Amyotrophic lateral sclerosis (ALS) is a devastating disease leading to degeneration of motor neurons and skeletal muscles, including those required for swallowing. Tongue weakness is one of the earliest signs of bulbar dysfunction in ALS, which is attributed to degeneration of motor neurons in the hypoglossal nucleus in the brainstem, the axons of which directly innervate the tongue. Despite its fundamental importance, dysphagia (difficulty swallowing) and strategies to preserve swallowing function have seldom been studied in ALS models. It is difficult to study dysphagia in ALS models since the amount and rate at which hypoglossal motor neuron death occurs cannot be controlled, and degeneration is not limited to the hypoglossal nucleus. Here, we report a novel experimental model using intralingual injections of cholera toxin B conjugated to saporin (CTB-SAP) to study the impact of only hypoglossal motor neuron death without the many complications that are present in ALS models. Hypoglossal motor neuron survival, swallowing function, and hypoglossal motor output were assessed in Sprague-Dawley rats after intralingual injection of either CTB-SAP (25 g) or unconjugated CTB and SAP (controls) into the genioglossus muscle. CTB-SAP treated rats exhibited significant (p ≤ 0.05) deficits vs. controls in: (1) lick rate (6.0 ± 0.1 vs. 6.6 ± 0.1 Hz; (2) hypoglossal motor output (0.3 ± 0.05 vs. 0.6 ± 0.10 mV); and (3) hypoglossal motor neuron survival (398 ± 34 vs. 1018 ± 41 neurons). Thus, this novel, inducible model of hypoglossal motor neuron death mimics the dysphagia phenotype that is observed in ALS rodent models, and will allow us to study strategies to preserve swallowing function.

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.

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by degeneration of motor neurons and muscles, and death is usually a result of impaired respiratory function due to loss of motor neurons that control upper airway muscles and/or the diaphragm. Currently, no cure for ALS exists and treatments to date do not significantly improve respiratory or swallowing function. One cause of ALS is a mutation in the superoxide dismutase-1 (SOD1) gene; thus, reducing expression of the mutated gene may slow the progression of the disease. Our group has been studying the SOD1 G93A transgenic mouse model of ALS that develops progressive respiratory deficits and dysphagia. We hypothesize that solely treating the tongue in SOD1 mice will preserve respiratory and swallowing function, and it will prolong survival. At 6 weeks of age, 11 SOD1 G93A mice (both sexes) received a single intralingual injection of gene therapy (AAVrh10-miR SOD1). Another 29 mice (both sexes) were divided into two control groups: (1) 12 SOD1 G93A mice that received a single intralingual vehicle injection (saline); and (2) 17 non-transgenic littermates. Starting at 13 weeks of age, plethysmography (respiratory parameters) at baseline and in response to hypoxia (11% O 2) + hypercapnia (7% CO 2) were recorded and videofluoroscopic swallow study testing were performed twice monthly until end-stage disease. Minute ventilation during hypoxia + hypercapnia and mean inspiratory flow at baseline were significantly reduced (p < 0.05) in vehicle-injected, but not AAVrh10-miR SOD1-injected SOD1 G93A mice as compared with wild-type mice. In contrast, swallowing function was unchanged by AAVrh10-miR SOD1 treatment (p > 0.05). AAVrh10-miR SOD1 injections also significantly extended survival in females by *1 week. In conclusion, this study indicates that intralingual AAVrh10-miR SOD1 treatment preserved respiratory (but not swallowing) function potentially via increasing upper airway patency, and it is worthy of further exploration as a possible therapy to preserve respiratory capacity in ALS patients.

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.