Nerve Conduction Studies of the Larynx: Porcine Model and Human Application.

OBJECTIVE: There is a paucity of information in the literature about how electromyography (EMG) with nerve conduction studies (NCS) can be utilized in the field of neurolaryngology. The goal of this study was two-part: (1) to identify the NCS test parameters that best reflect underlying neurolaryngeal pathophysiology in a porcine model, and (2) to determine if comparable NCS parameters can be used to assess clinical laryngeal denervation injuries in patients. METHODS: Yukatan minipigs underwent general anesthesia with EMG and recurrent laryngeal NCS assessment. Recurrent laryngeal nerve (RLN) transection injury and uninjured RLN cases were included to determine NCS neuropathologic and normative findings, respectively. Our porcine analytic model was then applied to a clinical scenario of seven patients with RLN injury. RESULTS: The porcine model was able to differentiate and characterize normative and neuropathologic NCS changes. Clinical outcomes suggest that RLN NCS can be performed clinically and is overall well tolerated by in patients. Findings may serve as a useful tool to differentiate normal nerve function, neuropraxia, axonotmesis, and irreversible RLN injuries. CONCLUSION: This novel study is the first to demonstrate the feasibility and applicability of EMG with NCS in the evaluation of vocal fold function in a porcine model in addition to the clinical setting. Further studies are needed to better understand the clinical utility and predictive value in recovery of vocal fold paralysis.

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

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. LEVEL OF EVIDENCE: NA Laryngoscope, 134:272-282, 2024.

Enhanced Abductor Function in Bilateral Vocal Fold Paralysis with Muscle Stem Cells.

INTRODUCTION: Patients with bilateral vocal fold paralysis (BVFP) experience airway obstruction because of loss of abductor function of posterior cricoarytenoid (PCA) muscles. We previously reported that implantation of autologous muscle progenitor (stem) cells into thyroarytenoid muscles during reinnervation resulted in improved adductor function. In this study, that same approach was applied to treating PCA muscles in a canine model of BVFP. DESIGN: Animal study. METHODS: Two canines underwent baseline measures of glottal resistance (GR), then complete transection and suture repair of both recurrent laryngeal nerves. Muscle stem cells were isolated from skeletal muscle and cultured. Two months later, GR was measured, and then 107 stem cells were implanted into one PCA muscle of each animal. After four more months, GR and glottal opening force (GOF) were measured and the muscles were harvested for histologic study. One control dog underwent the same procedures without stem cell implantation, for comparison. RESULTS: GR increased by 21%-25% over baseline at 2 months, but after stem cell implantation, improved to 10%-14% over baseline at 6 months. PCA muscle strength, as determined by GOF, was 61%-65% on control sides (no stem cells), and 78%-83% on treated sides (with stem cells). Histology confirmed survival of stem cells and a 50% higher rate of innervation of motor endplates in the stem cell treated sides. CONCLUSION: Autologous muscle progenitor (stem) cells show promise as a potential new therapy for patients with bilateral vocal fold paralysis. Additional studies are needed to determine the optimal number of cells, timing of implantation, and other variables before launching a clinical trial. LEVEL OF EVIDENCE: NA (animal study) Laryngoscope, 134:324-328, 2024.

Amyloid Deposition in the Upper Aerodigestive Tract, A Single Institute Experience.

Objectives: The purpose of this study is to evaluate clinical information, laboratory results, and pathologic findings of patients with amyloidosis involving larynx, oral cavity, and pharynx from our institute. Methods: A total of 39 specimens from 28 patients were retrieved from 2000 to 2020. Data collection included clinical presentation, radiographic, laboratory results, and pathologic findings. Results: A total of 12 patients had laryngeal amyloidosis and true vocal cord was the most common location. Protein electrophoresis detected monoclonal protein in 10% (1/10) of patients tested. Two patients had hematopoietic disorder (2/12, 17%) and another patient had a peptide profile consistent with amyloid transthyretin (ATTR) detected by mass spectrometry. Twelve patients showed amyloidosis in the oral cavity with 75% involving the tongue. Monoclonal protein was found in 89% of cases tested. Nine patients (9/12, 75%) had systematic involvement including 6 with hematopoietic malignancy and 3 with biopsy-confirmed systemic light chain amyloidosis. Compared to the laryngeal amyloidosis, amyloid deposition in oral cavity had a significant higher association with systematic disease (P < .01). Pharyngeal amyloidosis was seen in 7 patients. Three of 6 patients tested (3/6, 50%) were found to have biopsy-confirmed hematopoietic malignancy. Conclusions: Laryngeal amyloidosis is mostly a localized disease. Amyloidosis involving oral cavity is associated with significantly higher risk of systematic involvement which warrants a comprehensive laboratory, radiographic, and pathologic workup. There is limited data about pharynx amyloidosis. Oropharynx and hypopharynx amyloidosis appear to be more likely associated with underlying hematologic malignancy compared to nasopharynx involvement.

Impact of Needle Selection on Survival of Muscle-Derived Cells When Used for Laryngeal Injections.

Objective: To describe how differing injector needles and delivery vehicles impact Autologous Muscle-Derived Cell (AMDC) viability when used for laryngeal injection. Methods: In this study, adult porcine muscle tissue was harvested and used to create AMDC populations. While controlling cell concentration (1 × 107 cells/ml), AMDCs including Muscle Progenitor Cells (MPCs) or Motor Endplate Expressing Cells (MEEs) were suspended in either phosphate-buffered saline or polymerizable (in-situ scaffold forming) type I oligomeric collagen solution. Cell suspensions were then injected through 23- and 27-gauge needles of different lengths at the same rate (2 ml/min) using a syringe pump. Cell viability was measured immediately after injection and 24- and 48-hours post-injection, and then compared to baseline cell viability prior to injection. Results: The viability of cells post-injection was not impacted by needle length or needle gauge but was significantly impacted by the delivery vehicle. Overall, injection of cells using collagen as a delivery vehicle maintained the highest cell viability. Conclusion: Needle gauge, needle length, and delivery vehicle are important factors that can affect the viability of injected cell populations. These factors should be considered and adapted to improve injectable MDC therapy outcomes when used for laryngeal applications.

Eliciting and Characterizing Porcine Vocalizations: When Pigs Fly.

BACKGROUND/OBJECTIVES: While voice-related therapeutic interventions are often researched preclinically in the porcine model, there are no well-established methods to induce porcine glottic phonation. Described approaches, such as training animals to phonate for positive reinforcement are time-consuming and plagued by inherent variability in the type of phonation produced and contamination of background noise. Thus, a reliable method of assessing glottic phonation in the porcine model is needed. METHODS: In this study, we have created a novel pulley-based apparatus with harness for "pig-lifting" with surrounding acoustic insulation and high-directional microphone with digital recorder for recording phonation. Praat and Matlab were used to analyze all porcine vocalizations for fundamental frequency (F0), intensity, duration of phonation and cepstral peak prominence (CPP). Glottic phonation was detected using F0 (≥2000 hz), duration (≥3 seconds) and researcher perceptual judgment. Partial-glottic phonations were also analyzed. Reliability between researcher judgment and acoustic measures for glottic phonation detection was high. RESULTS: Acoustic analysis demonstrated that glottic and partial-glottic phonation was consistently elicited, with no formal training of the minipigs required. Glottic vocalizations increased with multiple lifts. Glottic phonation continued to be elicited after multiple days but became less frequent. Glottic and partial-glottic phonations had similar CPP values over the 6 experimental days. CONCLUSION: Our cost-effective, reliable method of inducing and recording glottic phonation in the porcine model may provide a cost effective, preclinical tool in voice research.

Globus Pallidus Interna and Ventral Intermediate Nucleus of the Thalamus Deep Brain Stimulation for Adductor Laryngeal Dystonia: a Case Report of Blinded Analyses of Objective Voice Outcomes in 2 Patients.

BACKGROUND: Adductor laryngeal dystonia (ADLD) is a substantially debilitating focal progressive neurological voice disorder. Current standard of care is symptomatic treatment with repeated injections of botulinum toxin into specific intrinsic laryngeal muscles with extremely variable and temporary benefits. We report the use of bilateral deep brain stimulation (DBS) of globus pallidus (GPi) for long-term improvement of ADLD voice symptoms. OBJECTIVE: To investigate the effects of bilateral DBS of the GPi and ventral intermediate nucleus (VIM) of the thalamus on vocal function in 2 patients with ADLD associated with voice and hand tremor. METHODS: Blinded objective and quantitative analyses of voice were conducted before and after treatment in 2 female patients (70 and 69 years). Paired t-tests were conducted to compare voice measurements pre-GPi and post-GPi and VIM-DBS. A 2-way analysis of variance was conducted to determine the interaction between target (GPi/VIM) and time (pre/post) for each voice measure. RESULTS: Although the follow-up period differed between patients, the GPi-DBS implanted patient had notable improvement in vowel voicing (%), extent of tremor intensity (%), and overall speech intelligibility (%), compared with preoperative status. GPi-DBS also resulted in significant improvement in cepstral peak prominence (dB). VIM-DBS resulted in a significantly greater change in the tremor rate (Hz). CONCLUSION: Changes in phonatory function provide preliminary support for the use of bilateral GPi-DBS for treatment of ADLD and bilateral VIM-DBS for vocal tremor predominant ADLD. Future studies with larger sample sizes and standardized follow-up periods are needed to better assess the role of DBS for ADLD.

Determining Medical Urgency of Voice Disorders Using Auditory-Perceptual Voice Assessments Performed by Speech-Language Pathologists.

OBJECTIVE: This study examined whether speech-language pathologist auditory-perceptual voice assessments can predict the medical urgency of voice disorders. METHODS: Twenty speech-language pathologists (SLPs) evaluated 25 voice samples recorded during initial voice evaluations. Voice samples represented a range of dysphonia severity (mild-severe) balanced across patient diagnoses. Diagnoses included: benign lesions, laryngeal cancer, non-organic voice disorders, laryngeal edema (associated with LPR), and laryngeal paralysis or paresis. Laryngeal cancer and severe unilateral laryngeal paralysis were considered urgent disorders. While blinded to patient information, SLPs rated severity of voice quality, predicted patient diagnosis, and determined whether the patient should be seen urgently by a laryngologist. SLPs were then given basic medical history information and rated medical urgency of voice disorder a second time. RESULTS: On average, SLPs correctly identified 65% of urgent voices and 87% of nonurgent voices when blinded to patient information. Accuracy improved significantly to 86% for urgent voices with medical history information (P < .001) and decreased to 77% for nonurgent voices. Accuracy was better when severity of voice quality was severe for urgent voices and mild for nonurgent voices (P < .001). SLPs indicated that patient smoking history and severity of dysphonia were most influential in their decision making. Diagnostic accuracy of auditory-perceptual assessments was poor. CONCLUSIONS: SLPs identified 86% of medically urgent voice disorders when auditory perceptual assessments were combined with medical history information. Further work is needed to determine what medical history information is most crucial to rating accuracy and what speech tasks might best separate urgent and nonurgent patients.

Can Resident Auditory-Perceptual Voice Assessments Predict Medical Urgency of Voice Disorders?

BACKGROUND/OBJECTIVES: Growing reliance on telemedicine has created new triaging challenges. This study investigated how effectively otolaryngology resident auditory-perceptual voice assessments performed via telemedicine determined the need for urgent in-person clinic visits. METHODS: Twelve otolaryngology resident physicians (PGY1-PGY5) performed auditory-perceptual assessments on 25 voice samples recorded during initial voice evaluations. Voice samples were balanced in severity and taken in equal numbers from patients with the following diagnoses: benign laryngeal lesions, laryngeal cancer, functional voice disorders, laryngeal edema (associated with LPR), and laryngeal paralysis/paresis. Urgent diagnoses were defined as laryngeal cancer and severe unilateral laryngeal paralysis. For each voice sample, residents were initially blinded to patient medical history. Residents rated severity of voice disorder, predicted patient diagnosis, and determined the urgency of seeing the patient in clinic. Residents then reviewed information from the patient's medical history and again rated urgency of voice disorder. RESULTS: On average, residents identified urgent voice disorders in 56% of cases. After reviewing medical history, this number significantly increased to 77% (P = 0.001). Voice severity, smoking history, time since onset, and course of symptoms were considered most influential when determining medical urgency of voice patients. Year in residency program had no effect on rating accuracy. As expected, diagnostic accuracy of auditory-perceptual assessments was low, ranging from 40% for laryngeal paralysis/paresis to 5% for laryngeal edema. CONCLUSION: Auditory-perceptual voice assessment, combined with medical history, predicted most medically urgent voice disorders. Further work should investigate if task-specific training might improve these results and which medical history items are most critical. Until accuracy of auditory-perceptual assessment of medical urgency is improved, these data underscore the importance of laryngeal examination in identifying medical urgency and etiology of dysphonia.

Safety practices for in-office laryngology procedures during clinical reintroduction amidst COVID-19.

Objective: Describe safety practices for performing in-office laryngology procedures during clinical re-introduction amidst the coronavirus disease 2019 (COVID-19) pandemic. Methods: An anonymous survey in Qualtrics was created to evaluate demographics, preprocedure testing, practice settings, anesthesia, and personal protective equipment (PPE) use for five procedure categories (non-mucosal-traversing injections, mucosal-traversing injections, endoscopy without suction, endoscopy with suction/mucosal intervention via working channel, and laser via working channel). The survey was emailed to the Fall Voice Community on Doc Matter and to members of the American Broncho-Esophagological Association (ABEA) from May to June 2020. Results: Eighty-two respondents were analyzed (response rate: 10%). Respondents represented diverse locations, including international. Most reported academic (71%) or private practices (16%), laryngology fellowship training (76%), and a significant practice devotion to laryngology and broncho-esophagology. During the early re-introduction, most continued to perform all procedure categories. The office was preferred to the OR setting for most, though 36% preferred the OR for laser procedures. There was a preference for preprocedural SARS-Cov2 testing for procedures involving a working channel (>67%), and these procedures had the highest proportion of respondents discontinuing the procedure due to COVID-19. Various types of topical anesthesia were reported, including nebulizer treatments. The most common forms of personal protective equipment utilized were gloves (>95%) and N95 masks (>67%). Powered-air purifying respirators and general surgical masks were used infrequently. Conclusions: During the early re-introduction, respondents reported generally continuing to perform office laryngology procedures, while greater mucosal manipulation affected decisions to stop procedures due to COVID-19, perform preprocedural SARS-Cov2 testing, and alter topical anesthesia. Gloves and N95 masks were the predominate PPE. Level of Evidence: N/A.

Laryngeal Reconstruction Using Tissue-Engineered Implants in Pigs: A Pilot Study.

OBJECTIVE/HYPOTHESIS: There are currently no treatments available that restore dynamic laryngeal function after hemilaryngectomy. We have shown that dynamic function can be restored post hemilaryngectomy in a rat model. Here, we report in a first of its kind, proof of concept study that this previously published technique is scalable to a porcine model. STUDY DESIGN: Animal study. METHODS: Muscle and fat biopsies were taken from three Yucatan minipigs. Muscle progenitor cells (MPCs) and adipose stem cells (ASCs) were isolated and cultured for 3 weeks. The minipigs underwent a left laterovertical partial laryngectomy sparing the left arytenoid cartilage and transecting the recurrent laryngeal nerve. Each layer was replaced with a tissue-engineered implant: 1) an acellular mucosal layer composed of densified Type I oligomeric collagen, 2) a skeletal muscle layer composed of autologous MPCs and aligned oligomeric collagen differentiated and induced to express motor endplates (MEE), and 3) a cartilage layer composed of autologous ASCs and densified oligomeric collagen differentiated to cartilage. Healing was monitored at 2 and 4 weeks post-op, and at the 8 week study endpoint. RESULTS: Animals demonstrated appropriate weight gain, no aspiration events, and audible phonation. Video laryngoscopy showed progressive healing with vascularization and re-epithelialization present at 4 weeks. On histology, there was no immune reaction to the implants and there was complete integration into host tissue with nerve and vascular ingrowth. CONCLUSIONS: This pilot study represents a first in which a transmural vertical partial laryngectomy was performed and successfully repaired with a customized, autologous stem cell-derived multi-layered tissue-engineered implant. LEVEL OF EVIDENCE: NA Laryngoscope, 131:2277-2284, 2021.

Motor endplate-expressing cartilage-muscle implants for reconstruction of a denervated hemilarynx.

OBJECTIVE: Tissue engineering of the larynx requires a complex, multiple tissue layer design. Additionally, spontaneous reinnervation of the larynx after recurrent laryngeal nerve (RLN) injury is often disorganized, resulting in subpar function. This study investigates use of tissue-engineered cartilage and motor endplate-expressing (MEE) tissue-engineered skeletal muscle implants for laryngeal reconstruction and the promotion of organized reinnervation after RLN injury. METHODS: F344 rat primary muscle progenitor cells (MPCs) were isolated. Three-dimensional muscle constructs were created by encapsulating MPCs in type I oligomeric collagen under passive tension. Constructs were then cultured in differentiation medium (MPC control constructs) or induced to form motor endplates (MEE constructs) with neurotrophic agents. Three-dimensional cartilage constructs were created with adipose stem cells differentiated in chondrocyte medium. The muscle and cartilage constructs were implanted into surgically created myochondral defects in the F344 rat larynx with injured or intact (control) RLN. At 1-, 3-, and 6-month timepoints, videolaryngoscopy, electromyography (EMG), histology, and immunohistochemistry were used to assess outcomes. RESULTS: At all timepoints, cartilage-muscle implants were well integrated into host tissue. Functionally, there was increased vocal fold adduction and EMG activity in nerve-injured rats treated with the MEE constructs when compared to those treated with the MPC control constructs. Motor endplate-expressing constructs had increased myofiber cross-sectional area compared to MPC control constructs. CONCLUSION: Although our laboratory previously demonstrated that muscle and cartilage constructs could be used separately for hemilaryngeal reconstruction, this study suggests combining them with the modification of MEEs rather than MPCs, resulting in improved muscle recovery after recurrent laryngeal nerve injury. LEVEL OF EVIDENCE: NA Laryngoscope, 129:1293-1300, 2019.

Current Indications for Transnasal Esophagoscopy: An American Broncho-Esophagological Association Survey.

INTRODUCTION:: The aim of this study was to evaluate the current indications for and clinical factors influencing the use of transnasal esophagoscopy (TNE). METHODS:: An online survey was sent to American Broncho-Esophagological Association members, including questions on demographics, indications, and factors influencing the use of TNE. RESULTS:: Sixty of 251 members (24%) completed the survey. Ninety-three percent of respondents reported academic practices, while 98% practice in medium to large urban settings. Thirty-five (58%) completed laryngology fellowships. Mean monthly TNE procedure count was 7.15 (range, <1-35). The most common indications were dysphagia (82%), biopsy (50%), and laryngopharyngeal reflux (47%). Chronic cough, head and neck cancer screening, gastroesophageal reflux (GER), and tracheoesophageal puncture were also commonly reported indications (44% each). For laryngopharyngeal reflux and GER, most respondents perform TNE for recalcitrant disease following a medical trial of at least 3 months. Long-standing GER symptoms, documentation of GER on pH and impedance testing, and abnormal findings on previous esophagoscopy lead to greater TNE use. Specific dysphagia indications included abnormal esophagographic findings (70%), history or examination localizing to the esophagus (60%), solid dysphagia only (53%), and solid and/or liquid dysphagia (40%). The primary sites most likely to prompt TNE use for head and neck cancer surveillance were the esophagus (92.3%) and hypopharynx (84.6%). Balloon dilation was the most common indication for which respondents do not currently perform TNE but would like to (n = 8). CONCLUSIONS:: TNE indications have not been well established. According to respondents from the American Broncho-Esophagological Association, TNE is most commonly used for dysphagia and laryngopharyngeal reflux and slightly less so for GER and head and neck cancer screening and surveillance. Several clinical indicators were identified that influence the decision to perform TNE.

Three-dimensional tissue-engineered skeletal muscle for laryngeal reconstruction.

OBJECTIVE: There is an unmet need for tissue-engineered three-dimensional (3D) muscle constructs for laryngeal reconstruction. Functional engineered muscle could be used to repair postoncologic or traumatic defects or to medialize the vocal fold in cases of paresis/paralysis. Autologous, organized, engineered muscle that has adequate bulk integrates into host tissue and restores function currently does not exist. METHODS: Primary skeletal muscle progenitor cells (MPCs) were isolated from F344 rats. Three-dimensional muscle constructs were created by encapsulating MPCs via flow alignment in a customized collagen formulation and cultured under passive tension. Muscle-specific immunohistochemistry and confocal microscopy were used to evaluate muscle tissue differentiation. After 2 weeks of culture, muscle constructs were implanted into surgically created defects in the rat larynx. Postmortem function testing and histology was performed at 1 and 3 months. RESULTS: Immunohistochemistry with confocal microscopy demonstrated well-differentiated myotubes, which were well aligned and distributed throughout the engineered construct in vitro. There was evidence of restoration of normal laryngeal function at 1 month postoperative, as indicated by safe swallow (no aspiration events), weight gain, and excellent animal survival. Postmortem specimens demonstrated functional muscle contraction on ex vivo testing, and histology confirmed integration into host tissue. CONCLUSION: This is the first study to demonstrate that functional, 3D tissue-engineered skeletal muscle can be developed from primary MPCs and standardized oligomeric collagen. Collectively, these findings may have tremendous clinical implications for autologous laryngeal muscle repair and reconstruction. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:603-609, 2018.

Use of autologous adipose-derived mesenchymal stem cells for creation of laryngeal cartilage.

OBJECTIVES/HYPOTHESIS: Adipose-derived mesenchymal stem cells (ASCs) are an exciting potential cell source for tissue engineering because cells can be derived from the simple excision of autologous fat. This study introduces a novel approach for tissue-engineering cartilage from ASCs and a customized collagen oligomer solution, and demonstrates that the resultant cartilage can be used for laryngeal cartilage reconstruction in an animal model. STUDY DESIGN: Basic science experimental design. METHODS: ASCs were isolated from F344 rats, seeded in a customized collagen matrix, and cultured in chondrogenic differentiation medium for 1, 2, and 4 weeks until demonstrating cartilage-like characteristics in vitro. Large laryngeal cartilage defects were created in the F344 rat model, with the engineered cartilage used to replace the cartilage defects, and the rats followed for 1 to 3 months. Staining examined cellular morphology and cartilage-specific features. RESULTS: In vitro histological staining revealed rounded chondrocyte-appearing cells evenly residing throughout the customized collagen scaffold, with positive staining for cartilage-specific markers. The cartilage was used to successfully repair large cartilaginous defects in the rat model, with excellent functional results. CONCLUSIONS: This study is the first study to demonstrate, in an animal model, that ASCs cultured in a unique form of collagen oligomer can create functional cartilage-like grafts that can be successfully used for partial laryngeal cartilage replacement. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E123-E129, 2018.

Improved adductor function after canine recurrent laryngeal nerve injury and repair using muscle progenitor cells.

OBJECTIVE: Muscle progenitor cells (MPCs) can be isolated from muscle samples and grown to a critical mass in culture. They have been shown to survive and integrate when implanted into rat laryngeal muscles. In this study, the ability of MPC implants to enhance adductor function of reinnervated thyroarytenoid muscles was tested in a canine model. STUDY DESIGN: Animal study. METHODS: Sternocleidomastoid muscle samples were harvested from three canines. Muscle progenitor cells were isolated and cultured to 107 cells over 4 to 5 weeks, then implanted into right thyroarytenoid muscles after ipsilateral recurrent laryngeal nerve transection and repair. The left sides underwent the same nerve injury, but no cells were implanted. Laryngeal adductor force was measured pretreatment and again 6 months later, and the muscles were harvested for histology. RESULTS: Muscle progenitor cells were successfully cultured from all dogs. Laryngeal adductor force measurements averaged 60% of their baseline pretreatment values in nonimplanted controls, 98% after implantation with MPCs, and 128% after implantation with motor endplate-enhanced MPCs. Histology confirmed that the implanted MPCs survived, became integrated into thyroarytenoid muscle fibers, and were in close contact with nerve endings, suggesting functional innervation. CONCLUSION: Muscle progenitor cells were shown to significantly enhance adductor function in this pilot canine study. Patient-specific MPC implantation could potentially be used to improve laryngeal function in patients with vocal fold paresis/paralysis, atrophy, and other conditions. Further experiments are planned. LEVEL OF EVIDENCE: NA. Laryngoscope, 2017.

Sulforaphane suppresses the growth of glioblastoma cells, glioblastoma stem cell-like spheroids, and tumor xenografts through multiple cell signaling pathways.

OBJECTIVE Defects in the apoptotic machinery and augmented survival signals contribute to drug resistance in glioblastoma (GBM). Moreover, another complexity related to GBM treatment is the concept that GBM development and recurrence may arise from the expression of GBM stem cells (GSCs). Therefore, the use of a multifaceted approach or multitargeted agents that affect specific tumor cell characteristics will likely be necessary to successfully eradicate GBM. The objective of this study was to investigate the usefulness of sulforaphane (SFN)-a constituent of cruciferous vegetables with a multitargeted effect-as a therapeutic agent for GBM. METHODS The inhibitory effects of SFN on established cell lines, early primary cultures, CD133-positive GSCs, GSC-derived spheroids, and GBM xenografts were evaluated using various methods, including GSC isolation and the sphere-forming assay, analysis of reactive oxygen species (ROS) and apoptosis, cell growth inhibition assay, comet assays for assessing SFN-triggered DNA damage, confocal microscopy, Western blot analysis, and the determination of in vivo efficacy as assessed in human GBM xenograft models. RESULTS SFN triggered the significant inhibition of cell survival and induced apoptotic cell death, which was associated with caspase 3 and caspase 7 activation. Moreover, SFN triggered the formation of mitochondrial ROS, and SFN-triggered cell death was ROS dependent. Comet assays revealed that SFN increased single- and double-strand DNA breaks in GBM. Compared with the vehicle control cells, a significantly higher amount of γ-H2AX foci correlated with an increase in DNA double-strand breaks in the SFN-treated samples. Furthermore, SFN robustly inhibited the growth of GBM cell-induced cell death in established cell cultures and early-passage primary cultures and, most importantly, was effective in eliminating GSCs, which play a major role in drug resistance and disease recurrence. In vivo studies revealed that SFN administration at 100 mg/kg for 5-day cycles repeated for 3 weeks significantly decreased the growth of ectopic xenografts that were established from the early passage of primary cultures of GBM10. CONCLUSIONS These results suggest that SFN is a potent anti-GBM agent that targets several apoptosis and cell survival pathways and further preclinical and clinical studies may prove that SFN alone or in combination with other therapies may be potentially useful for GBM therapy.

A Systematic Review.

Objectives (1) To systematically identify studies evaluating the use of intralesional cidofovir or bevacizumab as an adjunct in adult recurrent respiratory papillomatosis, determine disease severity and functional outcomes, and assess study quality. (2) To compare outcomes between the 2 adjuncts. Data Sources Ovid Medline, EMBASE, Scopus, and Clinical-Trials.gov . Review Methods Data sources were systematically searched. A priori inclusion and exclusion criteria were instituted. Quality was evaluated with the Newcastle-Ottawa Quality Assessment Scale. A priori criteria were instituted to select studies suitable for comparison. Results A total of 254 identified studies led to 16 for full-text review, including 14 for cidofovir and 2 for bevacizumab. Disease severity outcomes were reported in all studies, including remission rate, Derkay scores, time interval between operations, and/or lesion volume reduction. Remission rate was the most commonly reported (14 studies). Functional outcomes were reported in 5 studies (36%), including quality-of-life questionnaires, acoustic/aerodynamic analysis, and perceptual voice analysis. Voice-related quality of life was the most commonly reported (2 studies). Of 16 studies, 12 (75%) were rated poor quality. Reports almost invariably showed improved disease severity and functional outcomes following treatment; however, variable outcome measures and inadequate follow-up disallowed direct comparison of adjuncts. Conclusion Remission rate was the most commonly reported disease severity outcome, and voice-related quality of life was the most commonly reported functional outcome. Most studies were of poor quality. No studies met criteria for comparative analysis between adjuncts. Future research would be improved by reporting consistent and comparable disease severity and functional outcomes, treatment protocols, and follow-up.

Microarray Analysis Gene Expression Profiles in Laryngeal Muscle After Recurrent Laryngeal Nerve Injury.

OBJECTIVES: The pathophysiology of recurrent laryngeal nerve (RLN) transection injury is rare in that it is characteristically followed by a high degree of spontaneous reinnervation, with reinnervation of the laryngeal adductor complex (AC) preceding that of the abducting posterior cricoarytenoid (PCA) muscle. Here, we aim to elucidate the differentially expressed myogenic factors following RLN injury that may be at least partially responsible for the spontaneous reinnervation. METHODS: F344 male rats underwent RLN injury (n = 12) or sham surgery (n = 12). One week after RLN injury, larynges were harvested following euthanasia. The mRNA was extracted from PCA and AC muscles bilaterally, and microarray analysis was performed using a full rat genome array. RESULTS: Microarray analysis of denervated AC and PCA muscles demonstrated dramatic differences in gene expression profiles, with 205 individual probes that were differentially expressed between the denervated AC and PCA muscles and only 14 genes with similar expression patterns. CONCLUSIONS: The differential expression patterns of the AC and PCA suggest different mechanisms of reinnervation. The PCA showed the gene patterns of Wallerian degeneration, while the AC expressed the gene patterns of reinnervation by adjacent axonal sprouting. This finding may reveal important therapeutic targets applicable to RLN and other peripheral nerve injuries.