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Published in final edited form as: Physiol Behav. 2018 Nov 26;199:273–281. doi: 10.1016/j.physbeh.2018.11.031

Early impacts of modified food consistency on oromotor outcomes in mouse models of Down syndrome

Tiffany J Glass 1,*, Sara L Twadell 1, Luke C Valmadrid 1, Nadine P Connor 1,2
PMCID: PMC6358162  NIHMSID: NIHMS1515556  PMID: 30496741

Abstract

Down syndrome (DS) in humans is associated with differences of the central nervous systemand oromotor development. DS also increases risks for pediatric feeding challenges, which sometimes involve the use of altered food consistencies. Therefore, experimental food consistency paradigms are of interest to oromotor investigations in mouse models of Down syndrome (DS). The present work reports impacts of an altered food consistency paradigm on the Ts65Dn and Dp(16)1Yey mouse models of DS, and sibling control mice. At weaning, Ts65Dn, Dp(16)1Yey and respective controls were assigned to receive either a hard food or a soft food (eight experimental groups, n=8–10 per group). Two weeks later, mice were assessed for mastication speeds and then euthanized for muscle analysis. Soft food conditions were associated with significantly smaller weight gain (p = .003), significantly less volitional water intake through licking (p=.0001), and significant reductions in size of anterior digastric myofibers positive for myosin heavy chain isoform (MyHC) 2b (p=.049). Genotype was associated with significant differences in weight gain (p =.004), significant differences in mastication rate (p=.001), significant differences in a measure of anterior digastric muscle size (p=.03), and significant reductions in size of anterior digastric myofibers positive for MyHC 2a (p=.04). In multiple measures, the Ts65Dn model of DS was more affected than other genotype groups. Findings indicate a soft food consistency condition in mice is associated with significant reductions in weight gain and oromotor activity, and may impact digastric muscle. This suggests extended periods of food consistency modifications may have impacts that extend beyond their immediate roles in facilitating deglutition.

Keywords: Down syndrome, deglutition, food consistency, mouse model, Ts65Dn, Dp(16)1Yey

Graphical abstract

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1. Introduction

Down syndrome (DS) is caused by a trisomy of the 21st chromosome[1], and involves intellectual disability, differences of brain development, and differences in motor function [2, 3]. DS is also associated with risks for difficulties with eating, drinking, or swallowing, especially during childhood [4–7]. This may involve developmental delays in progressions to a range of food textures and consistencies, or the use of softened foods, or thickened liquids [4, 5, 8, 9]. Modified food consistencies can reduce motoric demands necessary for deglutition, thereby permitting safe oral intake. However, there has been little research addressing the potential for adaptations in development associated with extended periods of food consistency modification specifically in the context of DS. Because childhood is a time of rapid oromotor development, and the postnatal development of cranial muscles are tuned to the motoric demands of daily function[10–13], extended food consistency modifications may influence postnatal development of muscles and oromotor functions involved in deglutition. Animal models of feeding disorders offer opportunities to address these research questions in controlled studies.

In routine care for mice, weaning involves moving young mice from the maternal cage (where nourishment and hydration are achieved through suckling), into a separate cage (where nourishment is derived from food pellets, and hydration is achieved through licking water). The transition from suckling to chewing food coincides with shifts in myosin heavy chain isoform (MyHC) compositions of cranial muscles[14–16]. These shifts are believed to occur as adaptations to new oromotor demands. One of these adaptations is an increase in relative abundance of MyHC 2b in the digastric muscle, which is involved in jaw opening during chewing[14, 15]. This time period also coincides with increases in muscle myofiber size[17]. These measures provide indicators of postnatal cranial muscle development in mice. We applied an agarose-based soft food paradigm[18] to investigate cranial muscle and oromotor outcomes in mouse models of DS.

The Ts65Dn and Dp(16)1Yey mouse models are both used for studies of DS. While the Dp(16)1Yey/+ model duplicates the entire portion of mouse chromosome 16 that is syntenic to the human chromosome 21[19], the Ts65Dn model of DS has a partial trisomy involving a portion of the mouse chromosome 16 that is syntenic to the human chromosome 21[20]. This extra chromosome also includes a centromeric portion of mouse chromosome 17 that is not implicated in DS in humans[21]. Many aspects of oromotor function remain to be characterized in these models. We tested the hypotheses that hard vs soft food consistencies elicit differences in digastric muscle outcomes in mice, and that mouse models of DS undergo cranial muscle adaptations to food consistency changes that differ from those of typically developing mice. We anticipated that Ts65Dn and Dp(16)1Yey would show lower MyHC 2b levels than controls, and that MyHC profiles would be affected by food consistency in control mice, but unaffected by food consistency in the mouse models of DS.

2. Materials and Methods

2.1. Mice

This study was in accordance with a protocol approved by the University of Wisconsin School of Medicine and Public Health Animal Care and Use Committee. Ts65Dn and sibling euploid controls were propagated from female trisomic mice (Jackson strain #005252; B6EiC3Sn.BLiA-Ts(1716)65Dn/ DnJ), bred to B6EiC3Sn.BLiAF1/J males (Jackson #003647). Dp(16)1Yey/+ were raised in the same mixed background as the Ts65Dn, as achieved by at least five backcrosses to B6EiC3Sn.BLiAF1. All Dp(16)1Yey and sibling WT controls used in this study were propagated from Dp(16)1Yey mated to F1 partners (Jackson #003647). Group sizes of 8–10 mice per condition were arrived at with interests in permitting detection of digastric muscle phenotypes, as estimated from previous work[22]. Data were collected from 74 male mice; distributed between eight groups comprised of two food consistency conditions administered to Ts65Dn, their euploid sibling controls, as well as Dp(16)1Yey, and their WT sibling controls. Mice were kept on a 12:12 reverse light cycle, weaned at 21 days of age, and genotyped by Transnetyx, Inc (Cordova, TN) as previously reported [22], or by separated PCR. Mice were euthanized through CO2. Mouse genotypes were masked to workers conducting and analyzing assays . All sample data points were reported for each experiment, with the exceptions of incidental loss or exclusions due to technical artifact.

2.2. Food Preparation

Hard food pellets were the Harlan Teklad diet #7913. Soft food was prepared using powder from pellets of the #7913 diet ground with a mortar and pestle, and mixed with agar gel as previously suggested[18, 23]. At the time of soft food preparation, dry food powder was evenly distributed into small plastic cups. For every 9 g of dry food powder, 1 g of agar powder (now® Real Food) was mixed with 90 ml of water, which was boiled and then permitted to cool slightly. This agar preparation was added to the dry food powder, mixed, and placed at 4° C. The mixture was stirred occasionally and then permitted to solidify. This produced a food gel that retained its shape until deformed by physical pressure (Figure 1a). Freshly prepared soft food was provided daily to the mice in sufficient quantity for one day of ad libitum consumption as estimated from a prior study[24]. The IDDSI (International Dysphagia Diet Standardisation Initiative) Framework [25, 26] was used to characterize this soft food recipe. The recipe does not exceed 2 mm particle size restrictions and approximates a Stage 5 food (minced and moist), as determined from fork pressure test, fork drip test, finger test, and spoon tilt test.

Figure 1: Impacts of food consistency conditions and genotype on weight:

Figure 1:

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a: Soft food (left) and hard food pellets (right). b: Ts65Dn, Dp(16)1Yey and respective sibling controls were distributed into food condition groups after weaning at 21 days of age. Mice were then weighed daily for two weeks. n = 8–10 mice per group. c: Weight 12 days after weaning reveals significant main effects for both food consistency condition and genotype. d: Analysis of weight gain between day 0 and day 12 after weaning indicates significantly less weight gain in the soft food condition. Graphs indicate mean and SD. * indicates p ≤ .05, ** indicates p ≤ .01, *** indicates p ≤ .001

2.3. Food Consistency Conditions

At weaning, young mice were assigned to receive either hard food ad libitum, or soft food ad libitum, with ad libitum access to water, and daily weight monitoring. Social housing was ensured during the food consistency condition, such that no mice were housed alone during the study period. Accordingly, the assignment of each mouse to a food consistency condition was made arbitrarily, as determined by the constraints imposed by the number of male mice in each litter, interests in distributing littermates and genotypes evenly between food consistency conditions, and social housing requirements. After two weeks of daily food maintenance and weighing, mice were assessed for mastication measures, and then euthanized for muscle analyses.

2.4. Daily assessment of volitional water licking

Mice were provided ad libitum access to water through a water spout and bottle with volume markings in 1 ml increments (Ancare, Bellmore, NY). Water levels in the bottles were recorded daily in the afternoon. Each cage contained a minimum of two and a maximum of four mice, with mice from one or more genotype groups in each cage as determined by the logistical and social housing parameters described above. To avoid the use of single housing, the volume of water licked per mouse was determined by dividing daily cage water volume reduction by the total number of mice in the cage.

2.5. Mastication Assays

A food consistency condition duration of two weeks post weaning was selected due to the association of this age span with rapid growth rates of cranial bones and masticatory muscles in mice[27]. At a time-point of two weeks of food consistency conditions, mice were assessed for mastication rates with their respective food consistency conditions. The evening before data collection, mice were transferred to individual cages from which food and water were withheld overnight, for 16–18 hours, with an assay time limit of 21 hours. The following morning, food and water were offered. Mastication was videotaped at 60 frames per second while the video camera recorded the mouse in lateral profile. Two workers manually analyzed videos to quantify mastication rates[28]. The pertinent behavior is comprised of initial incision into the food, followed by subsequent chewing cycles (mastication). Chewing cycle rates were analyzed beginning at a starting frame in which the jaw was maximally closed, by counting the number of subsequent chewing cycles (comprised of maximal jaw opening and full jaw closure), within 1 second. Five separate 1-second chewing episodes were analyzed for each mouse, and the five episodes were averaged to generate one data point per mouse. Analyzed chewing episodes were randomly selected for re-analysis to permit inter-rater and intra-rater reproducibility verification. Intra-rater and inter-rater intra-class correlation coefficients (ICC) were determined using no fewer than 10% of the data points. Intra-rater reliability was determined to be at or above .97, and inter-rater reliability was determined to be .84. This indicated acceptable reproducibility of these analyses.

2.6. SDS-PAGE

Digastric muscles (chosen due to previously reported adaptations to weaning [14, 15]) and limb muscles (used as controls) were isolated from euthanized mice. Protein was analyzed with a 6% acrylamide, 30% glycerol separation gel and a 4% acrylamide, 30% glycerol stacking gel [29, 30]. Protein (400 ng) from each muscle was separated through sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) for 29–32 hours, using a SE900 gel system(Hoefer, Inc), then silver stained (SilverQuest Staining Kit, Thermo Fisher Scientific). Band optical density (average pixel value), obtained using UN-SCAN-IT software (Silk Scientific), was used to determine relative percentage of MyHC isoform composition of each muscle.

2.7. Immunofluorescence and image analysis

Specimens were identified throughout processing with alphanumeric codes to blind workers to sample cohort identities. Muscles were isolated in their entirety from euthanized mice and frozen in OCT medium in cassettes suspended in isopentane cooled with liquid nitrogen. Frozen OCT blocks were manually cut in half with a razor blade at a location grossly estimated to bisect the central region of the muscle, and one of the two resulting block halves was mounted to obtain up to five 10-micron tissue cross sections from the cut muscle face. Slides were blocked in 1% BSA and .1% Triton-X 100 in PBS. Anti-Laminin (Sigma) was applied at 1:200, and DSHB antibodies BF-F3 and SC-71 were applied at 1:1, overnight, at 4°C. The following day, slides were rinsed, and incubated for 1 hour in secondary antibodies in 10% normal goat serum in PBS. Secondary antibodies used were Alexa Fluor 350 (1:50), Alexa Fluor 568 (1:300), and Alexa Fluor 488 (1:1000). Soleus muscle was used as a biological negative control for MyHC 2b, and extensor digitorum longus (EDL) muscle was used as a biological positive control for MyHC 2b. Technical negative controls were comprised of sections with the primary antibodies omitted. For each mouse, one tissue section in its entirety was imaged at 20X using an Olympus BX53 epifluorescence microscope. Tissue sections were chosen for imaging with interests in eschewing those with incidental mechanical defects.

The entire imaged muscle section was used for section area analysis and myofiber analysis. Muscle section area was quantified by Image J. Myofiber Minimum Feret’s diameter measures were generated using the Matlab App SMASH as previously described [31]. Minimum Feret’s diameter is a measure of myofiber size that offers improved accuracy relative to the cross-sectional area measurement, in the context of myofibers that may present as somewhat oblique[31]. Acquired image sections were excluded from all analyses if they were comprised of only 250 or fewer analyzable myofibers, or included a substantial proportion of myofibers in excessively oblique or longitudinal orientations. Sections were additionally excluded from specific MyHC-specific myofiber analysis if they included fewer than five myofibers positive for the MyHC isoform of interest. Myofibers compromised by incidental artifacts were determined not to be analyzable and were omitted from analysis.

2.8. Statistical Analysis

Statistical analysis was performed with GraphPad Prism version 6.05 (La Jolla, CA) for all murine assays. Statistical significance was defined at α=0.05. Data were analyzed by 2-way ANOVA and Tukey’s post hoc tests. In experiments in which weight and licking data were collected over time for individual mice, outcomes were analyzed at a single timepoint, just prior to the timepoints of overnight food regulation preceding mastication assays, euthanasia, and collection of muscles for analysis. IBM SPSS Statistics v24 was used for ICC analysis.

3. Results

3.1. Both Food Consistency and Genotype Affect Weight

All groups gained weight during the food consistency condition (Fig 1b). Analysis of mouse weight on day 12 post weaning revealed significant effects for both genotype and food consistency , in the absence of significant interactions. Soft food groups showed lower weight than hard food groups [F(1,64) = 6.54, p = .01]. Genotype also accounted for significant weight differences [F(3,64) = 19.12, p < .0001]. Post hoc tests indicated Ts65Dn showed significantly lower weights than euploid controls for both hard food conditions (p ≤ .01) and soft food conditions (p ≤ .05). Ts65Dn also showed lower weights than Dp(16)1Yey for both hard food conditions (p ≤ .01) and soft food conditions (p ≤ .001) (Fig 1c). Analysis of weight gain (Fig 1d) similarly indicated that soft food groups showed less weight gain than hard food groups. There were significant main effects for food consistency [F(1, 63) = 9.29, p = 0.003], and genotype [F(3, 63) = 4.93, p = 0.004], in the absence of significant interactions.

Six mice developed health concerns during the study. These comprised of one euploid mouse in the hard food condition with malocclusion, one Dp(16)1Yey mouse in the soft food condition that died of unknown causes, two Ts65Dn littermates in the soft food condition that were severely lethargic after overnight food regulation preceding mastication assessment, and two additional Ts65Dn littermates in the soft food condition that were euthanized two days after weaning due to hunched posture and poor weight gain. Necropsy of the latter two Ts65Dn mice suggested concerns with the GI, including possible immaturity or hypoplasia of the mucosa in the cecum, intestinal blunting i.e. villous atrophy, and a possible early enterocolitis due to overgrowth and loss of the normal flora balance in the GI tract. Two euploid sibling cage-mates of these Ts65Dn mice continued to receive the soft food condition for the duration of the study and remained in apparent good health.

3.2. Soft food induced significant changes in oromotor activity, as measured by daily amounts of water consumed through licking

Incidental observations after initiation of the study suggested that mice in the hard food group often licked water, while mice in the soft food group rarely licked water. Subsequent daily monitoring of water consumption confirmed that mice in the soft food condition had significant reductions in water intake through ad libitum licking at a water spout, as compared with mice in the hard food condition (Figure 2). Analysis at 12 days after weaning demonstrated significant decrements in water consumption (ml) in the soft food group [t(10) = 6.06, p=.0001].

Figure 2: Impact of food consistency on licking behavior:

Figure 2:

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Mice in mixed-genotype cages had access to water ad libitum. Water levels in the bottles were recorded daily. The ml of water consumed per mouse was determined by dividing daily cage water consumption values by the number of mice in the cage. n=14 mice per food consistency condition, distributed across 5–7 cages per food consistency condition. Icons indicate mean and SD.

3.3. Ts65Dn showed reduced mastication rate

Mice were assessed for mastication rates after two weeks of food consistency conditions. In the absence of significant interactions, 2-way ANOVA showed significant main effects for genotype [F(3, 51) = 6.03, p=.001] but no main effects for food consistency. In post hoc tests, Ts65Dn showed reduced rates relative to Dp(16)1Yey in both hard and soft food conditions (p≤.05), and reduced mastication rates relative to euploid controls in soft food conditions (p≤.05) (Figure 3).

Figure 3: Impact of food consistency on mastication rates:

Figure 3:

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After two weeks of food consistency conditions, mice were assessed for mastication rates. n = 6–9 mice per group. Bars indicate means and SD. * indicates p ≤ .05

3.4. Food consistency did not induce early differences in digastric muscle MyHC isoform profiles

Muscles assessed following two weeks of food consistency conditions showed no significant differences between food conditions in relative percentages of the MyHC 2b isoform. In the Anterior Digastric (ADG), neither genotype [F(3, 58)=1.42, p=.25] nor food consistency [F(1,58)=.18, p=.67] were identified to have significant main effects on the relative percentages of MyHC 2b. Similarly, in the Posterior Digastric (PDG), neither genotype [F(3, 58)=.54, p=.67] nor food consistency [F(1,58)=3.24, p=.08], were identified to have significant main effects on relative percentages of the MyHC 2b isoform (Figure 4).

Figure 4: Impact of food consistency on digastric muscle MyHC composition:

Figure 4:

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Silver-stained SDS-PAGE MyHC analysis of total protein from digastric muscle homogenates. a: Anterior digastric muscle, b: Posterior digastric muscle. Graphs indicate mean and SD. n = 8–10 mice per group.

3.5. Food consistency and genotype were associated with alterations in measures of digastric muscle size

Because Ts65Dn appeared more affected than other experimental groups in preceding experiments , the muscles of Ts65Dn and respective euploid controls were assessed by microscopy (Figure 5a). The ADG had microscopy results suggesting differences in the Ts65Dn genotype as well as in the soft food condition. In analysis of section area (Figure 5b), ADG showed significantly smaller sections (mm2) associated with the Ts65Dn genotype [F(1,23)=5.37, p=.03], and suggested a trend of smaller sections associated with the soft food consistency [F(1,23)=3.82, p=.06]. In analyses of myofiber Minimum Feret’s diameter, ADG showed significantly smaller MyHC 2a-positive fibers in the Ts65Dn genotype [F(1,21)=4.64, p=.04], and smaller MyHC-2b positive fibers in the soft food condition [F(1,21)=4.34, p=.049]; whereas inclusive analyses of all myofibers (including fibers negative for either MyHC 2a or 2b expression) showed no significant differences between groups. No tissue section or myofiber measures in the ADG showed significant interactions between food condition and genotype. In contrast, PDG showed no significant differences between groups in either section analysis or myofiber analyses.

Figure 5: Impact of food consistency on digastric muscle size.

Figure 5:

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a: Immunolabeling of thin tissue sections from anterior and posterior digastric muscles. Scale indicates 200 microns. b: Quantitative analysis of muscle sections: Minimum Feret’s diameter of myofibers in each muscle section, and muscle section area. Graphs indicate mean and SD. n=4–9 per group, as shown. * indicates p ≤ .05

4. Discussion

We hypothesized that a hard food consistency vs soft food consistency would elicit differences in digastric muscles in mice, and that mouse models of DS would undergo digastric muscle adaptations to food consistency that differed from those of control mice. We found that the soft food consistency had significant impacts irrespective of genotype. These included reductions in measures of weight gain, licking behavior, and reductions in size of MyHC 2b-positive anterior digastric myofibers. Additionally, irrespective of food consistency condition, Ts65Dn demonstrated reduced weights, reduced mastication rates, and reduced anterior digastric muscle sizes as compared to other groups. However, neither food consistency nor genotype were associated with quantitative differences of MyHC 2b levels in the digastric muscle as evaluated by SDS-PAGE.

Feeding difficulties in humans with DS can involve an array of factors, including differences of craniofacial morphology[32, 33], oral sensory and oral motor differences [4, 5, 34], gastrointestinal disorders and malformations [35–37], and other medical comorbidities[7, 38]. Because Ts65Dn and Dp(16)1Yey are genetically distinct models of DS, concurrent investigation of the two models provide opportunities to consider relationships between underlying mechanisms of impairment and functional outcomes related to feeding. The Ts65Dn model features a partial trisomy comprised of a portion of the mouse chromosome (Mmu) Mmu16 and a centromeric region of Mmu17[20, 21], whereas the Dp(16)1Yey model has normal karyotype and a duplication of the entirety of Mmu16 that is syntenic with the human 21st chromosome[19]. Prior work has found that Ts65Dn and Dp(16)1Yey differ in the extent to which they model some of the phenotypes associated with DS [39, 40]. For example, compared to Ts65Dn, Dp(16)1Yey have been reported to show a relative absence of embryonic brain growth deficits, and may be less affected in acquisition of some early-acquired developmental milestones[39, 40]. However, it has been suggested that Dp(16)1Yey and Ts65Dn both show similar skull and craniofacial dysmorphology (such as relatively smaller mandibles) relative to controls[41, 42]. That is of interest for the present study because craniofacial differences may be pertinent to feeding challenges in DS[33]. Although the craniofacial phenotypes of the two models were not evaluated at the 5-week age point in the current study, prior reports of craniofacial similarities between the two models may encourage the speculation that craniofacial phenotypes are unlikely to be the primary cause of reductions in mastication rates of Ts65Dn relative to Dp(16)1Yey. Furthermore, reduced mastication rates in Ts65Dn as compared to Dp(16)1Yey incidentally coincided with significantly reduced weights in Ts65Dn as compared to Dp(16)1Yey in this study. Prior work has attributed reduced weights in Ts65Dn to global cell proliferation deficits caused by the trisomy[43], however, it is possible to speculate that in mouse models of DS, as in humans with DS, feeding phenotypes may also influence postnatal body weight. Finally, it would be beneficial for future studies to include female mice, as sex-specific differences may be anticipated in outcomes related to DS[44].

The food consistency conditions elicited significant differences in oromotor activity. While mice weaned with a hard food licked water, mice weaned with the soft food rarely licked water. Although this experiment design collected data averaged between multiple genotype groups in each cage, striking reductions in the amount of liquid water consumed by mice receiving soft food suggests that all genotype groups were similarly affected in this measure by the food consistency condition. The water intrinsic to the soft food composition used in this study likely meets hydration needs of the mice, thus removing the need to lick water[45]. Drinking in mice entails cycles of movement comprised of jaw opening, tongue protrusion, and tongue retrusion [28, 46]. Therefore, this absence of licking in mice raised on the soft food implies meaningful alterations of daily oromotor activity.

While SDS-PAGE indicated no significant differences across groups in digastric muscle MyHC profiles, microscopy measures suggested a trend of reductions in anterior digastric muscle size in soft food groups, and significant reductions in anterior digastric MyHC 2b-positive myofiber size in soft food groups. Although it is interesting to consider the possibility that altered oromotor activity levels were relevant to digastric muscle outcomes in this study, an alternative possibility is that the biological significance of these muscle outcomes may relate to the finding that the soft food caused significant reductions in weight gains. It is possible that soft food incurred global differences of growth that superceded differences of functional specialization in the digastric muscles. Present findings of reduced weight gain in the soft food condition are in contrast to a prior report of an agar-based food modification which had negligible impacts on weight in rats; however, that prior study employed food prepared with agar in a paste form[47], as opposed to agar in gel form as in the present study. Therefore, a speculative explanation of consistency-induced weight reductions observed in the present study is that the soft food consistency was associated with greater satiety. Thickening can produce greater perceptions of satiety relative to the volume consumed, resulting in reduced total consumption[48]. Notably, previous studies have reported reduced muscle fiber number and size in murine models of restricted nutrition; sometimes with specific fiber types predominately affected [49–52]. Risks for undernutrition and reduced muscle mass in older adults have also been described in circumstances of food consistency modifications [48, 53, 54]. However, a limitation of this study is that it was not designed to measure the amount of food consumed and thus cannot evaluate whether weight reductions in the soft food condition were caused by reduced food intake. Despite this limitation, findings that a diet consistency modification negatively impacted weight gain in mice suggest that extended use of some consistency modifications may be associated with risks, and that animal models may permit improved understanding or anticipation of those risks.

An additional limitation of this study is that the specific soft food formulation used was in many ways different from soft food formulations typically used for human populations. Altered food and liquid consistencies encompass a wide array of strategies that can be integral to management of dysphagia and feeding challenges. Fluid thickeners can be an effective adaptation for children with DS who have pharyngeal phase dysphagia[4, 5], and have further been reported by families to improve quality of life and health[55]. However, there have been reports of adverse outcomes in the use fluid consistency modifications for some populations, including necrotizing enterocolitis in infants[56, 57], which supports interest in weaning from thickeners as soon as medically advisable[58]. In this vein, opportunities for future work using murine models of DS include characterization of outcomes following the applications of specific thickener formulations in current clinical use, which would permit conclusions with greater potential for translational applicability to the human population.

Impact of the Ts65Dn genotype on MyHC 2b content and inclusive myofiber Minimum Feret’s diameter in the digastric muscles were not significant in this study, which is in contrast to our findings of reduced myofiber cross-sectionalarea (CSA) in Ts65Dn in a prior study[22]. Because significant findings of phenotypes varied between studies, it is of value to highlight methodological differences and potential sources of variability. First, the two studies differed in the nutritional composition of the diet. Diet composition has been identified as a key factor in management of biological variation in pre-clinical studies of murine muscle pathology[59]. Both hard and soft food conditions in the current study used Harlan Teklad diet #7913, in which wheat, corn, and oats predominate, whereas the previous study used Harlan Teklad diet #8604; in which soybean meal predominates. Diets that are soy-based have been reported to significantly impact MyHC and myofiber phenotypes in a mouse model of cardiomyopathy[60], and have also been reported to significantly impact seizure phenotypes in Ts65Dn [61, 62]. Given this, it appears appropriate for future investigation of muscle development in murine models of DS to give careful consideration to diet composition. A second variable that bears mentioning is that this study involved daily handling of the mice as necessitated by collection of weight data, whereas the prior study involved no routine interactions with the mice other than weekly cage changes. It has been proposed that stress attributable to handling merits consideration in studies murine models of muscle pathology[59], and handling has been suggested to be pertinent to experimental outcomes in Ts65Dn specifically[63]. Thirdly, the present study evaluated 5-week-old mice, whereas the prior study pooled 5–6 week old mice. While an age range of a week would likely be negligible in studies of adult mice, rapid cranial growth and muscle maturation at these younger ages are compatible with the possibility that an age range of a week is not inconsequential for the study of cranial muscle in juvenile mice[27, 59]. Finally, the potential for technical variability in section-based studies of mouse cranial muscles is worth noting. Prior studies of rat digastric muscles have reported central vs peripheral heterogeneity of muscle fibers [64]. This type of spatial heterogeneity was accommodated in the present study design through acquisition and analysis of entire cross-sections, rather than of limited fields of view as has historically been a common approach in studies of murine muscle. However, anterior-posterior differences within a muscle may be studied through sectioning the entire muscle. Sectioning muscles in their entirety would also technically optimize empirical selection of the center of the muscle as evidenced in tissue sections, with greater precision than the manual bisection approach used in the present study. The potential for significant anatomical regionalization of different myofiber types within cranial muscles[29, 65] may introduce variability to section-based studies, and may justify approaches in future work that accommodate interests in regional specialization within cranial muscles that may differ with sex, developmental status, and age.

Conclusions:

Compared to a hard food condition, a nutritionally comparable soft food consistency was associated with significant reductions in weight gain, significant reductions in licking, and significant changes in measures of digastric myofibers in both mouse models of DS and typical control mice. These findings suggest opportunities for refinement of basic murine experimental paradigms used to study oromotor challenges associated with DS.

Highlights:

  • A soft food consistency causes reduced weight gain in mouse models of Down syndrome and control mice

  • A soft food consistency causes significant reductions in licking behavior

  • Ts65Dn mice have slower mastication rates than other genotypes

  • Both genotype and food consistency may impact digastric muscle

Acknowledgements:

This work was supported through the NIH, National Institute on Deafness and Other Communication Disorders (NIDCD) F32 DC014885, R01DC008149, and R01DC014358. We would like to thank Dr. Annette Gendron-Fitzpatrick DVM, Ph.D., DACVP, and The Comparative Pathology Laboratory, University of Wisconsin-Madison, for necropsy analysis of Ts65Dn mice. We appreciate the expertise and resources provided by Dr. Alberto C. Costa and Melissa Stasko, Case Western Reserve University, who supplied initial breeding pairs of Dp(16)1Yey mice in the mixed background used to generate mice used in this research. The SC-71 and BF-F3 antibodies developed by Stefano Schiaffino were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH, and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242.

Footnotes

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

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