performed. SC proliferation and migration increased in a PRP dose-dependent manner, with the PRP increasing the levels of neurotrophic factors, myelin-associated glycoprotein, and ERK. In vivo, the PRP group showed significantly better vocal cord mobility and less vocalis muscle atrophy than the non-loaded NGC group. Histologically, the ingrowth of nerve endings occurred more rapidly in the PRP group, and acetylcholinesterase, neurofilament, and S-100 expression in neural endings were significantly higher in the PRP group. Furthermore, transmission electron microscopy showed that myelinated axons were more tightly packed in the PRP group. This study shows that PRP-loaded NGCs provide a favorable environment for neural regeneration and suggests that this technique has therapeutic potential for promoting RLN recovery.

Fabrication of microgroove poly(lactic-co-glycolic acid) nerve guide conduit using dry-jet wet spinning for rat laryngeal recurrent nerve regeneration

Recurrent laryngeal nerve (RLN) injury is a serious complication of thyroidectomy. Here, the degradable poly(lactic-co-glycolic acid) (PLGA) nerve guide conduits (NGCs) with microgroove or smooth inner surface were fabricated using a phase inversion-based dry-jet wet spinning method for RLN regeneration. The morphology and mechanical properties of NGCs were observed by scanning electron microscope (SEM) and a universal testing machine, showing that the porous NGC has good mechanical properties and is suitable for implantation. Further, in vitro PC12 cells cultured on the NGCs showed that microgroove conduit (GNGC) exhibited better cell adhesion, proliferation, and cell orientation. Both smooth and microgroove conduit (SNGC and GNGC) were transplanted into Sprague-Dawley (SD) rat models for assessing the repair effect on the 5-mm RLN gap. The results showed that GNGC was superior to SNGC in nerve regeneration and had similar effects to the autograft group in reducing muscle atrophy and restoring target organ function. Therefore, the degradable NGCs combined with groove structure could guide the regeneration of injured RLN, promote the partial functional recovery of target organs and prevent secondary surgical injury. This study contributes to the design and fabrication of nerve guide conduits and the application of tissue engineering in the field of RLN injury repair.

A narrative review of current therapies in unilateral recurrent laryngeal nerve injury caused by thyroid surgery.

To summarize and compare current common treatments in recurrent laryngeal nerve injury (RLNI). In addition, we introduced nerve tissue engineering technology in RLNI animal models. This review is a comprehensive summary of current therapies for unilateral RLNI. RLNI is a common complication in thyroid surgery. Although preoperative imaging and intraoperative nerve monitoring are widely applied, some damage to nerves is still inevitable. Currently, advances in nerve repair and regeneration have been made, but relatively few studies have focused on RLNI. In this review, we summarized and compared current common treatments in RLNI. We searched the literature on PubMed and Web of Science, and chose studies about RLNI in thyroid surgery. Keywords included the following terms: "recurrent laryngeal nerve regeneration", "injection laryngoplasty", "type I thyroplasty", "arytenoid adduction", and "nerve tissue engineering technology". Only English language studies were included. In the section on nerve tissue engineering technology, we described the application in detail in the table below. Currently, the majority of treatments could obtain different effects to some extent, but there are still shortcomings that need to be overcome further overcome. Therefore, potential exploration such as nerve tissue engineering technology is worthy of attention.

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.

Functional recurrent laryngeal nerve regeneration using a silicon tube containing a collagen gel in a rat model.

In this study, we examined the effect of differing gap lengths on regeneration of transected recurrent laryngeal nerves using silicon tubes containing type I collagen gel and the ability of this regeneration to result in restoration of vocal fold movements in rats. We simulated nerve gaps in Sprague-Dawley rats by transecting the left recurrent laryngeal nerves and bridged the nerve stumps using silicon tubes containing type 1 collagen gel. Three experimental groups, in which the gap lengths between the stumps were 1, 3, or 5 mm, were compared with a control group in which the nerve was transected but was not bridged. After surgery, we observed vocal fold movements over time with a laryngoscope. At week 15, we assessed the extent of nerve regeneration in the tube, histologically and electrophysiologically. We also assessed the degree of atrophy of the thyroarytenoid muscle (T/U ratio). Restoration of vocal fold movements was observed in 9 rats in the 1-mm group, in 6 rats in the 3-mm group, and in 3 rats in the 5-mm group. However, in most rats, restoration was temporary, with only one rat demonstrating continued vocal fold movements at week 15. In electromyograph, evoked potentials were observed in rats in the 1-mm and 3-mm groups. Regenerated tissue in the tube was thickest in the 1-mm group, followed by the 3-mm and 5-mm groups. The regenerated tissue showed the presence of myelinated and unmyelinated nerve fibers. In assessment of thyroarytenoid muscle atrophy, the T/U ratio was highest in the 1-mm group, followed by the 3-mm and 5-mm groups. We successfully regenerated the nerves and produced a rat model of recurrent laryngeal nerve regeneration that demonstrated temporary recovery of vocal fold movements. This rat model could be useful for assessing novel treatments developing in the future.

Recurrent laryngeal nerve transection in mice results in translational upper airway dysfunction.

The recurrent laryngeal nerve (RLN) is responsible for normal vocal-fold (VF) movement, and is at risk for iatrogenic injury during anterior neck surgical procedures in human patients. Injury, resulting in VF paralysis, may contribute to subsequent swallowing, voice, and respiratory dysfunction. Unfortunately, treatment for RLN injury does little to restore physiologic function of the VFs. Thus, we sought to create a mouse model with translational functional outcomes to further investigate RLN regeneration and potential therapeutic interventions. To do so, we performed ventral neck surgery in 21 C57BL/6J male mice, divided into two groups: Unilateral RLN Transection (n = 11) and Sham Injury (n = 10). Mice underwent behavioral assays to determine upper airway function at multiple time points prior to and following surgery. Transoral endoscopy, videofluoroscopy, ultrasonic vocalizations, and whole-body plethysmography were used to assess VF motion, swallow function, vocal function, and respiratory function, respectively. Affected outcome metrics, such as VF motion correlation, intervocalization interval, and peak inspiratory flow were identified to increase the translational potential of this model. Additionally, immunohistochemistry was used to investigate neuronal cell death in the nucleus ambiguus. Results revealed that RLN transection created ipsilateral VF paralysis that did not recover by 13 weeks postsurgery. Furthermore, there was evidence of significant vocal and respiratory dysfunction in the RLN transection group, but not the sham injury group. No significant differences in swallow function or neuronal cell death were found between the two groups. In conclusion, our mouse model of RLN injury provides several novel functional outcome measures to increase the translational potential of findings in preclinical animal studies. We will use this model and behavioral assays to assess various treatment options in future studies.

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

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. Laboratory experiment with rat model. 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. 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. 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. NA. Laryngoscope, 129:2537-2542, 2019.

Recurrent laryngeal nerve regeneration using an oriented collagen scaffold containing Schwann cells.

Regeneration of the recurrent laryngeal nerve (RLN), which innervates the intrinsic laryngeal muscles such that they can perform complex functions, is particularly difficult to achieve. Synkinesis after RLN neogenesis leads to uncoordinated movement of laryngeal muscles. Recently, some basic research studies have used cultured Schwann cells (SCs) to repair peripheral nerve injuries. This study aimed to regenerate the RLN using an oriented collagen scaffold containing cultured SCs. Preliminary animal experiment. A 10-mm-long autologous canine cervical ansa was harvested. The nerve tissue was scattered and subcultured on oriented collagen sheets in reduced serum medium. After verifying that the smaller cultivated cells with high nucleus-cytoplasm ratios were SCs, collagen sheets with longitudinally oriented cells were rolled and inserted into a 20-mm collagen conduit. The fabricated scaffolds containing SCs were autotransplanted to a 20-mm deficient RLN, and vocal fold movements and histological characteristics were observed. Scaffolds containing cultured SCs were successfully fabricated. Immunocytochemical examination revealed that these isolated and cultured cells, identified as SCs, expressed S-100 protein and GFAP but not vimentin. The orientation of SCs matched that of the oriented collagen sheet. Two months after successful transplantation, laryngeal endoscopy revealed coordinated movement of the bilateral vocal folds by external stimulation under light general anesthesia. Hematoxylin and eosin staining showed that the regenerated RLN lacked epineurium surrounding the nerve fibers and was interspersed with collagen fibers. Myelin protein zero was expressed around many axons. Partial regeneration of RLN was achieved through the use of oriented collagen scaffolding. NA Laryngoscope, 127:1622-1627, 2017.

Changes in neurotrophic factors of adult rat laryngeal muscles during nerve regeneration

Injury to the recurrent laryngeal nerve (RLN) leads to the loss of ipsilateral laryngeal fold movement, with dysphonia, and occasionally dysphagia. Functional movement of the vocal folds is never restored due to misrouting of regenerating axons to agonist and antagonist laryngeal muscles. Changes of neurotrophic factor expression within denervated muscles occur after nerve injury and may influence nerve regeneration, axon guidance and muscle reinnervation. This study investigates the expression of certain neurotrophic factors in the laryngeal muscles during the course of axonal regeneration using RT-PCR. The timing of neurotrophic factor expression was correlated to the reinnervation of the laryngeal muscles by motor axons. Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF) and Netrin-1 (NTN-1) increased their expression levels in laryngeal muscles after nerve section and during regeneration of RLN. The upregulation of trophic factors returned to control levels following regeneration of RLN. The expression levels of the neurotrophic factors were correlated with the innervation of regenerating axons into the denervated muscles. The results suggest that certain neurotrophic factor expression is strongly correlated to the reinnervation pattern of the regenerating RLN. These factors may be involved in guidance and neuromuscular junction formation during nerve regeneration. In the future, their manipulation may enhance the selective reinnervation of the larynx.

Value of a novel PGA-collagen tube on recurrent laryngeal nerve regeneration in a rat model.

Nerbridge (Toyobo Co., Ltd., Osaka, Japan) is a novel polyglycolic acid (PGA) tube that is filled with collagen fibers and that facilitates nerve fiber expansion and blood vessel growth. It is biocompatible and commercially available, with governmental approval for practical use in Japan. We hypothesized that the PGA-collagen tube would promote regeneration of the recurrent laryngeal nerve (RLN). This hypothesis was examined in a rat axotomy model of the RLN. Prospective animal study. The axotomy model was established by transection of the left RLN in adult Sprague-Dawley rats. The cut ends of the nerve were bridged using Nerbridge (Toyobo Co., Ltd.) with a 1-mm gap (tube-treatment group) or direct sutures (sutured-control group). Left vocal fold mobility, nerve conduction velocity, morphology, and histology were assessed after 15 weeks. Fifteen weeks after treatment, nerve fiber connections were observed macroscopically in both groups, and more clear myelinated fibers and better prevention of laryngeal muscle atrophy were observed in the tube-treatment group compared with the sutured-control group. However, vocal fold movement recovery was not observed in either group, and the conduction velocity of the RLN did not differ between the two groups. Better nerve regeneration was observed in the tube-treatment group. The combination therapy with molecular or gene therapy might be an effective strategy to improve vocal fold movement. The PGA-collagen tube has the potential to promote regeneration of the RLN and to be a scaffold for drug administration in these combination therapies. N/A. Laryngoscope, 126:E233-E239, 2016.

Functional Regeneration of Recurrent Laryngeal Nerve Injury During Thyroid Surgery Using an Asymmetrically Porous Nerve Guide Conduit in an Animal Model

Vocal cord paralysis (VCP) caused by recurrent laryngeal nerve (RLN) damage during thyroidectomy commonly results in serious medico-legal problems. The purpose of this study was to evaluate the usefulness of an asymmetrically porous polycaprolactone (PCL)/Pluronic F127 nerve guide conduit (NGC) for functional regeneration in a RLN injury animal model. A biodegradable, asymmetrically porous PCL/F127 NGC with selective permeability was fabricated for use in this study. A 10-mm segment of left RLN was resected in 28 New Zealand white rabbits, and then an asymmetrically porous NGC or a nonporous silicone tube was interposed between both stumps and securely fixed. Vocal cord mobility was endoscopically evaluated at one, four, and eight weeks postoperatively. Nerve growth through NGCs was assessed by toluidine blue staining, and thyroarytenoid (TA) muscle atrophy was evaluated by hematoxylin and eosin staining. Immunohistochemical stainings for acetylcholinesterase (AchE), anti-neurofilament (NF), and anti-S100 protein were also conducted, and transmission electron microscopy (TEM) was used to evaluate functional nerve regeneration. At eight weeks postoperatively, endoscopic evaluations showed significantly better recovery from VCP in the asymmetrically porous PCL/F127 NGC group (6 of 10 rabbits) than in the silicone tube group (1 of 10 rabbits). Continued nerve growth on the damaged nerve endings was observed with time in the asymmetrically porous PCL/F127 NGC-interposed RLNs. TA muscle dimensions and AchE expressions in TA muscle were significantly greater in the asymmetrically porous PCL/F127 NGC group than in the silicone tube group. Furthermore, immunohistochemical staining revealed the expression of NF and S100 protein in the regenerated nerves in the asymmetrically porous PCL/F127 NGC group at eight weeks postoperatively, and at this time, TEM imaging showed myelinated axons in the regenerated RLNs. The study shows that asymmetrically porous PCL/F127 NGC provides a favorable environment for RLN regeneration and that it has therapeutic potential for the regeneration of RLN damage.

Restoration of Recurrent‐Laryngeal‐Nerve Function after Injury in a Rat Model

Objectives: Recurrent laryngeal nerve (RLN) injury occurs in 4-10% of thyroid surgeries. Spontaneous RLN regeneration takes months and is often incomplete. Lithium promotes neuronal growth in vitro. We developed methods to objectively detect vocal-fold impairment in the rat and tested whether systemic lithium enhances recovery after RLN injury. Methods: The right RLN was crushed in eight adult rats, and osmotic pumps releasing either lithium or water were implanted. Eight sham-operated animals were also implanted with either lithium or water pumps. Vocal-fold motion was captured by microsuspension laryngoscopy and was measured quantitatively by image analysis at the time of surgery and at one and four weeks. Vocalizations, breathing patterns, and laryngeal electromyography were assessed at four weeks. Results: Cessation of unilateral vocal-fold motion was observed simultaneously with every RLN crush. One week after surgery, 50% of “Crush/Water” animals exhibited full vocal-fold motion, compared to 75% of “Crush/Lithium” and 100% of the sham-operated animals. In animals with unilateral vocal-fold immobility, the quantitative analysis also revealed a subtle decrease of vocal-fold movement on the non-operated side. At four weeks after surgery, vocalizations, breathing patterns, vocal-fold motion, and electromyograms were indistinguishable among all groups. Lithium-treated animals weighed 5% less than water-treated controls. Conclusions: Quantitative endoscopy is a sensitive and efficient tool for detecting vocal-fold impairment in rats. While our results might suggest enhanced recovery in lithium treated animals, all animals showed a rapid return of vocal-fold motion. Perhaps more severe nerve injury is necessary to uncover an effect of lithium on nerve regeneration.

Recurrent Laryngeal Nerve Regeneration through a Silicone Tube Produces Reinnervation without Vocal Fold Mobility in Rats

We established an animal model of recurrent laryngeal nerve reinnervation with persistent vocal fold immobility following recurrent laryngeal nerve injury. In 36 rats, the left recurrent laryngeal nerve was transected and the stumps were abutted in a silicone tube with a 1-mm interspace, facilitating regeneration. The mobility of the vocal folds was examined endoscopically 5, 10, and 15 weeks later. Electromyography of the thyroarytenoid muscle was performed. Reinnervation was assessed by means of a quantitative immunohistologic evaluation with anti-neurofilament antibody in the nerve both proximal and distal to the silicone tube. The atrophy of the thyroarytenoid muscle was assessed histologically. We observed that all animals had a fixed left vocal fold throughout the study. The average neurofilament expression in the nerve both distal and proximal to the silicone tube, the muscle area, and the amplitude of the compound muscle action potential recorded from the thyroarytenoid muscle on the treated side increased significantly (p < 0.05) over time, demonstrating regeneration through the silicone tube. Recurrent laryngeal nerve regeneration through a silicone tube produced reinnervation without vocal fold mobility in rats. The efficacy of new laryngeal reinnervation treatments can be assessed with this model.

Potential for promoting recurrent laryngeal nerve regeneration by remote delivery of viral gene therapy

The aims of this study were to demonstrate the ability to enhance nerve regeneration by remote delivery of a viral vector to the crushed recurrent laryngeal nerve (RLN), to demonstrate the usefulness of a crushed RLN model to test the efficacy of viral gene therapy, and to discuss future potential applications of this approach. Animal study. Adult Sprague-Dawley rats were assigned to two groups. In the experimental group, an adeno-associated viral (AAV) vector carrying a zinc-finger transcription factor, which stimulates endogenous insulinlike growth factor I production (AAV2-TO-6876vp16), was injected into the crushed RLN. In the control group, an AAV vector carrying the gene for green fluorescent protein was injected into the crushed RLN. Unilateral RLN paralysis was confirmed endoscopically. At 1 week, laryngeal endoscopies were repeated and recorded. Larynges were cryosectioned in 15-μm sections and processed for acetylcholine histochemistry (motor endplates) followed by neurofilament immunoperoxidase (nerve fibers). Percentage nerve-endplate contact (PEC) was determined and compared. Vocal fold motion was evaluated by blinded reviewers using a visual analogue scale (VAS). The difference between PEC on the crushed and uncrushed sides was statistically less in the experimental group (0.54 ± 0.18 vs. 0.30 ± 0.26, P = .0006). The VAS score at 1 week was significantly better in the experimental group (P = .002). AAV2-TO-6876vp16 demonstrated a neurotrophic effect when injected into the crushed RLN. The RLN offers a conduit for viral gene therapy to the brainstem that could be useful for the treatment of RLN injury or bulbar motor neuron disease.

Spontaneous regeneration of recurrent laryngeal nerve following long‐term vocal fold paralysis in humans: Histologic evidence

To validate the occurrence of spontaneous regeneration of the recurrent laryngeal nerve (RLN) in patients with symptomatic vocal fold paralysis (VFP). Histologic study. Between June 2008 and February 2009, 29 patients with symptomatic unilateral VFP caused by thyroid surgery were continuously enrolled in this study, with 30 normal RLNs serving as the control. The denervation course was from 7 to 74 months. Intraoperative exploration was performed to identify the sites and types of RLN lesions. The status of RLN regeneration was detected by histologic examination, and the number of myelinated axons was counted. Intraoperative exploration showed that RLN was transected in 24 cases, and the continuity of the RLN stump was recognized in the five other cases. Connective tissue connected the distal and the proximal ends of all transected RLNs under a surgical microscope. Light microscopy showed varying numbers (17-259) of abnormal myelinated axons in these injured RLNs; the maximum was less than 40% of those of normal RLNs (658 ± 79). Electron microscopy also demonstrated that abnormal myelinated axons with small diameters commonly existed in all injured RLNs, with dark myelin clumps without axons (Bungner bands) and fibrotic components. The present study demonstrates that a varying degree of spontaneous RLN regeneration is substantially engaged in the process of subclinical reinnervation following laryngeal denervation in humans, which may account for different clinical characteristics and outcomes of VFP.

Effects of Nerve-Muscle Pedicle on Immobile Rat Vocal Folds in the Presence of Partial Innervation

We investigated whether implantation of an ansa cervicalis nerve (ACN)-muscle pedicle into the thyroarytenoid (TA) muscle is efficacious in the presence of partial recurrent laryngeal nerve (RLN) innervation. We studied a total of 36 rats. Twelve of the rats served as positive and negative control animals. In the remaining 24 rats, the left RLN was transected, a 1-mm piece of nerve was removed, and the stumps were abutted in silicone tubes (STs), inducing partial RLN regeneration. Twelve of the ST-treated rats underwent this procedure alone, and the other 12 rats had a nerve-muscle pedicle (NMP) implanted into the left TA muscle 5 weeks after ST treatment. At 15 weeks, reinnervation was assessed by histologic evaluation of the TA muscle and by electromyography with stimulation of the RLNs and ACNs. The muscle area, the number of nerve terminals, the number of acetylcholine receptors, and the ratio of nerve terminals to acetylcholine receptors were significantly greater (p < 0.05) in the NMP group than in the ST group. Electromyography elicited TA muscle compound action potentials upon stimulation of the RLNs and ACNs. In rats, NMP implantation is efficacious for reducing atrophic changes in the TA muscle in the presence of partial RLN innervation.

Tissue engineering strategies for reinnervation of the larynx

Objectives. Injury to the recurrent laryngeal nerve (RLN) results in synkinesis and a dysfunctional larynx. Novel nerve repair techniques might enhance the speed of RLN regeneration and encourage accurate muscle reinnervation. Our first approach has been to apply neurotrophins (NT) to the site of repair using artificial nerve conduits. Currently we are investigating whether adult stem cells can be differentiated to provide a source of Schwann cells for future cell‐based therapies. Method. In a pig model, the phrenic nerve was anastomosed with the RLN abductor branch1 and repaired using poly‐hydroxybutyrate wrap‐around conduits either untreated or impregnated with NT‐3 (500 ng/mL). Markers of nerve regeneration (neurofilament) and muscle denervation/reinnervation (NCAM) were assessed to determine the efficiency of repair. Stem cells were isolated from adipose tissue and treated with glial growth factors for 2 weeks before phenotypic characterisation. Results. Compared with controls, NT‐3 treated animals showed significantly elevated levels of neurofilament protein in the distal nerve segment at 2 months (n = 5, P &lt; 0.01). Injury increased the number of denervated, NCAM positive abductor muscle fibres from 1.3 ± 0.68% to 33.9 ± 5.8% at 2 months and 3.7 ± 2.3% at 4 months. NT‐3 did not significantly reduce these numbers. Differentiated adipose derived stem cells adopted a bi‐polar morphology similar to Schwann cells and expressed S100 and GFAP glial proteins. Conclusions. NT‐3 treatment promotes early nerve regeneration but is insufficient to enhance long‐term muscle reinnervation. Differentiated adipose derived stem cells may represent a novel source of Schwann cells for future RLN repair studies. Reference 1 Kingham P.J., Birchall M.A., Burt R., et al. (2005) Reinnervation of laryngeal muscles: a study of changes in myosin heavy chain expression. Muscle Nerve. 32(6), 761‐‐766

1219 Inhibition of synaptogenesis between cultured cortical neurons by map1b antisense oligodeoxynucleotides

Injury to the recurrent laryngeal nerve (RLN) leads to the loss of ipsilateral laryngeal fold movement, with dysphonia, and occasionally dysphagia. Functional movement of the vocal folds is never restored due to misrouting of regenerating axons to agonist and antagonist laryngeal muscles. Changes of neurotrophic factor expression within denervated muscles occur after nerve injury and may influence nerve regeneration, axon guidance and muscle reinnervation. This study investigates the expression of certain neurotrophic factors in the laryngeal muscles during the course of axonal regeneration using RT-PCR. The timing of neurotrophic factor expression was correlated to the reinnervation of the laryngeal muscles by motor axons. Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF) and Netrin-1 (NTN-1) increased their expression levels in laryngeal muscles after nerve section and during regeneration of RLN. The upregulation of trophic factors returned to control levels following regeneration of RLN. The expression levels of the neurotrophic factors were correlated with the innervation of regenerating axons into the denervated muscles. The results suggest that certain neurotrophic factor expression is strongly correlated to the reinnervation pattern of the regenerating RLN. These factors may be involved in guidance and neuromuscular junction formation during nerve regeneration. In the future, their manipulation may enhance the selective reinnervation of the larynx.

Experiments in laryngeal reinnervation.

Laryngeal reinnervation surgical procedures were performed in 26 dogs. Nineteen animals comprise the data reported in this thesis. Two different operative procedures were investigated, the ansa hypoglossi neuromuscular pedicle in 5 dogs, and the split phrenic nerve graft in 14 dogs. The studies were designed to evaluate whether either procedure resulted in inspiratory abduction of the vocal cord: and, if so, the mechanism by which abduction was produced. Five dogs in Experiment 1 demonstrated similar results from the ansa hypoglossi neuromuscular pedicle procedure. Apparent vocal cord abduction was seen during hyperpnea from airway obstruction, but was abolished by superior laryngeal nerve transection, or detachment of the sternothyroid muscle. None of the nerves in the neuromuscular pedicles was electrically excitable. Fourteen dogs in Experiment 2 underwent the split phrenic nerve graft operation. Vocal cord abduction, synchronized with inspiration, was noted in 5 animals. Two dogs had inspiratory abduction in quiet respiration. Electromyography, nerve action potentials, endoscopic motion pictures, and histologic study confirmed that posterior cricoarytenoid muscle reinnervation had occurred via the nerve graft from the phrenic nerve. Vocal cord abduction and electromyographic activity in the posterior cricoarytenoid muscle were abolished by transection of the nerve graft or the phrenic nerve. Conclusions of the study include the following: 1. The neuromuscular pedicle procedure does not result in reinnervation of the posterior cricoarytenoid muscle the pedicle's nerve and muscle block. 2. The phrenic procedure may result in reinnervation of the posterior cricoarytenoid via the nerve graft, and inspiratory abduction of the paralyzed vocal cord. Failure of the phrenic procedure to produce reinnervation appeared to be due to a. recurrent laryngeal nerve regeneration, or b. avulsion of the nerve graft due to swallowing and other laryngeal movements.