Dysphagia, or a disorder in swallowing, can be associated with critical problems in the safety and efficiency of eating and drinking that may lead to malnutrition, dehydration, aspiration pneumonia, and increased risk of death (1, 2). Risks for these problems increase with age. Adults with Down syndrome (DS) may experience dysphagia across oral, pharyngeal, and esophageal phases of swallowing (3) along with gastro-esophageal reflux disease, choking, and aspiration (2, 3). These issues can contribute to behavioral differences such as food refusal. Factors that may contribute to problems at the oral preparatory stage of the swallow in DS may include atypical tongue protrusion from the mouth during eating and drinking, inefficient mastication, and dental differences (4–7). Hypotonicity is often believed to be present and may be implicated in oromotor discoordination, inadequate lip sealing, and impaired tongue movement (8, 9). Pharyngeal phase swallowing problems include timing delays, which lead to a high risk of aspiration (4). In addition, uncoordinated patterns of breathing and swallowing can expose some individuals to higher risk of aspiration, presumably due to temporal incongruities that may result in presence of the bolus near an open airway (4). DS is also associated with primary or secondary esophageal motor disorders (10, 11). As adults with DS age, dysphagia might also be observed at earlier time points than in the general aging population (3). Accordingly, studies of putative mechanisms of dysphagia associated with adulthood and aging in DS are critical to development of effective management of care for individuals with DS experiencing signs of dysphagia.

Dysphagia in adults with DS may involve many underlying anatomical and functional factors including sensorimotor, orofacial, and developmental differences. Anatomically, craniofacial differences including an underdeveloped maxilla and shorter length and width of the palate in adults with DS may impact the safety and efficiency of feeding and swallowing (8, 12). People with DS may have differences in palatal morphology compared with people without DS, and it has been proposed that neuromotor dysfunction incurred by DS may result in less capacity to adjust tongue movement to accommodate these differences (8). For these and other reasons, studies of dysphagia in adults without DS cannot easily generalize to adults with DS. This population must be studied directly.

Mouse models of DS provide a means to study biomedical problems through experimental paradigms that are highly controlled for many variables, and that may involve mechanistic hypotheses and methodology that are not possible to perform in studies with human subjects. The Ts65Dn mouse model of DS displays a remarkable number of phenotypes applicable to characteristics of individuals with DS (13). The craniofacial phenotype of Ts65Dn has been extensively studied and has been reported to include relative reductions in size of the palate, as well as reduced size of the mandibles, which parallels aspects of craniofacial differences in many humans with DS (14). Applicable phenotypes also include traits indicative of muscle differences associated with DS, such as reduced grip strength, altered motor coordination, and reduced running and swimming speeds (15, 16). Genotype-specific differences have also been reported in a variety of measures of muscle biology (17–19). This suggests Ts65Dn is an appropriate model for studying DS-related craniofacial muscle impairments. However, the Ts65Dn mouse model does not entirely replicate the trisomy in humans with DS. The Ts65Dn partial trisomy is comprised of a portion of mouse chromosome 16 (Mmu16) and a section of chromosome 17, leading to a failure to model triplication of some of the genes found on Hsa21, in addition to including the triplication of other genes that are not applicable to DS (13, 20). Consequently, while this mouse model displays numerous phenotypes of interest, it may not encompass all of the genetic and molecular aspects of specific DS features. While work is on-going to compare phenotypes of Ts65Dn to phenotypes of newer mouse models that are anticipated to have superior molecular verisimilitude to DS, Ts65Dn is presently the only mouse model that has been both characterized for feeding and swallowing phenotypes, as well as having been found to demonstrate feeding and swallowing phenotypes that are translationally applicable to DS (21, 22). Ts65Dn exhibits differences from controls in body weight, chewing rates under some conditions, and some biological measures of specific muscles involved in swallowing and jaw movement (21, 23).

Although muscle weakness and hypotonia are typically assumed to be present in adults with DS and are often believed to impact muscles of the tongue, there are relatively few clinical studies and quantitative measures available for this area of study (9). Evaluation of pressure exerted by the tongue during swallowing in adults with DS has been studied through intra-oral pressure sensors (8). This has demonstrated that DS is associated with reductions in the duration and magnitude of tongue pressure on the median palate during swallowing, and that some of these reductions can be explained by differences of palate anatomy that are unique to DS. However, while some DS-specific tongue function phenotypes may occur as adaptations to anatomical differences of the palate, tongue function phenotypes may also be partly or wholly attributable to physiological, sensorimotor, or neuromotor differences of the tongue muscle system (8). Further, despite the fact that tongue strength is believed to be relevant to some aspects of swallowing ability under some circumstances, it is unclear if tongue muscle weakness coincides with significant swallowing phenotypes in DS. Murine models of DS provide access to a wide range of options for investigation of tongue muscle function that can circumnavigate some of these challenges, and which may provide some indication of whether differences in tongue muscle function coincide with differences in swallow function in DS.

The Videofluoroscopic Swallow Study (VFSS) is one of the gold standard diagnostic tests in humans for assessing oropharyngeal stages of swallowing. To adapt this method for mice and small rodents, a murine VFSS protocol was developed (24). This includes a reliable step-by-step test protocol for quantifying swallow metrics (24, 25). While VFSS has been used to identify significant differences in swallowing in the Ts65Dn model (21), VFSS alone gives incomplete information about muscle function during swallowing. Tongue muscle contractile properties can be directly evaluated in murine models as a function of neuromuscular electrical stimulation (NMES), either directly to muscle or with stimulation of the hypoglossal nerves (26). These methods allow quantification of twitch and tetanic forces, rate of force development, and half-decay times. These measures, indicative of tongue muscle force, temporal features of muscle contraction, and tongue muscle fatigue resistance can offer valuable insights into tongue muscle function. Thus, NMES and VFSS methods complement each other to evaluate structure and function of swallowing more precisely. The present study used VFSS and hypoglossal nerve stimulation in the adult Ts65Dn model of DS to test the hypothesis that the Ts65Dn genotype is associated with atypical measures of tongue muscle contractile properties, as well as atypical swallow function.

The goal of this work was to test the hypothesis that the Ts65Dn genotype is associated with atypical measures of swallow function and tongue muscle contractile properties. Our findings were compatible with this hypothesis. We found that during continuous eating of a puree texture, adult Ts65Dn demonstrated significantly slower swallow rates and significantly longer time intervals between consecutive swallows. In addition, Ts65Dn demonstrated slower rates of tongue force development and a more rapid onset of tongue muscle fatigue during evoked retrusive actions. While Ts65Dn had lower values of retrusive tongue forces than euploid control, there was also a significant relationship between animal size and tongue force in both Ts65Dn and control groups, such that presumably smaller muscles generated commensurately lower forces. This is an important factor to consider because DS is often associated with slower growth and thus smaller body size than euploid. Also, sex-specific differences in body size occur in DS as well as in those without DS (29–31). When tongue forces were normalized to body size for analysis, such that tongue forces were analyzed in the context of body weight, no significant differences in tongue force were observed between Ts65Dn and control. Collectively, these findings suggest that while tongue forces are appropriate to animal size in Ts65Dn, Ts65Dn have significant increases in fatiguability of tongue muscles and are slower to develop force in tongue muscles. While it is plausible that these muscle contraction phenotypes may be implicated in increased ISI and decreased swallow rates in Ts65Dn, future studies are required to determine whether there is a causal relationship between these tongue muscle contractile phenotypes and swallowing phenotypes in Ts65Dn.

Properties of swallowing characterized in this study independently replicated previous findings of the adult Ts65Dn swallowing phenotype (24). While the causes of this phenotype remain unknown, slower swallow rates do not coincide with significantly slower jaw movements during food procurement, as evaluated through the JER measure. Compared to controls, Ts65Dn had significantly more jaw excursion cycles prior to each swallow as demonstrated in the JSR measures. A possible explanation is that despite maintaining typical jaw cycle rates, Ts65Dn may have reduced efficiency of oral processing for collection and transfer the bolus prior to initiation of the swallow. Oral processing depends upon sensorimotor coordination of the tongue muscle system. To examine the possibility that function of the tongue muscle system is altered in Ts65Dn, a hypoglossal nerve stimulation paradigm was used to quantify muscle contractile properties during experimentally elicited tongue retrusion. While there was no evidence of genotype-specific weakness in the tongue muscle system upon initial stimulation, Ts65Dn showed significant reductions in the rates of retrusive tongue muscle force development. In addition to having slower rates of force development, Ts65Dn also showed significant increases in fatiguability of tongue retrusion upon repeated stimulation, manifested as significant reductions in the ability to maintain retrusive tongue muscle force upon repeated muscle contraction demands. Heightened susceptibility to muscle fatigue in the tongue may be particularly concerning for deglutition efficacy in Ts65Dn because the swallowing phenotype in this model entails significantly greater oral processing times prior to each swallow than for controls. In some older human populations the consumption of even a typical meal may cause subsequent delays in swallowing initiation, possibly due to fatigue incurred by consuming the meal (32). However, it is worth noting that tongue fatigue in our experimental system did not occur immediately upon stimulation, but rather, occurred only after stimulation for over 2 minutes, which may not have been entirely applicable to the duration of tongue activity elicited during VFSS sessions which typically lasted only several seconds. Therefore, one may speculate that findings of precocious tongue fatigue in Ts65Dn may be more applicable to more naturalistic deglutition settings, such as in ad libitum feeding occurring in standard husbandry conditions. In those settings, more naturalistic eating bouts may unfold over much long periods of time. It is also reasonable to consider that longer oral processing times in Ts65Dn may exacerbate tongue muscle fatigue during eating. In humans, it has been reported that oral processing demands of consuming meals may elicit fatigue-induced reductions in tongue strength in some populations (33, 34). From the standpoint of translational DS research, future work to better understand the causes and the consequences of tongue muscle fatigue in DS may support improved understanding of deglutition phenotypes in this syndrome.

In both humans and murine models, tongue protrusion and potentially co-activation of tongue muscles are involved in successful deglutition (35–37). The puree food used in this study was acquired by mice through licking, which requires at least protrusion of the tongue from the oral cavity to procure the food, retrusion of the tongue into the oral cavity, and collection of a cohesive food bolus followed by a number of key muscle actions that alter pressures within the upper aerodigestive tract for posterior transport of the bolus leading the swallow. Tongue protrusion is accomplished through the genioglossus (an extrinsic tongue muscle), and may be aided through activity of the transverse and verticalis (intrinsic tongue muscles), which are thought to promote elongation of the intrinsic tongue (36). Conversely, tongue retrusion is accomplished through the styloglossus and hyoglossus (extrinsic tongue muscles), and contraction of the superior longitudinal and inferior longitudinal (intrinsic tongue muscles) that may act to shorten the intrinsic tongue when contracted (37, 38). Because of the complex interdigitation of these tongue muscles and the fact that they work synergistically during tongue movement and shape changes of the intrinsic tongue, it is not uncommon for physiological studies of the tongue to interrogate the tongue muscle system intact. This strategy was used in the present study, in which bilateral stimulation of the hypoglossal nerves in the intact tongue muscle system elicited contraction of both protrusive and retrusive tongue muscles, ultimately resulting in net retrusive forces, which were then quantified. The benefit of this approach is it generates information about the physiological properties of the intact muscle system in the mice. The substantial limitation of this approach is that it is unable to clearly identify which components of the tongue muscle system may be primarily responsible for contractile phenotypes of interest. Therefore, the precise muscular etiologies of the genotype-specific differences in muscle fatigue in tongue retrusion identified in this study remain somewhat unclear, as described below.

It is possible that susceptibility to muscle fatigue in Ts65Dn may be related to differences of basic muscle biology associated with this syndrome, including phenotypes related to constitutive oxidative stress, energy metabolism, and muscle bioenergetics, as characterized previously (19, 39, 40). A prior study of the soleus muscle in Ts65Dn identified typical contraction forces in Ts65Dn soleus without fatigue, but found reduced muscle forces during recovery from fatigue (19). Since both a prior study of limb muscle as well as the current study of tongue muscle found no evidence of muscle weakness under typical conditions, but did find genotype-specific reductions in force related to fatigue conditions, it can be speculated that Ts65Dn may have global muscle phenotypes specific to fatigue.

Because the present study measured net retrusive forces that occurred as a result of co-activation of tongue protrusors and retrusors, an alternative possibility to consider is that precocious fatigue in tongue retrusion in this context could also result from an imbalance in the contribution of tongue protrusor muscles relative to retrusor muscles, or a larger proportion of force generated by tongue protrusors in Ts65Dn relative to controls. There are presently few, if any, studies elucidating the relative contributions of tongue protrusor muscles and tongue retrusor muscles to the anatomy and function of the tongue muscle system in DS. However, there is extensive information in the literature that DS may entail atypical co-activation of agonist and antagonistic muscles, and it is possible to speculate that this may occur in the tongue muscle system (9). In conjunction with further studies of tongue physiology, future work may consider avenues through which such an imbalance between tongue protrusors and tongue retrusors could occur in DS. One example of an avenue for future investigation may be the somatotopic organization of the hypoglossal motor nucleus, the anatomical region in which motor neurons in the brainstem responsible for the control of intrinsic and extrinsic tongue muscles of tongue protrusion and tongue retrusion. This somatotopic organization is directed in part through expression of the transcription factor Runx1, which is located on the 21st human chromosome and has been reported to have atypical expression levels in DS (39). Runx1 has been reported to promote expansion of hypoglossal motor neurons controlling tongue protrusor muscles specifically (41). Several studies have characterized a preponderance of tongue protrusion phenotypes in DS, or tendencies to protrude the tongue in situations that would not typically elicit tongue protrusion in individuals without DS (7, 42, 43). Historically, it has been widely recognized that many differences in tongue movement and positioning in DS may be at least partly attributable to craniofacial etiologies, including differences in palate morphology and a disproportionately smaller oral cavity, which could impose substantial anatomical constraints on tongue movement (8, 44). However, the present study evaluated retrusion of the tongue muscle system through a nerve stimulation paradigm in which the tongue was not constrained within the oral cavity, and in which there were neither behavioral nor volitional influences on tongue movement. Therefore, it is possible that the retrusive phenotype reported here could be attributable to differences of neuromuscular anatomy or physiology of the tongue in Ts65Dn.

Using information gained from basic studies of this area to ultimately improve outcomes for individuals with DS will require engagement with a variety of challenges. The clinical study of tongue muscle function and swallowing disorders in adults with DS has some logistical limitations. These include limited geographical availability and capacities of specialized multidisciplinary clinical care centers for DS, which creates barriers for some adults with DS to receive health care services aligned with best practices (45, 46), accessibility barriers that disproportionately disenfranchise individuals with communication support needs who may require special accommodation in order to communicate with medical providers (47), which may be applicable to many individuals with DS (44, 48, 49), and risks for disconnects between researcher expectations and the lived experiences of some disabled study participants (50). In light of these systemic challenges, the continued use of animal models of DS for basic discovery of underlying mechanisms of dysphagia is one of many strategies that can be used to ultimately advance more equitable inclusion of people with DS in the benefits of basic dysphagia research.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

The animal study was approved by University of Wisconsin, Madison, School of Medicine and Public Health Institutional Animal Care and Use Committee and the Roswell Park Comprehensive Cancer Center Institutional Animal Care and Use Committee. The study was conducted in accordance with the local legislation and institutional requirements.

TG: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. JR: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – original draft, Writing – review & editing. EF: Data curation, Investigation, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing. MO: Validation, Writing – original draft, Writing – review & editing. NA: Investigation, Writing – original draft, Writing – review & editing. YY: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Writing – original draft, Writing – review & editing. NC: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing.