Effects of Tongue Exercise Frequency on Tongue Muscle Biology and Swallowing Physiology in a Rat Model

Abstract

Age-related changes in muscle composition and function are often treated using exercise, including muscles of the tongue to treat swallowing impairments (dysphagia). Although tongue exercise is commonly prescribed, optimal tongue exercise doses have not been determined. The purpose of this study was to evaluate effects of varying tongue exercise frequency on tongue force, genioglossus muscle fiber size, composition and metabolism, and swallowing in a rat model. We randomized 41 old and 40 young adult Fischer 344/Brown Norway rats into one of four tongue exercise groups: 5 days/week; 3 days/week; 1 day/week; or sham. Tongue force was higher following all exercise conditions (vs sham); the 5 day/week group had the greatest change in tongue force (p < 0.001). There were no exercise effects on genioglossus (GG) fiber size or MyHC composition (p > 0.05). Significant main effects for age showed a greater proportion of Type I fibers in (p < 0.0001) and increased fiber size of IIa fibers (p = 0.026) in old. There were no significant effects of citrate synthase activity or PGC-1α expression. Significant differences were found in bolus speed and area (size), but findings were potentially influenced by variability. Our findings suggest that tongue force is influenced by exercise frequency; however, these changes were not reflected in characteristics of the GG muscle assayed in this study. Informed by findings of this study, future work in tongue dose optimization will be required to provide better scientific premise for clinical treatments in humans.

Keywords: Deglutition; Deglutition disorders; Rat model; Tongue exercise.

Conflict of interest statement

Conflict of Interest: The authors have no conflicts of interest to declare.

Figures

Figure 1.

Experimental flow chart demonstrating randomization of groups, experimental protocol, euthanasia, and experiments performed. IHC=Immunohistochemistry, CSA=Cross Sectional Area, MyHC=Myosin Heavy Chain.

Figure 2.

Tongue Exercise Schedule. Each box represents a single day in the acclimation and tongue exercise protocol. After 7 days of acclimation, rats underwent Maximum Voluntary Tongue Force (MVTF) testing to determine maximum tongue forces prior to training. This value was used to calculate daily threshold values during exercise and was re-evaluated at the end of Week 4 for the exercise animals only (5-day, 3 day, 1 day/week groups only). Exercise days occurred Monday through Friday, where rats entered the Tongue Exercise Apparatus and completed either sham exercise (less than 20 presses at 2 mN force threshold) or tongue exercise (average 100 presses at set threshold for that training week). Exercise days for each group are indicated in the figure. At the end of each exercise day Monday – Friday, all rats received 3 hours of water ad libitum in the watering cages.

Figure 3.

Exercise Apparatus and Watering Cages. Watering cages are used to prime rats for tongue exercise training using classical conditioning principles, providing a reinforcement (water) in the same location as in the training apparatus. The Training Apparatus dispenses water onto the disc when the rat presses with adequate force as measured through the force transducer. Adapted from Connor et al. 2009.

Figure 4.

Representative example from 1 genioglossus (GG) muscle: TIFF files for Semi-Automatic Muscle Analysis (SMASH) after ImageJ processing. A) Blue outlines laminin, Red indicates MyHC Type I isoform B) Blue outlines fiber laminin, Green indicates MyHC Type IIa isoform, Red indicates MyHC Type IIb isoform C) Blue outlines fiber laminin, Green indicates MyHC Type IIa isoform, Red indicates MyHC IIx isoform. Representative examples for staining validation: D) 1 extensor digitorus longum (EDL) muscle demonstrating Type IIb in Red; and E) 1 soleus (SO) muscle demonstrating Type I in Red.

Figure 5.

Average Difference in Daily Force Threshold. Error bars represent standard deviation. The average difference between the assigned force threshold (% Maximum Voluntary Tongue Force) and the actual force threshold achieved (tongue force during exercise) per exercise day are displayed. Average force per session across groups ranged between 10.2 – 12.4 mN above threshold. 5 day/week rats did have slightly higher average daily forces (above threshold) as compared to the 1 day/week exercise group (p=0.047). Y = Young Adult; O = Old; 1 = 1 day/week exercise; 3 = 3 days/week exercise; 5 = 5 days/week exercise.

Figure 6.

Change in Maximum Voluntary Tongue Force (MVTF) after 8-weeks of tongue exercise at different dose conditions (sham control, 1 day/week, 3 days/week, and 5 days/week of exercise). There was not a significant main effect for age (F1,73=3.306, p=0.295), so age conditions are combined here. There was a significant main effect for exercise condition (F3,73=45.271, p<0.0001): 5 days/week of exercise had the greatest increase in MVTF. Both 1 and 3 days/week of exercise had greater increases in MVTF than the sham control condition, but less than 5 days/week of exercise.

Figure 7.

MyHC Isoform Composition (%) and Fiber Size (CSA). Exercise groups are collapsed here by age, since there was no exercise effect across all MyHC Fiber types. There were significant main effects for age: 1) Increased percentage MyHC Type I in old (F1,54=44.927, p<0.0001); 2) Increased percentage of MyHC Type IIb in young adult (F1,54=13.467, p=0.001); 3) Fiber size in old MyHC Type IIa fibers was greater than young adult (F1,54=5.242, p=0.026).

Figure 8.

Change in bolus speed, bolus area, and mastication rate after tongue exercise. For change in bolus speed, there was an interaction effect (F3,66=6.007 p=0.001), where old 5 day/week rats had faster speeds after tongue training than old 3 day/week rats (p=0.004). However, there was a pre-existing difference between these groups at baseline (3 day/week old group > bolus speed at baseline compared with 5 day/week old group p=0.026). Change in bolus area after tongue exercise showed an interaction effect (F3,69=3.02, p=0.036), 5 day/week old animals had larger boluses sizes after tongue exercise as compared to young adult 5 day/week. Change in mastication rate after tongue exercise showed no main effects for age, exercise, or interaction effects (F1,60=1.529, p=0.221; F3,60=0.302, p=0.824; F3,60=1.273, p=0.292, respectively). Y= Young Adult; O= Old; C= Sham control; 1= 1 day/week exercise; 3= 3 days/week exercise; 5= 5 days/week exercise.