Airway Protection-A Role for Vagal P2RY1 Receptors.

Recent events bring the importance of respiratory health to the forefront of our collective attention. In this issue of Cell, a new study by Prescott and Umans et al. reveals how a dedicated laryngeal sensory motor reflex circuit protects our airways from aspirated foods or liquids.

Multi-institutional search for patient factors associated with adverse events following tracheotomy.

BACKGROUND: Tracheotomy is a common procedure for otolaryngologists. The risk of complications is difficult to predict. This study aims to identify measurable preoperative indicators associated with adverse events following tracheotomy. METHODS: The charts of adults undergoing tracheotomy for respiratory failure at one of four university-affiliated hospitals between 1/2012 and 8/2018 were reviewed. Complications were analyzed in the context of demographics, physiologic parameters, and comorbidities. RESULTS: Among 507 tracheotomies performed, the most common complications included infection, bleeding, and cardiac arrest. Mortality was 39 % in patients with pulmonary hypertension, 42 % in those with ejection fraction ≤ 40 and 32 % in those with abnormal right ventricular function, double the rates in patients without each of these findings. CONCLUSION: Many critically ill tracheotomy patients experience significant rates of adverse events. Risk factors for mortality include ejection fraction ≤ 40, pulmonary hypertension, and abnormal ventricular function. These should be considered for use in preoperative counseling.

Small Intestinal Submucosa Implantation for the Possible Treatment of Vocal Fold Scar, Sulcus, and Superficial Lamina Propria Atrophy.

OBJECTIVES: Evaluate the histologic effects of grafting porcine-derived small intestinal submucosa (SIS) into the vocal fold superficial lamina propria (SLP) layer for the potential treatment of vocal fold scar, sulcus and superficial lamina propria atrophy. METHODS: Small intestinal submucosa was implanted into the right vocal fold SLP of 6 mongrel dogs. The left vocal fold served as a sham surgical control. At 2, 4, and 6 weeks postoperative, bilateral vocal fold specimens were evaluated histologically. RESULTS: At 2 and 4 weeks, respectively, SIS-implanted vocal folds demonstrated moderate and mild inflammation and acute and chronic inflammation. At 6 weeks, inflammation was minimal and chronic. The 6-week specimens showed copious amounts of newly generated hyaluronic acid (HA) within the graft. There was no reactive fibrosis at 6 weeks. CONCLUSIONS: In the canine model, SIS appears safe for SLP grafting. Inflammation is similar to that of sham surgery. Small intestinal submucosa results in newly generated HA without concomitant fibrosis. Small intestinal submucosa has potential to be used in treatment of disorders with SLP, including vocal fold scar, sulcus, and atrophy. Studies evaluating the effect of SIS implantation on vocal fold function, as well as the ultimate fate of the graft, are required.

Extramedullary plasmacytoma of the cricoid cartilage with solitary plasmacytoma of the rib.

BACKGROUND: Extramedullary plasmacytoma is a rare plasma cell neoplasm of the soft tissues characterized by the presence of a single, discrete lesion without evidence of systemic disease. Extramedullary plasmacytoma may disseminate into multiple myeloma, a systemic plasma cell disease. METHODS: A rare case of extramedullary plasmacytoma of the cricoid cartilage with solitary plasmacytoma of the rib was reviewed. RESULTS: The patient was found to have two discrete lesions; one of the cricoid cartilage and one of the lateral left fifth rib. Despite the presence of multiple tumors, the patient was not diagnosed with multiple myeloma as the bone marrow appeared normal. CONCLUSIONS: Due to the rarity of these neoplasms and the unusual localization of the extramedullary plasmacytoma tumor, a definitive diagnosis was difficult to make in this case. This case may be instructive for the differential diagnosis of laryngeal lesions.

Lateral Transcervical In-office Botulinum Toxin Injection for Retrograde Cricopharyngeal Dysfunction.

OBJECTIVE: Operating room (OR) injection of botulinum toxin (BTX) injection is effective in the management of retrograde cricopharyngeal dysfunction (RCPD). This study aims to analyze the efficacy and safety of in-office (IO) 30 Unit BTX injection into the cricopharyngeus via lateral transcervical approach. METHODS: A retrospective chart review of patients who underwent BTX injection either in the OR or IO for RCPD was performed. Postoperative success, defined by patient-reported complete or near complete resolution of symptoms, side effects, and complication rates of each group was determined and compared. To determine the learning curve of IO injections, success rates of the injections performed in the early and late 6 months were compared. Chi-square test was used for determining the statistical significance. RESULTS: Overall, 78 injections (37 IO and 41 OR) for RCPD were performed by the senior author. The success rate of OR injections (90.2%) is significantly higher than IO injections (64.9%) at the first-month follow-up (p = 0.022). No significant difference was found in the side effect rates. Success and side effect rates were also similar in early and late injections (p > 0.05). CONCLUSIONS: IO lateral transcervical BTX injection for RCPD is a safe method that does not require general or topical anesthesia. While the side effects are similar and IO injections have many advantages, the success rates are lower than OR injections. LEVEL OF EVIDENCE: 3 Laryngoscope, 134:283-286, 2024.

Quality of Office-based Procedure Training During Laryngology Fellowship.

OBJECTIVES: The primary objective was to assess the perspectives of recent laryngology fellowship graduates on office-based procedure training, with a secondary objective to compare this with previous research on perspectives of fellowship directors. METHODS: Recent laryngology fellowship graduates were surveyed via an online survey platform regarding post-fellowship practice and various aspects of office-based procedure training, including perceived competence, mentorship, and barriers. RESULTS: There were 51 respondents. Seventy-six percent of respondents felt they "definitely" received adequate office procedure training. Number of procedures as primary surgeon was significantly associated with perception of adequate training (OR 1.54, 95% CI: 1.08-2.19, p = 0.018) and high post-fellowship office procedure volume (OR 1.56, 95% CI: 1.02-2.39, p = 0.040). Fellows reported a lower percentage of procedures as primary surgeons compared with program directors (46.8% vs. 61.9%, p = 0.028). Fellows and directors agreed that informal debriefs were more commonly employed than more structured training elements such as checklists and simulators. Of nine office procedures, laryngeal electromyography, KTP laser, and transnasal esophagoscopy had the greatest decreases in practice after training. CONCLUSION: Although most recent laryngology fellowship graduates endorse adequate office-based procedure training, a range of individual experiences exists, and office procedure volume, both overall and across individual procedures, may decrease after fellowship. Fellows performing office procedures as primary surgeons may be linked to perceived quality of training and post-fellowship volume. LEVEL OF EVIDENCE: NA Laryngoscope, 134:1802-1806, 2024.

Netrin-1 as A neural guidance protein in development and reinnervation of the larynx.

Neural guidance proteins participate in motor neuron migration, axonal projection, and muscle fiber innervation during development. One of the guidance proteins that participates in axonal pathfinding is Netrin-1. Despite the well-known role of Netrin-1 in embryogenesis of central nervous tissue, it is still unclear how the expression of this guidance protein contributes to primary innervation of the periphery, as well as reinnervation. This is especially true in the larynx where Netrin-1 is upregulated within the intrinsic laryngeal muscles after nerve injury and where blocking of Netrin-1 alters the pattern of reinnervation of the intrinsic laryngeal muscles. Despite this consistent finding, it is unknown how Netrin-1 expression contributes to guidance of the axons towards the larynx. Improved knowledge of Netrin-1's role in nerve regeneration and reinnervation post-injury in comparison to its role in primary innervation during embryological development, may provide insights in the search for therapeutics to treat nerve injury. This paper reviews the known functions of Netrin-1 during the formation of the central nervous system and during cranial nerve primary innervation. It also describes the role of Netrin-1 in the formation of the larynx and during recurrent laryngeal reinnervation following nerve injury in the adult.

Transcriptome Analysis of Left Versus Right Intrinsic Laryngeal Muscles Associated with Innervation.

OBJECTIVES/HYPOTHESIS: Recurrent laryngeal nerve injury diagnosed as idiopathic or due to short-term surgery-related intubation exhibits a higher incidence of left-sided paralysis. While this is often attributed to nerve length, it is hypothesized there are asymmetric differences in the expression of genes related to neuromuscular function that may impact reinnervation and contribute to this laterality phenomenon. To test this hypothesis, this study analyzes the transcriptome profiles of the intrinsic laryngeal muscles (ILMs), comparing gene expression in the left versus right, with particular attention to genetic pathways associated with neuromuscular function. STUDY DESIGN: Laboratory experiment. METHODS: RNA was extracted from the left and right sides of the rat posterior cricoarytenoid (PCA), lateral thyroarytenoid (LTA), and medial thyroarytenoid (MTA), respectively. After high-throughput RNA-Sequencing, 88 samples were organized into 12 datasets according to their age (P15/adult), sex (male/female), and muscle type (PCA/LTA/MTA). A comprehensive bioinformatics analysis was conducted to compare the left-right ILMs across different conditions. RESULTS: A total of 774 differentially expressed genes were identified across the 12 experimental groups, revealing age, sex, and muscle-specific differences between the left versus right ILMs. Enrichment analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways implicated several genes with a left-right laryngeal muscle asymmetry. These genes are associated with neuronal and muscular physiology, immune/inflammatory response, and hormone control. CONCLUSION: Bioinformatics analysis confirmed divergent transcriptome profiles between the left-right ILMs. This preliminary study identifies putative gene targets that will characterize ILM laterality. LEVEL OF EVIDENCE: N/A Laryngoscope, 2024.

Development and Application of Automated Vocal Fold Tracking Software in a Rat Surgical Model.

OBJECTIVE: The rat is a widely used model for studying vocal fold (VF) function after recurrent laryngeal nerve injury, but common techniques for evaluating rat VF motion remain subjective and imprecise. To address this, we developed a software package, called RatVocalTracker1.0 (RVT1.0), to quantify VF motion and tested it on rats with iatrogenic unilateral vocal fold paralysis (VFP). METHODS: A deep neural network was trained to identify the positions of the VFs and arytenoid cartilages (ACs) in transoral laryngoscope videos of the rat glottis. Software was developed to estimate glottic midline, VF displacement, VF velocity, and AC angle. The software was applied to laryngoscope videos of adult rats before and after right recurrent and superior laryngeal nerve transection (N = 15; 6M, 9F). All software calculated metrics were compared before and after injury and validated against manually calculated metrics. RESULTS: RVT1.0 accurately tracked and quantified VF displacement, VF velocity, and AC angle. Significant differences were found before and after surgery for all RVT1.0 calculated metrics. There was strong agreement between programmatically and manually calculated measures. Automated analysis was also more efficient than nearly all manual methods. CONCLUSION: This approach provides fast, accurate assessment of VF motion in rats with minimal labor and allows for quantitative comparison of lateral differences in movement. Through this novel analysis method, we can differentiate healthy movement from unilateral VFP. RVT1.0 is open-source and will be a valuable tool for researchers using the rat model for laryngology research. LEVEL OF EVIDENCE: NA Laryngoscope, 134:340-346, 2024.

Botulinum Toxin Injection for Retrograde Cricopharyngeal Dysfunction: A Prospective Cohort Study.

OBJECTIVES: Retrograde Cricopharyngeal Dysfunction (RCPD) is treated by botulinum toxin (BTX) injection into the cricopharyngeus. This prospective study compares the effectiveness and side effects of operating room (OR) and in-office (IO) injections. METHODS: Patients over 18 years of age with inability to burp, abdominal, thoracic, or cervical gurgling sounds, bloating, and excessive flatulence were diagnosed with RCPD and included in the study. Injections were performed in the OR (80U) or IO (30U) by the senior author. An RCPD questionnaire quantifying major and minor symptoms on a Likert scale, Eating Assessment Tool-10 (EAT-10), and Generalized Anxiety Score-7 (GAD-7), were completed preinjection; at 1, 2, and 3 weeks; and 3 months postoperatively. Linear mixed models were used to analyze effects of BTX injection on RCPD symptoms, the EAT-10, and the GAD-7. RESULTS: 108 (55 M/53F) patients completed the pretreatment survey, 53 (31 OR vs. 22 IO) completed the 3-week follow-up, and 36 (22 OR vs. 14 IO) completed the 3-month questionnaire. Average posttreatment RCPD scores were significantly lower in both groups at 3 weeks and 3 months (p < 0.0001), There was no difference between IO or OR (p = 0.4924). GAD-7 scores were significantly lower in both groups at week 3 (p = 0.0018) and month 3 (p = 0.0012). Postinjection EAT-10 scores were significantly higher in OR compared with IO (p = 0.0379). CONCLUSION: OR and IO injections are equally effective in the treatment of RCPD. Postinjection dysphagia is more severe after the OR injections which may be related to higher doses of BTX used. General anxiety levels decrease with treatment. LEVEL OF EVIDENCE: 2 Laryngoscope, 2024.

A comparison of confocal and epifluorescence microscopy for quantification of RNAScope and immunohistochemistry fluorescent images.

BACKGROUND: Quantification of RNA expression and protein production in fluorescent stainings provides critical information concerning neurodevelopment. A trustable independent quantification technique requires acquisition of reliable images prior to image processing. There is uncertainty in existing literature regarding the use of confocal microscopy compared to standard epifluorescence microscopy, especially in the context of RNA in situ hybridization protocols. NEW METHOD: The hindbrains of developing rat embryos from embryologic day 14 (E14) to E20 were sectioned and stained for expression of Hoxb1, Hoxb2, and Phox2b using both RNAScope and immunohistochemistry. Islet1 was used for identification of hindbrain motoneuron cell bodies. Slides were imaged using both confocal and epifluorescence microscopy. RESULTS: Expression patterns of both mRNA and protein were similar in both imaging modalities. Analyses of Hoxb1 and Hoxb2 mRNA expression were particularly concordant between-scopes, with similar p-values and posthoc differences between timepoints. Confocal imaging of Hoxb2 protein yielded a significant peak at E18, but this level of significance was not reached using epifluorescence microscopy. Although similar trends were observed, only Phox2b RNAScope results were statistically significant when analyzed with confocal microscopy. In contrast, Phox2b immunostaining analyses showed significant differences using both microscopes. COMPARISON WITH EXISTING METHODS: Researchers may save time and financial resources if epifluorescence microscopy provides comparable or equal results as confocal. CONCLUSIONS: Epifluorescence microscopy appears sufficient for quantification of RNAScope experiments with relatively low puncta per cell, while confocal microscopy gives clearer definition to immunohistochemical protein relationships and may be preferable especially in targets with low protein production.

Recurrent laryngeal nerve transection and anastomosis: rat laryngeal motoneuron survival and effect of the anastomosis site.

OBJECTIVES: We investigated the quantity of recurrent laryngeal nerve motoneurons (RLNMs) that survive after transection and anastomosis of the rat recurrent laryngeal nerve (RLN), as well as the impact of the anastomosis site on RLN regeneration. METHODS: Ten rats underwent right RLN transection and anastomosis. After 16 weeks, Fluoro-Ruby (FR) was applied to the RLN that was transected proximal or distal to the anastomosis site. The brain stems were harvested, and the nucleus ambiguus was evaluated for labeled RLNMs. The RLNM counts were compared to each other and to those from 3 control rats in which FR was applied to an acutely transected RLN. RESULTS: The number of RLNMs that were stained after RLN transection, anastomosis, and regeneration was consistent with the total number of RLNMs in the nucleus ambiguus of control rats. This finding confirms that most RLNMs survived after RLN transection and anastomosis. The quantity of labeled RLNMs was statistically similar whether the FR was applied proximal or distal to the anastomosis, implying that most of the viable axons that were present proximal to the anastomosis crossed into the distal nerve. CONCLUSIONS: Rat RLNMs survive nerve transection, anastomosis, and regeneration. The anastomosis site does not significantly impede axonal regeneration, and most of the axons traverse the anastomosis into the distal nerve.

A review of the peripheral proprioceptive apparatus in the larynx.

The larynx is an organ of the upper airway that participates in breathing, glutition, voice production, and airway protection. These complex functions depend on vocal fold (VF) movement, facilitated in turn by the action of the intrinsic laryngeal muscles (ILM). The necessary precise and near-instantaneous modulation of each ILM contraction relies on proprioceptive innervation of the larynx. Dysfunctional laryngeal proprioception likely contributes to disorders such as laryngeal dystonia, dysphagia, vocal fold paresis, and paralysis. While the proprioceptive system in skeletal muscle derived from somites is well described, the proprioceptive circuitry that governs head and neck structures such as VF has not been so well characterized. For over two centuries, researchers have investigated the question of whether canonical proprioceptive organs, muscle spindles, and Golgi tendon organs, exist in the ILM, with variable findings. The present work is a state-of-the-art review of the peripheral component of laryngeal proprioception, including current knowledge of canonical and possible alternative proprioceptive circuitry elements in the larynx.

Corrigendum: A review of the peripheral proprioceptive apparatus in the larynx.

[This corrects the article DOI: 10.3389/fnana.2023.1114817.].

Expression of Glial Cell-Derived Neurotrophic Factor Receptors Within Nucleus Ambiguus During Rat Development.

OBJECTIVE: The nucleus ambiguus (NAmb) is a column of neurons in the medulla oblongata, involved in bulbar functions. Expression of Glial Cell-Derived Neurotrophic Factor (GDNF) and its receptors (GDNFR) is observed within the cell bodies during reinnervation following recurrent laryngeal nerve (RLN) injury. Little is known regarding GDNFR expression in the formation of the NAmb and the laryngeal innervation during embryogenesis. Understanding the timing and pattern of GDNFR expression in embryogenesis versus after RLN injury may provide insights into therapeutic targets for regeneration after RLN injury. STUDY DESIGN: Laboratory experiment. METHODS: Rat brainstems at E14.5/E16.5/E18.5/E20.5/adult were stained for GDNFR: GFRα-1/GFRα-2/GFRα-3/Ret. Islet1 and choline acetyltransferase were used as cell body markers. Sections were observed using fluorescent microscopy and quantified through manual cell counting. RESULTS: Expression of GFRα-1, GFRα-3, and Ret was identified within the NAmb, hypoglossal, and facial nuclei of the adult medulla. During development, GFRα-1 immunoreactivity was seen at E20.5. GFRα-2 expression was not observed at any timepoint. GFRα-3 expression began at E16.5. Ret expression within nerve fibers in the NAmb were observed beginning at E14.5, but never in the cell bodies. CONCLUSION: Embryonic GDNFR expression in the NAmb differs from that of the adult after RLN injury. The developing brainstem experienced upregulation at discrete timepoints with signaling sustained through adulthood. In contrast, adult RLN-transected rats experienced patterns of up and down regulation. GFRα-1 may contribute to muscle targeting and neuromuscular junction maturation, GFRα-3 may contribute to both, as well as axon guidance. It is likely that GDNF is functioning via a Ret-independent pathway. LEVEL OF EVIDENCE: NA Laryngoscope, 133:2240-2247, 2023.

Transcriptome analysis of left versus right intrinsic laryngeal muscles associated with innervation.

Objectives/Hypothesis: Recurrent laryngeal nerve injury diagnosed as idiopathic or due to short-term surgery-related intubation exhibits a higher incidence of left-sided paralysis. While this is often attributed to nerve length, it is hypothesized there are asymmetric differences in the expression of genes related to neuromuscular function that may impact reinnervation and contribute to this laterality phenomenon. To test this hypothesis, this study analyzes the transcriptome profiles of the intrinsic laryngeal muscles (ILMs), comparing gene expression in the left versus right, with particular attention to genetic pathways associated with neuromuscular function. Study Design: Laboratory experiment. Methods: RNA was extracted from the left and right sides of the rat posterior cricoarytenoid (PCA), lateral thyroarytenoid (LTA), and medial thyroarytenoid (MTA), respectively. After high-throughput RNA-Sequencing (RNA-Seq), 88 samples were organized into 12 datasets according to their age (P15/adult), sex (male/female), and muscle type (PCA/LTA/MTA). A comprehensive bioinformatics analysis was conducted to compare the left-right ILMs across different conditions. Results: 774 differentially expressed genes (DEGs) were identified across the 12 experimental groups, revealing age, sex, and muscle-specific differences between the left versus right ILMs. Enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways implicated several genes with a left-right laryngeal muscle asymmetry. These genes are associated with neuronal and muscular physiology, immune/inflammatory response, and hormone control. Conclusion: Bioinformatics analysis confirmed divergent transcriptome profiles between the left-right ILMs. This preliminary study identifies putative gene targets that will characterize ILM laterality. Level of Evidence: N/A. LAY SUMMARY: Vocal fold paralysis is more common on the left. This study shows left versus right differences in gene expression related to innervation, suggesting the increased rate of left recurrent laryngeal nerve paralysis may be associated with genetic differences, not just nerve length.

The Effects of Endotracheal Tube Size During Bronchoscopy in Simulated Models of Intubated Patients.

OBJECTIVE: The aim is to use a simulation lung model to assess the possibility of performing bronchoscopy through endotracheal tubes (ETT) less than 8.0-mm while appropriately ventilating patients with normal and ARDS lungs in the setting of SARS-CoV-2. METHODS: Five SHERIDAN® ETTs were used to ventilate SimMan® 3G under respiratory compliance levels representing normal and severe ARDS lungs. Baseline measurements of peak pressure, plateau pressure, and auto-positive end expiratory pressure (auto-PEEP) were recorded at four different inspiratory times (Ti). Three different-sized disposable bronchoscopes were inserted, and all measurements were repeated. RESULTS: Normal lung model: Slim bronchoscopes in 6.0-mm ETTs resulted in plateau pressures <30 cm H2 O, and increasing Ti to minimize peak pressure resulted in low auto-PEEP. Regular bronchoscopes in 7.0-mm ETTs had similar results. Large bronchoscopes in 7.5-mm ETTs generated plateau pressures ranging from 28 to 35 cm H2 O with modest auto-PEEP. Severe ARDS lung model: Slim bronchoscopes in 6.0-mm ETTs resulted in plateau pressures of 46 and an auto-PEEP of 5 cm H2 O. Regular bronchoscopes in 7.0-mm ETTs generated similar results. Large bronchoscopes in 8.0-mm ETTs displayed plateau pressures of 44 and an auto-PEEP of 2 cm H2 O. CONCLUSION: To mitigate risk of laryngeal injury, larger ETTs during bronchoscopy should be avoided. Our data show bronchoscopy with any ETT causes auto-PEEP and high plateau pressures, especially in lungs with poor compliance; however, ETT less than 7.5 mm can be used when considering several factors. Our data also suggest similar studies in patients with varying degrees of ARDS would be informative. LEVEL OF EVIDENCE: NA Laryngoscope, 133:147-153, 2023.

Duration of Clinical Response After In-Office Steroid Injection for Vocal Fold Scar.

OBJECTIVE: To assess the duration of clinical response after in-office vocal fold steroid injection (VFSI) for vocal fold (VF) scar. METHODS: Demographic and clinical data for in-office VFSI occurring from 2017 to 2020 were collected. Two Speech-Language Pathologists (SLPs) used perceptual evaluation of voice and functional scales to evaluate blinded voice and laryngovideostroboscopy (LVS) samples collected pre- and post-injection across multiple timepoints. RESULTS: Blinded SLP ratings were used for 30 individual VFs undergoing initial injection in 18 patients. Persistent improvement in voice past 6 months was seen in 57% of patients after VFSI. Multiple measures of voice and amplitude, percent vibrating tissue, and closed phase predominance significantly improved at various follow-up timepoints on average. CONCLUSION: Accounting for patient heterogeneity and disease progression, in-office VFSI for VF scar is associated with sustained improvement in a subset of patients. Approximately half of patients can expect to experience a lasting improvement in voice. Future studies of larger scale are required to identify patient factors associated with long-term benefit. LEVEL OF EVIDENCE: 4 Laryngoscope, 133:2333-2339, 2023.

Temporal Expression of Hox Genes and Phox2b in the Rat Nucleus Ambiguus During Development: Implications on Laryngeal Innervation.

OBJECTIVES: Recurrent laryngeal nerve (RLN) injury results in synkinetic reinnervation and vocal fold paralysis. Investigation of cues expressed in the developing brainstem that influence correct selective targeting of intrinsic laryngeal muscles may elucidate post-injury abnormalities contributing to non-functional reinnervation. Primary targets of interest were Hoxb1 and Hoxb2, members of the Hox family that create overlapping gradients in the developing brain, and their target Phox2b, a transcription factor necessary for cranial nerve branchio- and visceromotoneuron survival. METHODS: Rat embryos at developmental days E14, E16, E18, and E20 (4 animals/age) were sectioned for RNA in situ hybridization to detect Hoxb1, Hoxb2, and Phox2b mRNA within the brainstem. Slides were costained with Islet1 antibody for identification of the nucleus ambiguus. Results were confirmed using immunohistochemistry. Sections were imaged on a confocal microscope. RNA and protein expressions were quantified using QuPath. Statistical analyses were performed using R. RESULTS: Hoxb1, Hoxb2, and Phox2b expressions varied according to embryologic age. Hoxb1 and Hoxb2 expression peaked at E16, with significant decreases at E18 and E20 (one-way ANOVA p = 0.001 for both). Phox2b expression was highest at E14 and trended downward with increased embryologic age (one-way ANOVA p = 0.005). CONCLUSION: Peak expression of Hoxb1 and Hoxb2 is observed at time points when the RLN arrives at the larynx and begins to branch toward individual muscles, positioning these gene products to be involved in cueing laryngeal motoneuron identity and target identification. Higher expression of Phox2b earlier in development suggests a role in laryngeal motoneuron formation. LEVEL OF EVIDENCE: NA Laryngoscope, 133:3462-3471, 2023.

Healthy Human Laryngopharyngeal Sensory Innervation Density Correlates with Age.

OBJECTIVE: Somatosensory feedback from upper airway structures is essential for swallowing and airway defense but little is known about the identities and distributions of human upper airway neurons. Furthermore, whether sensory innervation modifies with aging is unknown. In this study, we quantify neuronal and chemosensory cell density in upper airway structures and correlate with age. METHODS: Participants underwent biopsies from base of tongue, lateral and midline pharyngeal wall, epiglottis, and arytenoids (N = 25 13 female/12 male; 20-80 years, mean 51.4 years without clinical diagnosis of dysphagia or clinical indication for biopsy). Tissue sections were labeled with antibodies for all neurons, myelinated neurons, and chemosensory cells. Densities of lamina propria innervation, epithelial innervation, solitary chemosensory cells, and taste buds were calculated and correlated with age. RESULTS: Arytenoid had the highest density of innervation and chemosensory cells across all measures compared to other sites. Taste buds were frequently observed in arytenoid and epiglottis. Base of tongue, lateral pharynx, and midline posterior pharynx had minimal innervation and few chemosensory cells. Epithelial innervation was present primarily in close proximity to chemosensory cells and taste buds. Overall innervation and myelinated fibers in the arytenoid lamina propria decline with aging. CONCLUSION: Findings establish the architecture of healthy adult sensory innervation and demonstrate the varied distribution of laryngopharyngeal innervation, necessary steps toward understanding the sensory basis for swallowing and airway defense. We also document age-related decline in arytenoid innervation density. These findings suggest that sensory afferent denervation of the upper airway may be a contributing factor to presbyphagia. LEVEL OF EVIDENCE: NA Laryngoscope, 133:773-784, 2023.