Tongue and hypoglossal morphology after intralingual cholera toxin B–saporin injection

Abstract

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.

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

“…At the study endpoint (i.e., 9 days after tongue injection), a subset of 16 rats (1) control + sham exercise ( n = 5); (2) control + exercise ( n = 2); (3) CTB-SAP + sham exercise ( n = 6); and (4) CTB-SAP + exercise ( n = 3) underwent MRI of the brainstem and tongue to investigate corresponding structural changes that may correlate with behavioral findings. This study endpoint is the same as our previous studies ( Lind et al, 2018 , 2021 ), which was determined based on pilot data showing that other time points either did not result in dysphagia (4 days post tongue injection) or resulted in severe dysphagia (11–14 days post tongue injection). A summary of the 9-day experimental timeline is shown in Figure 1 .…”

“…Here, we hypothesized that structural degeneration in the brainstem and tongue would be detectable with in vivo MRI and present in sham exercise-treated CTB-SAP rats based on our previous findings (i.e., hypoglossal motor neuron degeneration and genioglossal myofiber atrophy; Lind et al, 2021), and would be prevented or decreased with tongue exercise. While our observations thus far are based on a small subset of rats and treatment groups, MRI did provide evidence of degeneration in the hypoglossal-tongue axis (i.e., a trend for 4th ventricle enlargement, significantly increased tongue volume and thickness, and marked hyperintensity of the tongue) in response to genioglossal myofiber atrophy (Lind et al, 2021), and these pathological MRI features were somewhat mitigated via tongue exercise in CTB-SAP rats. Importantly, fourth ventricle enlargement is a common feature of neurodegenerative diseases and is correlated with degeneration of the surrounding brain structures (Westeneng et al, 2015;Bede et al, 2019).…”

“…Our rat model was created as previously described (Lind et al, 2018(Lind et al, , 2021. In brief, rats were anesthetized via 5% isoflurane in an induction chamber and immobilized in ear bars in the supine position on a custom-built tilt table to stabilize the head during tongue injections.…”

“…Upon entering XII LMN cell bodies, CTB-SAP dissociates, leaving SAP free to bind to ribosomes and consequently halt protein synthesis, resulting in apoptotic cell death ( Llewellyn-Smith et al, 2000 ; Lujan et al, 2010 ). Our recent investigations with this model revealed that a single CTB-SAP injection into the midline genioglossus of adult rats resulted in ∼60% XII LMN cell death within 9 days ( Lind et al, 2018 ), with corresponding “downstream” degenerative changes in the XII nerve (i.e., denervation atrophy) and genioglossus (i.e., myofiber atrophy) ( Lind et al, 2021 ) as well as development of swallowing-related deficits (slower lick and swallow rates) ( Lind et al, 2018 ). In contrast, when control rats were tongue-injected with unconjugated CTB and SAP (CTB + SAP), only CTB was uptaken by XII nerve terminals for retrograde transport to XII LMNs, without resultant neuromuscular degenerative changes and corresponding swallowing-related deficits ( Lind et al, 2018 , 2021 ).…”

“…Our recent investigations with this model revealed that a single CTB-SAP injection into the midline genioglossus of adult rats resulted in ∼60% XII LMN cell death within 9 days ( Lind et al, 2018 ), with corresponding “downstream” degenerative changes in the XII nerve (i.e., denervation atrophy) and genioglossus (i.e., myofiber atrophy) ( Lind et al, 2021 ) as well as development of swallowing-related deficits (slower lick and swallow rates) ( Lind et al, 2018 ). In contrast, when control rats were tongue-injected with unconjugated CTB and SAP (CTB + SAP), only CTB was uptaken by XII nerve terminals for retrograde transport to XII LMNs, without resultant neuromuscular degenerative changes and corresponding swallowing-related deficits ( Lind et al, 2018 , 2021 ). Thus, our inducible rat model selectively involves the hypoglossal-tongue axis and provides a unique platform for studying the effects of targeted tongue exercise on tongue-related function, strength, and structure.…”