Impact of Limb Phenotype on Tongue Denervation Atrophy, Dysphagia Penetrance, and Survival Time in a Mouse Model of ALS.

Current treatments for dysphagia in ALS do not target the underlying tongue weakness and denervation atrophy that is prevalent in spinal and bulbar ALS cases. To address this clinical gap, we studied the low copy number SOD1-G93A (LCN-SOD1) mouse model of ALS to quantify the impact of limb phenotype on tongue denervation atrophy, dysphagia penetrance, and survival time in preparation for future treatment-based studies. Two male LCN-SOD1 breeders and 125 offspring were followed for limb phenotype inheritance, of which 52 (30 LCN-SOD1 and 22 wild-type/WT, both sexes) underwent characterization of dysphagia penetrance (via videofluoroscopic swallow study; VFSS) and survival time at disease end-stage (15-20% body weight loss). From these, 16 mice (8/genotype) underwent postmortem histological analysis of the genioglossus for evidence of denervation atrophy. Results revealed that both breeders displayed a mixed (hindlimb and forelimb) ALS phenotype and sired equal proportions of hindlimb vs. mixed phenotype offspring. Dysphagia penetrance was complete for mixed (100%) versus incomplete for hindlimb (64%) phenotype mice; yet survival times were similar. Regardless of limb phenotype, LCN-SOD1 mice had significantly smaller genioglossus myofibers and more centralized myonuclei compared to WT mice (p < 0.05). These biomarkers of denervation atrophy were significantly correlated with VFSS metrics (lick and swallow rates, p < 0.05) but not survival time. In conclusion, both LCN-SOD1 phenotypes had significant tongue denervation atrophy, even hindlimb phenotype mice without dysphagia. This finding recapitulates human ALS, providing robust rationale for using this preclinical model to explore targeted treatments for tongue denervation atrophy and ensuing dysphagia.

Advancing Laryngeal Adductor Reflex Testing Beyond Sensory Threshold Detection.

Flexible endoscopic evaluation of swallowing with sensory testing (FEESST) is a promising clinical tool to assess airway integrity via the laryngeal adductor reflex (LAR). The current clinical protocol relies on sensory threshold detection, as relatively little is known about the motor response of this sensorimotor airway protective reflex. Here, we focused on characterizing normative LAR motion dynamics in 20 healthy young participants using our prototype high-pressure syringe-based air pulse device and analytic software (VFtrack™) that tracks vocal fold (VF) motion in endoscopic videos. Following device bench testing for air pulse stimulus characterization, we evoked and objectively quantified LAR motion dynamics in response to two suprathreshold air pulse stimuli (40 versus 60 mm Hg), delivered to the arytenoid mucosa through a bronchoscope working channel. The higher air pressures generated by our device permitted an approximate 1 cm endoscope working distance for continual visualization of the bilateral VFs throughout the LAR. Post hoc video analysis identified two main findings: (1) there are variant and invariant subcomponents of the LAR motor response, and (2) only a fraction of suprathreshold stimuli evoked complete glottic closure during the LAR. While the clinical relevance of these findings remains to be determined, we have nonetheless demonstrated untapped potential in the current FEESST protocol. Our ongoing efforts may reveal LAR biomarkers to quantify the severity of laryngeal pathology and change over time with natural disease progression, spontaneous recovery, or in response to intervention. The ultimate goal is to facilitate predictive modeling of patients at high risk for dysphagia-related aspiration pneumonia.

Tongue and hypoglossal morphology after intralingual cholera toxin B-saporin injection.

INTRODUCTION: We recently developed an inducible model of dysphagia using intralingual injection of cholera toxin B conjugated to saporin (CTB-SAP) to cause death of hypoglossal neurons. In this study we aimed to evaluate tongue morphology and ultrastructural changes in hypoglossal neurons and nerve fibers in this model. METHODS: Tissues were collected from 20 rats (10 control and 10 CTB-SAP animals) on day 9 post-injection. Tongues were weighed, measured, and analyzed for microscopic changes using laminin immunohistochemistry. Hypoglossal neurons and axons were examined using transmission electron microscopy. RESULTS: The cross-sectional area of myofibers in the posterior genioglossus was decreased in CTB-SAP-injected rats. Degenerative changes were observed in both the cell bodies and distal axons of hypoglossal neurons. DISCUSSION: Preliminary results indicate this model may have translational application to a variety of neurodegenerative diseases resulting in tongue dysfunction and associated dysphagia.

Optimizing the Translational Value of Mouse Models of ALS for Dysphagia Therapeutic Discovery.

The goal of this study was to compare dysphagia phenotypes in low and high copy number (LCN and HCN) transgenic superoxide dismutase 1 (SOD1) mouse models of ALS to accelerate the discovery of novel and effective treatments for dysphagia and early amyotrophic lateral sclerosis (ALS) diagnosis. Clinicopathological features of dysphagia were characterized in individual transgenic mice and age-matched controls utilizing videofluoroscopy in conjunction with postmortem assays of the tongue and hypoglossal nucleus. Quantitative PCR accurately differentiated HCN-SOD1 and LCN-SOD1 mice and nontransgenic controls. All HCN-SOD1 mice developed stereotypical paralysis in both hindlimbs. In contrast, LCN-SOD1 mice displayed wide variability in fore- and hindlimb involvement. Lick rate, swallow rate, inter-swallow interval, and pharyngeal transit time were significantly altered in both HCN-SOD1 and LCN-SOD1 mice compared to controls. Tongue weight, tongue dorsum surface area, total tongue length, and caudal tongue length were significantly reduced only in the LCN-SOD1 mice compared to age-matched controls. LCN-SOD1 mice with lower body weights had smaller/lighter weight tongues, and those with forelimb paralysis and slower lick rates died at a younger age. LCN-SOD1 mice had a 32% loss of hypoglossal neurons, which differed significantly when compared to age-matched control mice. These novel findings for LCN-SOD1 mice are congruent with reported dysphagia and associated tongue atrophy and hypoglossal nucleus pathology in human ALS patients, thus highlighting the translational potential of this mouse model in ALS research.

A Surgical Mouse Model for Advancing Laryngeal Nerve Regeneration Strategies.

Iatrogenic recurrent laryngeal nerve (RLN) injury is a morbid complication of anterior neck surgical procedures. Existing treatments are predominantly symptomatic, ranging from behavioral therapy to a variety of surgical approaches. Though laryngeal reinnervation strategies often provide muscle tone to the paralyzed vocal fold (VF), which may improve outcomes, there is no clinical intervention that reliably restores true physiologic VF movement. Moreover, existing interventions neglect the full cascade of molecular events that affect the entire neuromuscular pathway after RLN injury, including the intrinsic laryngeal muscles, synaptic connections within the central nervous system, and laryngeal nerve anastomoses. Systematic investigations of this pathway are essential to develop better RLN regenerative strategies. Our aim was to develop a translational mouse model for this purpose, which will permit longitudinal investigations of the pathophysiology of iatrogenic RLN injury and potential therapeutic interventions. C57BL/6J mice were divided into four surgical transection groups (unilateral RLN, n = 10; bilateral RLN, n = 2; unilateral SLN, n = 10; bilateral SLN, n = 10) and a sham surgical group (n = 10). Miniaturized transoral laryngoscopy was used to assess VF mobility over time, and swallowing was assessed using serial videofluoroscopy. Histological assays were conducted 3 months post-surgery for anatomical investigation of the larynx and laryngeal nerves. Eight additional mice underwent unilateral RLN crush injury, half of which received intraoperative vagal nerve stimulation (iVNS). These 8 mice underwent weekly transoral laryngoscopy to investigate VF recovery patterns. Unilateral RLN injury resulted in chronic VF immobility but only acute dysphagia. Bilateral RLN injury caused intraoperative asphyxiation and death. VF mobility was unaffected by SLN transection (unilateral or bilateral), and dysphagia (transient) was evident only after bilateral SLN transection. The sham surgery group retained normal VF mobility and swallow function. Mice that underwent RLN crush injury and iVNS treatment demonstrated accelerated and improved VF recovery. We successfully developed a mouse model of iatrogenic RLN injury with impaired VF mobility and swallowing function that can serve as a clinically relevant platform to develop translational neuroregenerative strategies for RLN injury.

A mutation in the Warburg syndrome gene, RAB3GAP1, causes a similar syndrome with polyneuropathy and neuronal vacuolation in Black Russian Terrier dogs.

An autosomal recessive disease of Black Russian Terriers was previously described as a juvenile-onset, laryngeal paralysis and polyneuropathy similar to Charcot Marie Tooth disease in humans. We found that in addition to an axonal neuropathy, affected dogs exhibit microphthalmia, cataracts, and miotic pupils. On histopathology, affected dogs exhibit a spongiform encephalopathy characterized by accumulations of abnormal, membrane-bound vacuoles of various sizes in neuronal cell bodies, axons and adrenal cells. DNA from an individual dog with this polyneuropathy with ocular abnormalities and neuronal vacuolation (POANV) was used to generate a whole genome sequence which contained a homozygous RAB3GAP1:c.743delC mutation that was absent from 73 control canine whole genome sequences. An additional 12 Black Russian Terriers with POANV were RAB3GAP1:c.743delC homozygotes. DNA samples from 249 Black Russian Terriers with no known signs of POANV were either heterozygotes or homozygous for the reference allele. Mutations in human RAB3GAP1 cause Warburg micro syndrome (WARBM), a severe developmental disorder characterized by abnormalities of the eye, genitals and nervous system including a predominantly axonal peripheral neuropathy. RAB3GAP1 encodes the catalytic subunit of a GTPase activator protein and guanine exchange factor for Rab3 and Rab18 respectively. Rab proteins are involved in membrane trafficking in the endoplasmic reticulum, axonal transport, autophagy and synaptic transmission. The neuronal vacuolation and membranous inclusions and vacuoles in axons seen in this canine disorder likely reflect alterations of these processes. Thus, this canine disease could serve as a model for WARBM and provide insight into its pathogenesis and treatment.

Vestibular dysfunction, altered macular structure and trait localization in A/J inbred mice.

A/J mice develop progressive hearing loss that begins before 1 month of age and is attributed to cochlear hair cell degeneration. Screening tests indicated that this strain also develops early onset vestibular dysfunction and has otoconial deficits. The purpose of this study was to characterize the vestibular dysfunction and macular structural pathology over the lifespan of A/J mice. Vestibular function was measured using linear vestibular evoked potentials (VsEPs). Macular structural pathology was evaluated using light microscopy, scanning electron microscopy, transmission electron microscopy, confocal microscopy and Western blotting. Individually, vestibular functional deficits in mice ranged from mild to profound. On average, A/J mice had significantly reduced vestibular sensitivity (elevated VsEP response thresholds and smaller amplitudes), whereas VsEP onset latency was prolonged compared to age-matched controls (C57BL/6). A limited age-related vestibular functional loss was also present. Structural analysis identified marked age-independent otoconial abnormalities in concert with some stereociliary bundle defects. Macular epithelia were incompletely covered by otoconial membranes with significantly reduced opacity and often contained abnormally large or giant otoconia as well as normal-appearing otoconia. Elevated expression of key otoconins (i.e., otoconin 90, otolin and keratin sulfate proteoglycan) ruled out the possibility of reduced levels contributing to otoconial dysgenesis. The phenotype of A/J was partially replicated in a consomic mouse strain (C57BL/6J-Chr 17(A/J)/NaJ), thus indicating that Chr 17(A/J) contained a trait locus for a new gene variant responsible to some extent for the A/J vestibular phenotype. Quantitative trait locus analysis identified additional epistatic influences associated with chromosomes 1, 4, 9 and X. Results indicate that the A/J phenotype represents a complex trait, and the A/J mouse strain presents a new model for the study of mechanisms underlying otoconial formation and maintenance.

Videofluoroscopic Validation of a Translational Murine Model of Presbyphagia.

Presbyphagia affects approximately 40% of otherwise healthy people over 60 years of age. Hence, it is a condition of primary aging rather than a consequence of primary disease. This distinction warrants systematic investigations to understand the causal mechanisms of aging versus disease specifically on the structure and function of the swallowing mechanism. Toward this goal, we have been studying healthy aging C57BL/6 mice (also called B6), the most popular laboratory rodent for biomedical research. The goal of this study was to validate this strain as a model of presbyphagia for translational research purposes. We tested two age groups of B6 mice: young (4-7 months; n = 16) and old (18-21 months; n = 11). Mice underwent a freely behaving videofluoroscopic swallow study (VFSS) protocol developed in our lab. VFSS videos (recorded at 30 frames per second) were analyzed frame-by-frame to quantify 15 swallow metrics. Six of the 15 swallow metrics were significantly different between young and old mice. Compared to young mice, old mice had significantly longer pharyngeal and esophageal transit times (p = 0.038 and p = 0.022, respectively), swallowed larger boluses (p = 0.032), and had a significantly higher percentage of ineffective primary esophageal swallows (p = 0.0405). In addition, lick rate was significantly slower for old mice, measured using tongue cycle rate (p = 0.0034) and jaw cycle rate (p = 0.0020). This study provides novel evidence that otherwise healthy aging B6 mice indeed develop age-related changes in swallow function resembling presbyphagia in humans. Specifically, aging B6 mice have a generally slow swallow that spans all stages of swallowing: oral, pharyngeal, and esophageal. The next step is to build upon this foundational work by exploring the responsible mechanisms of presbyphagia in B6 mice.

Incidence and characterization of aerophagia in dogs using videofluoroscopic swallow studies.

BACKGROUND: Aerophagia (ingestion of air), is a functional aerodigestive disorder in people. Criteria for diagnosis of aerophagia in dogs are >1/3 of bolus volume containing air or ingested air resulting in gastric distention (>1/3 of end gastric volume). Aerophagia is highlighted during eating and drinking. Videofluoroscopic swallow studies (VFSS) document aerophagia in dogs, but the incidence, clinical signs (CS), and associated disorders are unknown. OBJECTIVES: Identify the incidence of aerophagia, compare CS between dogs with and without aerophagia, and identify associated and predisposing disorders using VFSS. ANIMALS: A total of 120 client-owned dogs. METHODS: Sequential VFSS and associated medical records from dogs presenting to veterinary teaching hospitals at Auburn University and the University of Missouri were retrospectively reviewed. Statistical comparisons were made using Mann-Whitney and chi-squared tests, odds ratios (OR), and multiple logistic regression (P < .05). RESULTS: The incidence (95% confidence interval [CI]) of aerophagia was 40% (31.7-48.9). Dogs with mixed CS (gastrointestinal [GI] and respiratory; P < .001, 58.3%) were more likely to have aerophagia than dogs with exclusively respiratory CS (25%). Aerophagia was significantly more common in brachycephalic dogs (P = .01; 45.8% vs 13.8%), dogs with nonbrachycephalic upper airway obstruction (P < .001; 33.3% vs 4.1%), pathologic penetration and aspiration (P-A) scores (P = .04; 41.6% vs 23.6%), and gagging (P < .001; 25% vs 11.7%). Mixed CS (P = .01), brachycephaly (P < .001), and upper airway obstruction (P < .001) were independent predictors of aerophagia. CONCLUSIONS AND CLINICAL IMPORTANCE: Aerophagia was common, particularly in dogs with mixed CS. Brachycephalic dogs and dogs with upper airway obstruction are predisposed. Aspiration risk was high, emphasizing overlapping upper aerodigestive pathways.

Minimally Invasive Murine Laryngoscopy for Close-Up Imaging of Laryngeal Motion during Breathing and Swallowing.

The larynx is an essential organ in mammals with three primary functions - breathing, swallowing, and vocalizing. A wide range of disorders are known to impair laryngeal function, which results in difficulty breathing (dyspnea), swallowing impairment (dysphagia), and/or voice impairment (dysphonia). Dysphagia, in particular, can lead to aspiration pneumonia and associated morbidity, recurrent hospitalization, and early mortality. Despite these serious consequences, existing treatments for laryngeal dysfunction are largely aimed at surgical and behavioral interventions that unfortunately do not typically restore normal laryngeal function, thus highlighting the urgent need for innovative solutions. To bridge this gap, we have been developing an experimental endoscopic approach to investigate laryngeal dysfunction in murine (i.e., mouse and rat) models. However, endoscopy in rodents is quite challenging due to their small size relative to current endoscope technology, anatomical differences in the upper airway, and the necessity for anesthesia to optimally access the larynx. Here, we describe a novel transoral laryngoscopy approach that permits close-up, unobstructed video imaging of laryngeal motion in mice and rats. Critical steps in the protocol include precise anesthesia management (to prevent overdosing that abolishes swallowing and/or risks respiratory distress-related mortality) and micromanipulator control of the endoscope (for stable video recording of laryngeal motion by a single researcher for subsequent quantification). Importantly, the protocol can be performed over time in the same animals to study the impact of various pathological conditions specifically on laryngeal function. A novel advantage of this protocol is the ability to visualize airway protection during swallowing, which is not possible in humans due to epiglottic inversion over the laryngeal inlet that obstructs the glottis from view. Rodents therefore provide a unique opportunity to specifically investigate the mechanisms of normal versus pathological laryngeal airway protection for the ultimate purpose of discovering treatments to effectively restore normal laryngeal function.

Radiation induced changes in profibrotic markers in the submental muscles and their correlation with tongue movement.

Swallowing impairment is a major complication of radiation treatment for oropharyngeal cancers. Developing targeted therapies that improve swallowing outcomes relies on an understanding of the mechanisms that influence motor function after radiation treatment. The purpose of this study was to determine whether there is a correlation between radiation induced changes in tongue movement and structural changes in irradiated submental muscles, as well as assess other possible causes for dysfunction. We hypothesized that a clinically relevant total radiation dose to the submental muscles would result in: a) quantifiable changes in tongue strength and displacement during drinking two months post treatment; and b) a profibrotic response and/or fiber type transition in the irradiated tissue. Sprague-Dawley adult male rats received radiation to the submental muscles at total dose-volumes known to provoke dysphagia in humans. A clinical linear accelerator administered 8 fractions of 8Gy for a total of 64Gy. Comparisons were made to sham-treated rats that received anesthesia only. Swallowing function was assessed using videofluoroscopy and tongue strength was analyzed via force lickometer. TGFß1 expression was analyzed via ELISA. The amount of total collagen was analyzed by picrosirius red staining. Immunofluorescence was used to assess fiber type composition and size. Significant changes in licking function during drinking were observed at two months post treatment, including a slower lick rate and reduced tongue protrusion during licking. In the mylohyoid muscle, significant increases in TGFß1 protein expression were found post radiation. Significant increases in the percentage of collagen content were observed in the irradiated geniohyoid muscle. No changes in fiber type expression were observed. Results indicate a profibrotic transition within the irradiated swallowing muscles that contributes to tongue dysfunction post-radiation treatment.

Prox2 and Runx3 vagal sensory neurons regulate esophageal motility.

Vagal sensory neurons monitor mechanical and chemical stimuli in the gastrointestinal tract. Major efforts are underway to assign physiological functions to the many distinct subtypes of vagal sensory neurons. Here, we use genetically guided anatomical tracing, optogenetics, and electrophysiology to identify and characterize vagal sensory neuron subtypes expressing Prox2 and Runx3 in mice. We show that three of these neuronal subtypes innervate the esophagus and stomach in regionalized patterns, where they form intraganglionic laminar endings. Electrophysiological analysis revealed that they are low-threshold mechanoreceptors but possess different adaptation properties. Lastly, genetic ablation of Prox2 and Runx3 neurons demonstrated their essential roles for esophageal peristalsis in freely behaving mice. Our work defines the identity and function of the vagal neurons that provide mechanosensory feedback from the esophagus to the brain and could lead to better understanding and treatment of esophageal motility disorders.

LARNet-STC: Spatio-temporal orthogonal region selection network for laryngeal closure detection in endoscopy videos.

The vocal folds (VFs) are a pair of muscles in the larynx that play a critical role in breathing, swallowing, and speaking. VF function can be adversely affected by various medical conditions including head or neck injuries, stroke, tumor, and neurological disorders. In this paper, we propose a deep learning system for automated detection of laryngeal adductor reflex (LAR) events in laryngeal endoscopy videos to enable objective, quantitative analysis of VF function. The proposed deep learning system incorporates our novel orthogonal region selection network and temporal context. This network learns to directly map its input to a VF open/close state without first segmenting or tracking the VF region. This one-step approach drastically reduces manual annotation needs from labor-intensive segmentation masks or VF motion tracks to frame-level class labels. The proposed spatio-temporal network with an orthogonal region selection subnetwork allows integration of local image features, global image features, and VF state information in time for robust LAR event detection. The proposed network is evaluated against several network variations that incorporate temporal context and is shown to lead to better performance. The experimental results show promising performance for automated, objective, and quantitative analysis of LAR events from laryngeal endoscopy videos with over 90% and 99% F1 scores for LAR and non-LAR frames respectively.

Axon and Myelin Sheath Segmentation in Electron Microscopy Images using Meta Learning.

Various neurological diseases affect the morphology of myelinated axons. Quantitative analysis of these structures and changes occurring due to neurodegeneration or neuroregeneration is of great importance for characterization of disease state and treatment response. This paper proposes a robust, meta-learning based pipeline for segmentation of axons and surrounding myelin sheaths in electron microscopy images. This is the first step towards computation of electron microscopy related bio-markers of hypoglossal nerve degeneration/regeneration. This segmentation task is challenging due to large variations in morphology and texture of myelinated axons at different levels of degeneration and very limited availability of annotated data. To overcome these difficulties, the proposed pipeline uses a meta learning-based training strategy and a U-net like encoder decoder deep neural network. Experiments on unseen test data collected at different magnification levels (i.e, trained on 500X and 1200X images, and tested on 250X and 2500X images) showed improved segmentation performance by 5% to 7% compared to a regularly trained, comparable deep learning network.

A Strength Endurance Exercise Paradigm Mitigates Deficits in Hypoglossal-Tongue Axis Function, Strength, and Structure in a Rodent Model of Hypoglossal Motor Neuron Degeneration.

The tongue plays a crucial role in the swallowing process, and impairment can lead to dysphagia, particularly in motor neuron diseases (MNDs) resulting in hypoglossal-tongue axis degeneration (e.g., amyotrophic lateral sclerosis and progressive bulbar palsy). This study utilized our previously established inducible rodent model of dysphagia due to targeted degeneration of the hypoglossal-tongue axis. This model was created by injecting cholera toxin B conjugated to saporin (CTB-SAP) into the genioglossus muscle of the tongue base for retrograde transport to the hypoglossal (XII) nucleus via the hypoglossal nerve, which provides the sole motor control of the tongue. Our goal was to investigate the effect of high-repetition/low-resistance tongue exercise on tongue function, strength, and structure in four groups of male rats: (1) control + sham exercise (n = 13); (2) control + exercise (n = 10); (3) CTB-SAP + sham exercise (n = 13); and (4) CTB-SAP + exercise (n = 12). For each group, a custom spout with adjustable lick force requirement for fluid access was placed in the home cage overnight on days 4 and 6 post-tongue injection. For the two sham exercise groups, the lick force requirement was negligible. For the two exercise groups, the lick force requirement was set to ∼40% greater than the maximum voluntary lick force for individual rats. Following exercise exposure, we evaluated the effect on hypoglossal-tongue axis function (via videofluoroscopy), strength (via force-lickometer), and structure [via Magnetic Resonance Imaging (MRI) of the brainstem and tongue in a subset of rats]. Results showed that sham-exercised CTB-SAP rats had significant deficits in lick rate, swallow timing, and lick force. In exercised CTB-SAP rats, lick rate and lick force were preserved; however, swallow timing deficits persisted. MRI revealed corresponding degenerative changes in the hypoglossal-tongue axis that were mitigated by tongue exercise. These collective findings suggest that high-repetition/low-resistance tongue exercise in our model is a safe and effective treatment to prevent/diminish signs of hypoglossal-tongue axis degeneration. The next step is to leverage our rat model to optimize exercise dosing parameters and investigate corresponding treatment mechanisms of action for future translation to MND clinical trials.

A Mouse Model of Dysphagia After Facial Nerve Injury.

OBJECTIVE: Dysphagia is common following facial nerve injury; however, research is sparse regarding swallowing-related outcomes and targeted treatments. Previous animal studies have used eye blink and vibrissae movement as measures of facial nerve impairment and recovery. The purpose of this study was to create a mouse model of facial nerve injury that results in dysphagia to enhance translational research outcomes. STUDY DESIGN: Prospective animal study. METHODS: Twenty C57BL/6J mice underwent surgical transection of the main trunk (MT) (n = 10) or marginal mandibular branch (MMB) (n = 10) of the left facial nerve. Videofluoroscopic swallow study (VFSS) assays for drinking and eating were performed at baseline and 14 days postsurgery to quantify several deglutition-related outcome measures. RESULTS: VFSS analysis revealed that MT transection resulted in significantly slower lick and swallow rates during drinking (P ≤ .05) and significantly slower swallow rates and longer inter-swallow intervals during eating (P ≤ .05), congruent with oral and pharyngeal dysphagia. After MMB transection, these same VFSS metrics were not statistically significant (P > .05). CONCLUSION: The main finding of this study was that transection of the facial nerve MT leads to oral and pharyngeal stage dysphagia in mice; MMB transection does not. These results from mice provide novel insight into specific VFSS metrics that may be used to characterize dysphagia in humans following facial nerve injury. We are currently using this surgical mouse model to explore promising treatment modalities such as electrical stimulation to hasten recovery and improve outcomes following various iatrogenic and idiopathic conditions affecting the facial nerve. LEVEL OF EVIDENCE: NA Laryngoscope, 131:17-24, 2021.

A mouse model of pharyngeal dysphagia in amyotrophic lateral sclerosis.

We recently established that the SOD1-G93A transgenic mouse is a suitable model for oral-stage dysphagia in amyotrophic lateral sclerosis (ALS). The purpose of the present study was to determine whether it could serve as a model for pharyngeal-stage dysphagia as well. Electrophysiological and histological experiments were conducted on end-stage SOD1-G93A transgenic mice (n = 9) and age-matched wild-type (WT) littermates (n = 12). Transgenic mice required a twofold higher stimulus frequency (40 Hz) applied to the superior laryngeal nerve (SLN) to evoke swallowing compared with WT controls (20 Hz); transgenic females required a significantly higher (P < 0.05) stimulus frequency applied to the SLN to evoke swallowing compared with transgenic males. Thus, both sexes demonstrated electrophysiological evidence of pharyngeal dysphagia but symptoms were more severe for females. Histological evidence of neurodegeneration (vacuoles) was identified throughout representative motor (nucleus ambiguus) and sensory (nucleus tractus solitarius) components of the pharyngeal stage of swallowing, suggesting that pharyngeal dysphagia in ALS may be attributed to both motor and sensory pathologies. Moreover, the results of this investigation suggest that sensory stimulation approaches may facilitate swallowing function in ALS.

Orthogonal Region Selection Network for Laryngeal Closure Detection in Laryngoscopy Videos.

Vocal folds (VFs) play a critical role in breathing, swallowing, and speech production. VF dysfunctions caused by various medical conditions can significantly reduce patients' quality of life and lead to life-threatening conditions such as aspiration pneumonia, caused by food and/or liquid "invasion" into the windpipe. Laryngeal endoscopy is routinely used in clinical practice to inspect the larynx and to assess the VF function. Unfortunately, the resulting videos are only visually inspected, leading to loss of valuable information that can be used for early diagnosis and disease or treatment monitoring. In this paper, we propose a deep learning-based image analysis solution for automated detection of laryngeal adductor reflex (LAR) events in laryngeal endoscopy videos. Laryngeal endoscopy image analysis is a challenging task because of anatomical variations and various imaging problems. Analysis of LAR events is further challenging because of data imbalance since these are rare events. In order to tackle this problem, we propose a deep learning system that consists of a two-stream network with a novel orthogonal region selection subnetwork. To our best knowledge, this is the first deep learning network that learns to directly map its input to a VF open/close state without first segmenting or tracking the VF region, which drastically reduces labor-intensive manual annotation needed for mask or track generation. The proposed two-stream network and the orthogonal region selection subnetwork allow integration of local and global information for improved performance. The experimental results show promising performance for the automated, objective, and quantitative analysis of LAR events from laryngeal endoscopy videos.Clinical relevance- This paper presents an objective, quantitative, and automatic deep learning based system for detection of laryngeal adductor reflex (LAR) events in laryngoscopy videos.

An Experimental Swallow Evoked Potential Protocol to Investigate the Neural Substrates of Swallowing.

Advancement in dysphagia intervention is hindered by our lack of understanding of the neural mechanisms of swallowing in health and disease. Evoking and understanding neural activity in response to normal and disordered swallowing is essential to bridge this knowledge gap. Building on sensory evoked potential methodology, we developed a minimally invasive approach to generate swallow evoked potentials (SwEPs) in response to repetitive swallowing induced by citric acid stimulation of the oropharynx in lightly anesthetized healthy adult rats. The SwEP waveform consisted of 8 replicable peaks within 10 milliseconds immediately preceding the onset of electromyographic swallowing activity. Methodology refinement is underway with healthy rats to establish normative SwEP waveform morphology before proceeding to models of advanced aging and age-related neurodegenerative diseases. Ultimately, we envision that this experimental protocol may unmask the pathologic neural substrates contributing to dysphagia to accelerate the discovery of targeted therapeutics.

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.