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. Author manuscript; available in PMC: 2024 Nov 1.
Published in final edited form as: Arch Oral Biol. 2023 Aug 1;155:105779. doi: 10.1016/j.archoralbio.2023.105779

Age-related Sex Differences in Tongue Strength and Muscle Morphometry in a Rat Model

Andrea Rohl 1, Nadine P Connor 2, John A Russell 3
PMCID: PMC10592197  NIHMSID: NIHMS1924240  PMID: 37556980

Abstract

Objective:

To investigate potential effects of sex on voluntary tongue strength, evoked twitch and tetanic tension, speed of contraction, and muscle fiber cross-sectional area in the muscles of the rat tongue. Additionally, we aimed to determine whether estrous cycle stage impacts any of the dependent variables as a pilot investigation into the use of female rats in a model of tongue exercise and aging.

Design:

Fischer 344-Brown Norway male and female rats in two age groups (16 middle-aged, 16 young-adult) were trained to use a tongue force operandum. Tongue muscle contraction, myosin heavy chain (MyHC) composition, and cross section area of the genioglossus and styloglossus muscles were examined. Vaginal lavage determined estrous cycle stage of the female rats daily.

Results:

The female group had significantly lower evoked twitch and tetanic tension, longer contraction times, and a smaller proportion of MyHC type IIa and MyHC type IIx in the styloglossus muscle. There was no significant sex effect in maximal voluntary tongue force (MVTF) despite a significant weight difference between the male and female groups. There were no significant age or sex effects in the genioglossus. Estrous cycle stage did not have a significant effect on any of the dependent variables.

Conclusions:

Sex and age both have a significant effect on tongue muscle structure and physiology. While the female group showed reduced contraction speed and maximal twitch and tetanic tension relative to the male group, differences in muscle morphology appeared to vary by muscle.

Keywords: Tongue, aging, sex differences, hypoglossal nerve, exercise, dysphagia

Introduction

Aging has a significant impact on the human neuromuscular system, with loss of muscle mass and strength (sarcopenia) affecting individuals as young as 60 years of age (Vandervoort, 2002). Because the aging population is growing so quickly (census.gov), it is critical to obtain an understanding of the how sarcopenia affects critical sensorimotor functions and effective methods of prevention and rehabilitation.

Sarcopenia affects oropharyngeal muscles active in swallowing (Kays & Robbins, 2006; Mortimore et al., 1999; Robbins et al., 2005), including the tongue (Van Daele et al., 2005). Muscular changes with age (Behan et al., 2012), have the potential to cause swallowing impairment, or dysphagia (Kays & Robbins, 2006; Mortimore et al., 1999), putting older adults at greater risk for airway penetration or aspiration.

Progressive resistance exercise of the tongue is used clinically to increase lingual strength and treat swallowing disorders in elderly people and those with neurological insults such as stroke (McKenna et al., 2017; Robbins et al., 2005; Robbins et al., 2007). Progressive resistance exercise has been shown to increase lingual strength and swallowing pressures (Robbins et al., 2005; Robbins et al., 2007; Rogus‐Pulia et al., 2016), improve swallowing function in stroke patients with dysphagia (Robbins et al., 2007), increase nutritional intake, and increase quality of life in patients with dysphagia (Rogus‐Pulia et al., 2016).The clinical data are compelling, but muscle mechanisms that underlie these changes must be studied to provide a biological basis for optimizing treatment parameters.

A rat model was developed to examine putative mechanisms of changes observed with clinical tongue exercise programs used in humans (Behan et al., 2012; Connor et al., 2009; Kletzien et al., 2013; Schaser et al., 2012). The use of rat models in preclinical research on the aging process is well-accepted as valid and reliable (Beery & Zucker, 2011; Perry, 2007; Turturro et al., 1999), with aging rats demonstrating muscle loss similar to that experienced by elderly humans (Cartee, 1995). However, a concern in the translation of animal models to clinical research is a potential sex bias in preclinical biomedical research (Beery & Zucker, 2011; Yoon et al., 2014). Biomedical research is dominated by single-sex studies of male animals, with as few as 3% of animal research studies including data on both sexes (Beery & Zucker, 2011; Yoon et al., 2014).

The effect of sex bias in behavioral research is poorly understood and must be studied to determine the degree to which both male and female animals must be used. Increased research cost is a frequently cited challenge for including both sexes in preclinical research (Beery & Zucker, 2011; Klein et al., 2015; Marts & Keitt, 2003). The additional variable of sex hormones and estrous cycles in females is another frequently considered factor (Holdcroft, 2007; Klein et al., 2015; Marts & Keitt, 2003; Yoon et al., 2014). In the female rat, estrous cycles (proestrus, estrus, metestrus, and diestrus) cycle every 4–5 days (Ajayi & Akhigbe, 2020). Estrous cycles and associated variability in estradiol levels have been shown to impact serotonin production and uptake in the hypoglossal nucleus (Seebart et al., 2007), performance on skilled motor tasks (Becker et al., 1987), learning and memory (Korol et al., 2004; Pompili et al., 2010; Stackman et al., 1997), motivation (Steiner et al., 1981), food and water intake (Jennings, 1969; Tarttelin & Gorski, 1971), and skeletal muscle contractile properties and muscle fiber size (McCormick et al., 2004; Moran et al.). However, it is not known how estradiol or estrous cycle stage may affect tongue muscle morphology and contractile properties.

There is a limited understanding of the effect of sex and aging on tongue strength, and several conflicting reports. For instance, there are reports of significantly higher maximal tongue forces in men than women (Adams et al., 2013; Guo et al., 2021), but there are also reports no significant differences (Abe et al., 2020). One study found differences in only in posterior tongue strength in men vs. women (Lin et al., 2021). Therefore, further exploration of potential sex differences in tongue strength and how age may impact these differences.

Most prior work in investigating swallowing disorders and the role of age-related changes in tongue muscle structure and physiology has used exclusively male rats (Fowler & Mortell, 1992; Guggenmos et al., 2009;; Nagai et al.; Plowman et al., 2013), with some exceptions that have included female rats (Ma et al., 2017). One recent study found that male rats demonstrated significantly higher twitch forces of the longitudinal muscles of the tongue, but found no sex differences in fatigue, twitch forces of other tongue muscles, muscle fiber cross sectional area, or fiber type (Fogarty & Sieck, 2021). No similar study has been done that accounts for estrous cycle stage. Research into sex dimorphism of the intrinsic laryngeal muscles of rats has shown differences in muscle fiber cross sectional area and muscle mass (Lenell et al., 2021).

Our purpose was to evaluate sex differences concerning tongue muscle training and measures in female rats. Because previous research has shown age-related decrements in tongue forces (Kletzien et al., 2013) and that male rats demonstrate higher skeletal muscle force outputs than female rats in muscles of the hind limb (Glenmark et al., 2004), we hypothesized that there would be a significant reduction in maximal voluntary tongue forces, tongue muscle twitch and tetanic tension evoked by maximal hypoglossal nerve stimulation, and reduced muscle fiber cross sectional area as a function of older age and female sex.

Methods

This study was performed in accordance with the NIH Guide for Care and Use of Laboratory Animals, 8th Edition, 2011. The University of Wisconsin School of Medicine and Public Health Animal Care and Use Committee approved all procedures. Thirty-two Fischer 344/Brown Norway rats were used for collection of data on muscle structure, strength, and contractile properties. The rats were grouped according to age and sex, resulting in four groups of eight: young adult male, middle-aged male, young adult female, and middle-aged female. Young adult rats were approximately 9 months of age while rats in the middle-aged groups were between 17 and 23 months of age. The Fischer 344/Brown Norway rat median life span is approximately 36 months of age (Turturro et al., 1999).

Tongue Exercise

Upon arrival to the research facility, rats were acclimated to the vivarium and our laboratory for 6–10 days. During this period, rats also underwent a 12:12 light cycle reversal to ensure that behavioral testing occurred when the rats were most alert. As in our previous research, rats were restricted access to water for 21 hours/day to allow use of water rewards for participation in the tongue force acquisition protocol (Cartee, 1995; Turturro et al., 1999).

To measure tongue force output, a custom-designed force acquisition instrument was used as described in prior reports from our laboratory (Connor et al., 2009; Kletzien et al., 2013). Tongue force measures were sampled using a force transducer connected to the disk at a sampling rate of 200 Hz. After an acclimation and training period, each rat underwent four days of maximal voluntary tongue force testing. During maximal force testing, each rat was placed in the force acquisition enclosure for up to 10 minutes and provided with a water reward for incrementally increasing tongue press forces. Each rat began at a threshold of 2 mN. The force acquisition system automatically dispensed water for each press over the current threshold. Thresholds automatically increased to each rat’s highest press, so that each successive press must increase in force until the rat was unable to reach the threshold.

Estrous Cycle

To account for any effects caused by changing estradiol levels, the estrous cycle phase of female rats was determined through vaginal lavage (Cora et al., 2015; Goldman et al., 2007). Samples were transferred by pipette directly onto a slide, air dried, and stained with a Wright’s Stain, Rapid Formula (Ricca Chemical Company, Arlington, TX). Two researchers separately examined vaginal smears by microscope and characterized them according to the presence, quantity, and type of epithelial cells and leukocytes. Each researcher then used these observations to classify samples as either estrus, metestrus, diestrus, or proestrus, according to previously published criteria (Cora et al., 2015; Paccola et al., 2018). Any slide for which there were discrepancies was re-examined and discussed until agreement was reached about the cycle phase for that slide.

Muscle Contractile Properties

After anesthesia with sodium pentobarbital IP (50–70 mg/kg), hypoglossal nerves were exposed bilaterally and nerve cuff electrodes were placed around each nerve. Supplemental anesthesia with pentobarbital injection was used if needed during the experiment. The rat’s tongue was manually protruded from the mouth and a small suture was placed in the tip to connect it to a force transducer. The whole hypoglossal nerve was stimulated bilaterally to evoke maximal retrusive tongue forces and muscle contractile properties were recorded. Following retrusive measures, the lateral branch was sectioned and the medial branch of the hypoglossal nerve was isolated and supramaximally stimulated to evoke maximal protrusive tongue forces. For both protrusive and retrusive tongue forces, the contractile properties assessed included maximum twitch tension (mN), maximum tetanic tension (mN), contraction time (ms), and decay time (ms).

Muscle Fiber Cross Sectional Area

Immediately following measurement of muscle contractile properties, rats were euthanized via a pentobarbital overdose (120 mg/kg IP) or an overdose of the barbiturate, Beuthanasia-D (0.2–0.3 cc/kg IP and/or IC). Genioglossus (protrusive muscle) and styloglossus (retrusive muscle) specimens were harvested and embedded in an optimal cutting temperature (OCT) compound. Tissues were then frozen in isopentane cooled over liquid nitrogen and stored at −80°C.

Myosin heavy chair (MyHC) is a strong determinant of muscle fiber type (Fry et al., 1994). Accordingly, muscle specimens were prepared to examine proportion of MyHC isoforms within each sample. Muscle cross-sections (10 μm) were cut from the midsection of the genioglossus and styloglossus muscles with a cryostat and mounted for staining. Sections were air dried at room temperature for 10 minutes, fixed in acetone at 4°C for 10 minutes, and blocking buffer was applied to the tissues for one hour (10% goat serum in phosphate buffered saline [PBS]). Primary antibodies against MyHC were then added to the tissues for 20 hours followed by four 7-minute PBS washes. Secondary antibodies were applied to the tissues for one hour followed by 8-minute PBS washes. Slides were then mounted with coverslips. Immunofluorescence analyses of MyHC expression were performed using primary antibodies against MyHCIIa (SC-71), and MyHCIIx (6H1) (Bloemberg & Quadrilatero, 2012). MyHCIIx and MyHCIIa are fast-contracting isoforms. The percent of muscle fibers within each sample positive for each MyHC isoform was calculated. Muscle fiber composition has been shown to be affected by both age (Kletzien et al., 2013) and sex (Haizlip et al., 2015).

Slides were photographed at 40x using a bright-field Nikon microscope linked to a PixeLink digital camera. Muscle cross-section images were analyzed via Semi-automatic Muscle Analysis using Segmentation of Histology software (SMASH) (Smith & Barton, 2014). A minimum of 250 fibers were analyzed in each muscle to ensure reliability of measures (Ceglia et al., 2013). SMASH analyzed the minimal Feret’s diameter (μm) of each muscle fiber for the most robust measure of muscle fiber area (Briguet et al., 2004). SMASH was also used for MyHC fiber typing of genioglossus and styloglossus muscle samples. SMASH created a mask for the original image according to intensity of staining within each cell, providing data on the fiber types present in the section. MyHC fiber type is reported as percentage of total fibers positive for MyHCIIa or MyHCIIx.

Statistical Analyses

We used a two-way analysis of variance (ANOVA) to examine the effects of age and sex and their interactions on all independent variables. Shapiro-Wilk tests were used to confirm normal distribution prior to running statistical analyses and normal distribution assumptions were met for all continuous variables. Post-hoc testing was completed on all significant interactions using a Fisher’s LSD analysis. A repeated-measures two-way ANOVA was performed to assess the effects of daily estrous cycle stage and age on voluntary tongue force. Eta-squared (η2) was calculated as a measure of effect size where appropriate. All analyses were performed using Stata statistical software (StataCorp, 2017). The α-level for statistical significance was set at < .05.

Results

Weight

Weight was significantly higher in the male groups (female: 236±30 grams; male: 435±74 grams; t=9.954, p<.001). Weight was also significantly higher with age for both the male groups (young male: 373±26 grams; middle-aged male: 498±44 grams; t=6.926, p<.001) and the female groups (middle-aged female: 258±21 grams; young female: 212±18 grams; t=4.061, p<.001). Pearson’s r was calculated to determine the relationship between dependent variables and animal weight. In addition, there was a significant correlation with maximal twitch tension (r=.5444, p=.0013), maximal tetanic tension (r=.8464, p<.001), styloglossus cross-sectional area (r=.7130, p<.001), and genioglossus cross-sectional area (r=.5069, p=.003). Weight was not significantly correlated with contraction time (r=−.3148, p=.0793), or decay time (r=−.1312, p=.4741). Moderate to strong relationships with weight were found for maximal voluntary tongue force (r=.3721, p=.0360; Figure 1).

Figure 1:

Figure 1:

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Scatterplot demonstrating the relationship of weight (gm) and maximal voluntary tongue force (mN) across age and sex groups. Each symbol represents an individual rat.

Maximal Voluntary Tongue Force

There was no significant effect of age (F(1,30)=.85, p=0.364, η2 =.030) or sex (F(1,30)=0.976, p=.332, η2 =.034) on maximal voluntary tongue force measurements and no significant interaction effect between age and sex (F(1,30)=4.004, p=0.055, η2 =.125). Figure 1 shows maximal voluntary tongue force measurements according to weight and group.

Tongue Muscle Contractile Properties

There was a significant main effect for sex for evoked retrusive tongue force measures. For both maximal twitch tension [F(1,30)=12.49, p=.0014, η2 =0.267] and maximal tetanic tension [F(1,29)=86.88, p=<.0001, η2=0.763], the male group had significantly higher tensions than the female group in the absence of age or interaction effects. There were no significant age, sex, or interaction effects for decay time. There was, however, a significant main effect for sex for contraction time [F(1,30)=8.2, p=.0076, η2 =0.228] with significantly longer contraction times found in the female group (Table 1).

Table 1:

Means and standard deviations of muscle contractile properties elicited during stimulation of the hypoglossal nerve.

Retrusive Force Data
Rat Group Maximal Twitch Tension, mN Contraction Time, ms Half-Decay Time, ms Maximal Tetanic Tension, mN
Young Female 283.5 (12.4)* 10.0 (0.66)* 37.4 (3.2) 827.2 (54.8)*
Middle-aged Female 267.3 (31.1)* 10.3 (0.6)* 36.7 (2.8) 801.5 (72.3)*
Young Male 301.2 (15.1)* 9.4 (0.5)* 36.0 (1.8) 1013.4 (51.2)*
Middle-aged Male 308.3 (29.2)* 9.8 (0.5)* 36.5 (2.6) 1053.6 (77.7)*
Protrusive Force Data
Rat Group Maximal Twitch Tension, mN Contraction Time, ms Half-Decay Time, ms Maximal Tetanic Tension, mN
Young Female 40.3 (3.3)* 11.6 (0.5)* 40.2 (1.3)a 116.0 (5.2)*
Middle-aged Female 33.5 (3.3)* 11.2 (0.5)* 35.1 (1.3)a 121.1 (5.2)*
Young Male 43.6 (3.1)* 10.2 (0.5)* 35.4 (1.3)a 150.5 (6.2)*
Middle-aged Male 47.2 (3.1)* 10.1 (0.5)* 34.7 (1.3)a 159.2 (8.0)*
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*

indicates a significant (p<.05) difference according to sex.

a

indicates a significant (p<.05) difference according to age. No age and sex interaction effects reached significance.

For evoked protrusive tongue force measures, similar effects were observed. For maximal twitch tension [F(1,29)=7.25, p=.012, η2 =0.218] and maximal tetanic tension [F(1,29)=32.128, p<.001, η2 =0.553], the male group demonstrated significantly higher tensions than the female group. No effects were seen for age or interaction effects. There was a significant main effect for sex for contraction time [F(1,29)=6.728, p=.015, η2 =0.206], but no age or interaction effects. Sex differences in decay time did not reach the level of significance [F(1,29)=3.952, p=.057, η2 =0.132], but there was a significant effect for age [F(1,29)=4.955, p=.035, η2 =0.16], with decay time significantly greater for the young group (Table 1).

Muscle Fiber Size and Type

Styloglossus

Analysis of styloglossus muscle fiber cross sectional area showed significant effects for sex [F(1,29)=14.2018, p=.0008, η2 =0.392] and age [F(1,29)=8.7478, p=.0063, η2 =0.223] with larger muscle fibers in the middle-aged than in the young adult group, and larger muscle fibers in the male than in the female group (Figure 2). There were no significant interaction effects for muscle fiber cross sectional area. Analysis of fiber type showed a significant sex effect [F(1,29)=10.7241, p=.0027, η2 =0.287] with the male group exhibiting a greater percentage of MyHCIIx fibers than the female group. Significant effects for both age [F(1,29)=6.4981, p=.0122, η2 =0.204] and sex [F(1,29)=7.8137, p=.0012, η2 =0.315] were found for percentage of MyHCIIa positive muscle fibers, with the male group and middle-aged group showing higher average percentages than their counterpart. Representative photomicrograms are shown in Figure 3 with quantitative data shown in Figure 2.

Figure 2:

Figure 2:

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Violin plot showing that A) in the genioglossus, there were no significant differences in cross-sectional area (μm) for either sex or age and B) in the styloglossus, the middle-aged group had significantly (p=.006) larger CSA than the young group and the male group had significantly (p<.001) larger CSA than the female group. Median is indicated by white dots and quartile data are indicated by dark boxes and lines. Plot width is representative of the distribution of datapoints.

Figure 3:

Figure 3:

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Immunofluorescence staining of styloglossus (SG) muscles cross sections. A: Middle-aged male. B: Young Female. MyHCIIx is stained red while MyHCIIa is stained blue. Laminin is green in the above images. Significantly larger cross-sectional area (p<. 05) was seen in the older group compared to the younger group, as well as in the male group compared to the female group. The relative frequency of MyHCIIx was also significantly higher in the male group.

Genioglossus

One genioglossus microgram was incorrectly measured using an erroneous scale bar and was eliminated from the analysis. Analysis of genioglossus muscle fiber size showed no significant effects for age [F(1,29)=2.787, p=.107, η2 =0.097] sex [F(1,29)=2.610, p=.118, η2 =0.091] or interaction between age and sex [F(1,29)=.011, p=.919, η2 =0.000] (Figure 2). No significant age or sex differences were found for percentage of MyHCIIx fibers and percentage MyHCIIa fibers in the genioglossus.

Estrous Cycle

Estrous cycle in both middle-aged and young females ranged from 4–6 days in length. As shown in Figure 4, estrous cycle stage did not have a significant effect on daily maximal voluntary tongue forces [F(3,60)=0.33, p=.803, η2 =0.017] nor was there an interaction effect of estrous cycle with age [(F(3,60)=0.65, p=.586, η2 =0.033]. Variability in maximal voluntary tongue force (as measured by the standard deviation of maximal voluntary tongue force measures over the four-day maximal tongue force testing period) did not show significant effects for sex [(F(1,30)=0.00, p=.96], age [F(1,30)=1.86, p=.1836] or interaction between sex and age [F(1,30)=.99, p=.3291]. Estrous cycle stage at time of specimen collection had no significant effects on tongue muscle contractile properties, muscle fiber cross sectional area, or fiber type.

Figure 4:

Figure 4:

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Scatterplot demonstrating the relationship of daily voluntary tongue force (mN) and estrous cycle stage in female rats across age. Each symbol represents an individual rat on one day during the four-day maximal force testing period. Metestrus was observed in only four instances, which is representative of the short relative length of that cycle stage (6–8 hours in total; Cora et al., 2015). No significant differences were found for estrous cycle stage.

Discussion

We hypothesized that there would be a significant reduction in maximal voluntary tongue force, maximal twitch tension, contraction speed, maximal tetanic tension, and muscle fiber cross sectional area as a function of older age and female sex. Our findings did not allow us to accept these hypotheses in full. Contrary to our hypothesis, age did not have a significant effect on maximal voluntary or evoked tongue forces, although evoked force measures tended to be lower in older rats. With regard to sex, we found significantly reduced maximal tetanic tension, maximal twitch tension, and contraction speeds in the female group than in the male group, in accord with our hypothesis. There was no significant difference in maximal voluntary tongue forces according to sex. Therefore, while age and sex influence tongue strength and muscle morphology in the rat, these effects are not consistent across all measures.

Body weight increased with aging in both males and females. However, the relationship between body weight and tongue force may not be straightforward across the age range in different rat strains (Zhang et al., 2008). In F344/BN rats, we found a moderate to strong correlations between weight and measures of tongue force and cross-sectional area. Previous research that has shown similar correlations between weight and measures of tongue force (Kletzien et al., 2013). Adjusting measures of strength for body weight is one well-documented method to account for variations in body size in humans (Jaric, 2003; Nevill et al., 1992). Research using animal models has been far less consistent in controlling for body weight. Without accounting for weight in statistical analyses, we risk differences in groups that vary in weight being significantly confounded by the “body-size effect” (Jaric, 2002). For example, researchers have found significantly higher cross sectional area and twitch forces in the soleus of male compared to female Fischer 344 rats, but after controlling for muscle mass those differences were no longer significant (Virgen-Ortiz et al., 2018). We opted not to weight-adjust the force data in this study and to present actual force data as they were observed in our study, which is consistent with prior findings in F344 rats where body weight varied across the 6–24 month age range in the absence of tongue force differences as a function of age (Zhang et al., 2008).

Our findings are consistent with previous research that found significantly greater muscle fiber cross-sectional area in males than in females in various skeletal muscles in humans, mice, and rats (Alway et al., 1990; Mierzejewska-Krzyżowska et al., 2011; Miller et al., 1993). Cantillon and Bradford (Cantillon & Bradford, 2000), however, found no sex differences in cross-sectional area of the geniohyoid and sternohyoid of the rat. One possible explanation for these inconsistencies is that sex and age may not affect all head and neck muscles in the same way, further highlighting the necessity of sex inclusion in preclinical research of this type.

Our findings are consistent with previous research in humans that has shown significantly higher evoked contraction strength in males than in females and slower contraction times in females than in males in the vastus lateralis in the quadriceps muscle group in the thigh (Wüst et al., 2008). The finding of slower contraction times in females is further supported by findings that female skeletal muscles have greater proportions of slow-contracting isoforms than male mammals where muscles are composed of a significantly greater proportion of MyHCIIx fibers (Haizlip et al., 2015). We found not only greater proportions of MyHCIIx in the styloglossus muscle of our male groups, but greater proportions of MyHCIIa (Figure 3). The fact that we did not find similar muscle fiber differences in the genioglossus is not entirely surprising. Differential expression of myosin heavy chain isoforms in various muscles has been documented (Haizlip et al., 2015), consistent with differing muscle fiber type findings in the genioglossus and styloglossus muscles studied here.

Our knowledge of sex differences in maximum voluntary tongue forces in humans is also limited. While a 2013 systematic review found significantly higher voluntary tongue force in adult men than in adult women (Adams et al., 2013), several recent studies have found no significant or inconsistent differences in maximal tongue force measures in men compared to women (Abe et al., 2020; Dietsch et al., 2013; Jones et al., 2021; Lin et al., 2021). These inconsistencies may indicate similarities with our findings in rats. We found no significant difference in maximal voluntary tongue force despite higher tongue force capacity in male group, as measured by evoked protrusive and retrusive twitch and tetanic tongue forces. This discrepancy may be explained by female rats routinely accessing a greater proportion of their maximal force capacity in the tongue when performing motor tasks than male rats. A motivational difference between male and female rats may explain this difference and could be attributed to differences in water intake or differences in density on serotonin receptors between male and female rats. Previous research has shown that female rats have higher levels of water intake than male animals (McGivern et al., 1996). Our voluntary tongue force acquisition paradigm relies on water as a primary reinforcer. It is possible that water is a stronger motivator for water-restricted female rats than for water-restricted male rats. There are also sex differences in motor control that stem from a greater density of serotonin receptors at the level of the hypoglossal nucleus in female rats than in male rats (Seebart et al., 2007). Research has shown that serotonin modulates motor activity and higher levels of serotonin in the nucleus accumbens can increase arousal and behaviors such as lever pressing in rats (Sasaki-Adams & Kelley, 2001). Higher levels of serotonin in the hypoglossal nucleus of female rats may contribute to increased motor output and response rates for oral motor exercises such as the voluntary tongue force task studied here. Further research is needed to examine potentially compounding motivational differences between male and female rats.

The finding that estrous cycle stage has no significant functional impact on voluntary tongue force, contractile properties, or morphological properties of tongue muscles in female rats is an important finding for future research incorporating female animals. This finding is consistent with research into the variability of physiological and behavioral measures in female mice (Prendergast et al., 2014) and physiological measures in female rats (Dayton et al., 2016) that have shown no significant sex differences in variability of measures, despite varying hormone levels in female rats. Note that only four data points were collected during a female rat’s metestrus stage. This is proportionate to the actual length of metestrus in the estrus cycle of the rat, with metestrus lasting only 6–8 hours in the female rats (Cora et al., 2015; Smith et al., 1994). Because varying hormone levels in female rats did not result in increased variability of tongue force measures, it should not be necessary to account for estrous cycle stage in future experiments of a similar nature.

We found minimal age effects in contractile properties and tongue strength, despite previous research showing significant decreases in tongue strength with age in both humans and rats (Adams et al., 2013; Connor et al., 2009; Nagai et al., 2010). Due to limited availability of older female rats, the older groups of rats included in this study were between 17–23 months of age, which we consider to be approaching middle age (Connor et al., 2013; Connor et al., 2009; Kletzien et al., 2013). It is likely that any age-related changes in strength, morphometry, and contractile properties of the tongue would be more evident in older animals that were not available to us in the completion of this work.

In addition to the limited availability of older female rats, there is another limitation in the methodology of this work. We were not able to blind experimenters to animal sex during the maximal tongue force testing phase of the study due to obvious differences in size and anatomy between male and female rats. Experimenters were, however, blinded to animal sex and age during analysis of fiber type and size. Thus, this limitation was controlled to the fullest extent possible.

There is significant debate about the appropriateness of modeling sex differences in rats. It may not be appropriate to model sex differences in tongue force in a rat model as the translation of sex differences in rats to sex differences in humans is not entirely clear, particularly when examining outcome measures that rely on rat behavior, like our tongue force training paradigm, as some of the most well documented sex differences in rats are in behavior, learning, and memory (Jonasson, 2005).

In summary, this investigation found that: 1) the female rats group demonstrated significantly reduced maximal tetanic tension, maximal twitch tension, and contraction speed compared to the male group, with no difference between the sexes in maximum voluntary tongue force, and 2) in the styloglossus, the female group demonstrated significantly lower proportions of MyHCIIa and MyHCIIx, but there were no significant sex or age differences in cross sectional area or myosin heavy chain composition within the genioglossus.

This study establishes essential baseline data for further investigation of contractile properties of the tongue, tongue training, and tongue muscle morphology in female rats. Due to sex differences in tongue strength, muscle morphology, and contractile properties of the tongue, future research of this kind should incorporate female rats to account for potential sex differences at the preclinical level. Failure to do so may negatively impact the efficacy of clinical dysphagia treatments in women. Further research into sex differences in effects of tongue exercise on evoked contractile properties and tongue muscle morphology may have direct implications for clinical tongue-strengthening exercise programs.

Highlights.

Both age and sex impact tongue strength and morphology in a rat model

Styloglossus cross sectional area was larger in the young adult and male groups

Males showed greater maximal evoked tensions than females

No sex differences in voluntary tongue forces

Estrous cycle stage had no effect on tongue force or morphology

Acknowledgments:

The authors gratefully acknowledge the assistance of Jared Cullen, and Dr. Annette Gendron, in the completion of this work. We also gratefully acknowledge Dr. Michelle Ciucci and Dr. Katie Hustad for their comments on an earlier version of this paper. This work was funded from the following sources: NIH grants R37CA225608, R01DC005935, R01 DC018071, and R01DC014358.

Footnotes

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Conflicts of Interest: None of the authors has any conflict of interest to disclose

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Contributor Information

Andrea Rohl, Department of Neurosurgery, University of Iowa.

Nadine P. Connor, Department of Surgery, University of Wisconsin-Madison, Madison Wisconsin; Department of Communication Sciences and Disorders, University of Wisconsin, Madison.

John A. Russell, Department of Surgery, University of Wisconsin-Madison, Madison Wisconsin; University of Wisconsin, Madison.

References

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