Tongue exercise ameliorates structural and functional upper airway deficits in a rodent model of hypoglossal motor neuron loss

Abstract

Introduction: Tongue weakness and atrophy can lead to deficits in the vital functions of breathing and swallowing in patients with motor neuron diseases (MNDs; e.g., amyotrophic lateral sclerosis (ALS) and pseudobulbar palsy), often resulting in aspiration pneumonia, respiratory failure, and death. Available treatments for patients with MNDs are largely palliative; thus, there is a critical need for therapies targeting preservation of upper airway function and suggesting a role for tongue exercise in patients with MNDs. Here, we leveraged our inducible rodent model of hypoglossal (XII) motor neuron degeneration to investigate the effects of a strength endurance tongue exercise program on upper airway structure and function. Our model was created through intralingual injection of cholera toxin B conjugated to saporin (CTB-SAP) into the genioglossus muscle of the tongue to induce targeted death of XII motor neurons.

Methods: Rats in this study were allocated to 4 experimental groups that received intralingual injection of either CTB-SAP or unconjugated CTB + SAP (i.e., control) +/- tongue exercise. Following tongue exercise exposure, we evaluated the effect on respiratory function (via plethysmography), macrostructure [via magnetic resonance imaging (MRI) of the upper airway and tongue], and ultrafine structure [via ex vivo magnetic resonance spectroscopy (MRS) of the tongue] with a focus on lipid profiles.

Results: Results showed that sham exercise-treated CTB-SAP rats have evidence of upper airway restriction (i.e., reduced airflow) and structural changes present in the upper airway (i.e., airway compression) when compared to CTB-SAP + exercise rats and control rats +/- tongue exercise, which was ameliorated with tongue exercise. Additionally, CTB-SAP + sham exercise rats have evidence of increased lipid expression in the tongue consistent with previously observed tongue hypertrophy when compared to CTB-SAP + exercise rats or control rats +/- tongue exercise.

Conclusion: These findings provide further evidence that a strength endurance tongue exercise program may be a viable therapeutic treatment option in patients with XII motor neuron degeneration in MNDs such as ALS. Future directions will focus on investigating the underlying mechanism responsible for tongue exercise-induced plasticity in the hypoglossal-tongue axis, particularly inflammatory associated factors such as BDNF.

Keywords: breathing; degeneration; dysphagia; motor neuron disease (MND); rat model; respiration.

Figure 1

Breathing parameters [inspiratory time (TI), mean inspiratory flow (VT/TI/100 g), peak inspiratory flow (PIF), and peak expiratory flow (PEF)] at baseline (B) and endline (E) timepoints during normoxia and maximum chemoreceptor stimulation (max) for all groups (control + sham exercise, control + exercise, CTB-SAP + sham exercise, and CTB-SAP + exercise). All breathing parameters (TI, VT/TI/100 g, PIF and PEF) were significantly impacted when comparing max to normoxia during both baseline and endline timepoints (denoted by *). Some breathing parameters were significantly impacted in CTB-SAP + sham exercise rats vs. CTB-SAP + exercise rats (TI) and control + exercise rats (TI, VT/TI/100 g, and PIF) during normoxia at the baseline timepoint and vs. control + sham exercise rats (TI and PIF) and CTB-SAP + exercise rats (PIF) during normoxia at the endline timepoint (denoted by &). In addition, breathing parameters were significantly affected in CTB-SAP + exercise rats vs. control + sham exercise rats (TI) and CTB-SAP + sham exercise rats (VT/TI/100 g and PIF) during normoxia at the baseline timepoint and vs. CTB-SAP + sham exercise rats (PIF and PEF), control + exercise rats (PIF and PEF), and control + sham exercise rats (PEF) during normoxia at the endline timepoint (denoted by #). Lastly, breathing parameters (TI, VT/TI/100 g, PIF, and PEF) were significantly different when comparing baseline to endline timepoints during normoxia for CTB-SAP + sham exercise (TI, VT/TI/100 g, and PIF) and CTB-SAP + exercise groups (TI, VT/TI/100 g, PIF, and PEF; denoted by +). Values are expressed as means ±1 S.E.M., and differences were considered significant if p < 0.05. The adjacent dots to the left or right of each bar represent individual animal values.

Figure 2

Ex vivo T2 weighted MRI of the tongue. Tongues from control + sham exercise (A), control + exercise (C), CTB-SAP + sham exercise (B), and CTB-SAP + exercise treated rats (D). Representative coronal slices are shown with lymphoid nodules and muscle indicated with white arrows (B). Lymphoid nodules in the tongue in CTB-SAP + sham exercise rats (B) appear more extensive vs. control + sham exercise rats (A).

Figure 3

Ex vivo magnetic resonance spectroscopy (MRS) of the tongue. MRS of the lymphoid nodules in the tongue was performed in a single voxel (2.73 × 1.33 × 3 mm = 10 mm³), indicated by red dashed box on T2 weighted MRI (A). Acquired MRS spectrum (black traces) and LC Model fitted lipid peaks (red traces) for each treatment group [control + sham exercise (B), control + exercise (D), CTB-SAP + sham exercise (A,C), and CTB-SAP + exercise rats (E)] are shown. The major lipid peaks with their chemical shifts are indicated in D and the provided table, respectively.

Figure 4

Relative lipid composition of the tongue lymphatic nodule tissue for all groups (control + sham exercise, control + exercise, CTB-SAP + sham exercise, and CTB-SAP + exercise). The relative concentration of Lip2.1 + Lip2.3 with respect to the Lip1.3 peak was significantly higher in CTB-SAP + sham exercise rats vs. control + sham exercise rats (denoted by *, p < 0.05). Values are expressed as means ±1 S.E.M. The adjacent dots to the right of each bar represent individual animal values.

Figure 5

In vivo sagittal airway MRI. Airway volume (denoted by double-sided white arrows) evaluated from the junction of the hard and soft palate (denoted by a white * in A) through the slice representing the tongue root (denoted by a white ^ in A). The CTB-SAP + sham exercise treated rat (B) appears to show evidence of a compressed airway when compared to rats from the other treatment groups [i.e., control + sham exercise (A), control + exercise (C), and CTB-SAP + exercise rats (D)].