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.

Keywords: denervation atrophy, dysphagia, motor neuron death, rodent models, ultrastructural changes.

FIGURE 1

Macroscopic tongue measurements in control and CTB-SAP–treated rats. There was no significant difference (P > 0.05) in size of control (black bars) and CTB-SAP–treated rats (gray bars) for any of the macroscopic measurements analyzed, including: body weight (A), tongue weight (B), tongue area (C), and tongue length and width (D). Data show mean ± standard error of the mean plus dot plots for individual data points. Abbreviation: CTB-SAP, cholera toxin B conjugated to saporin

FIGURE 2

Cross-sectional areas of myofibers in 3 parts of the posterior genioglossus for control and CTB-SAP–treated rats. Line graph depicts the mean ± standard error of the mean for each experimental group and location. A linear mixed effects model fitted to these data indicates significant effects (P < 0.05) for both experimental group and location. Control rats (dotted black line) had larger myofibers than CTB-SAP–treated rats (solid gray line) in locations 1 and 2 (*P < 0.05). For CTB-SAP–treated animals only, there were significant differences in CSAs between all pairs of locations, with fibers in location 3 being the largest and fibers in location 1 being the smallest (^#P < 0.05). Abbreviation: CTB-SAP, cholera toxin B conjugated to saporin

FIGURE 3

Ultrastructural changes in hypoglossal neurons and the hypoglossal nerve after intralingual CTB-SAP. Representative TEM images from the hypoglossal nucleus (A-C) and distal hypoglossal nerve (D,E) of a control (A,D) and CTB-SAP–treated rat (B,C,E). A, Normal motor neuron from a control rat is outlined in white. The white arrow indicates the nucleolus. B,C, Hypoglossal neurons in CTB-SAP–injected rats show progressive signs of degeneration including loss of the nucleolus (white arrow), decreased overall size, and increased nucleus:cytoplasm ratio. D, Nerve from the control rat consists of normal axons surrounded by compact myelin sheaths (white arrows). E, In the image from the CTB-SAP–treated rat, the axons appear smaller and are surrounded by thickened, irregular myelin sheaths, which are consistent with degeneration. Abbreviations: CTB-SAP, cholera toxin B conjugated to saporin; TEM, transmission electron microscopy