Altered tongue muscle contractile properties coincide with altered swallow function in the adult Ts65Dn mouse model of down syndrome.

Purpose: Down syndrome (DS) is a developmental disability associated with difficulties in deglutition. The adult Ts65Dn mouse model of DS has been previously shown to have differences in measures of swallowing compared with euploid controls. However, the putative mechanisms of these differences in swallowing function are unclear. This study tested the hypothesis that the Ts65Dn genotype is associated with atypical measures of tongue muscle contractile properties, coinciding with atypical swallow function. Methods: Adult (5-month-old) Ts65Dn (n = 15 female, 14 male) and euploid sibling controls (n = 16 female, 14 male) were evaluated through videofluoroscopy swallow studies (VFSS) to quantify measures of swallowing performance including swallow rate and inter-swallow interval (ISI). After VFSS, retrusive tongue muscle contractile properties, including measures of muscle fatigue, were determined using bilateral hypoglossal nerve stimulation. Results: The Ts65Dn group had significantly slower swallow rates, significantly greater ISI times, significantly slower rates of tongue force development, and significantly greater levels of tongue muscle fatigue, with lower retrusive tongue forces than controls in fatigue conditions. Conclusion: Tongue muscle contractile properties are altered in adult Ts65Dn and coincide with altered swallow function.

Tongue and laryngeal exercises improve tongue strength and vocal function outcomes in a Pink1-/- rat model of early Parkinson disease.

Parkinson disease (PD) causes voice and swallow dysfunction even in early stages of the disease. Treatment of this dysfunction is limited, and the neuropathology underlying this dysfunction is poorly defined. Targeted exercise provides the greatest benefit for offsetting voice and swallow dysfunction, and previous data suggest the hypoglossal nucleus and noradrenergic-locus coeruleus (LC) may be involved in its early pathology. To investigate relationships between targeted exercise and neuropathology of voice and swallow dysfunction, we implemented a combined exercise paradigm that included tongue force and vocalization exercises early in the Pink1-/- rat model. We tested the hypotheses that (1) tongue and vocal exercise improves tongue force and timing behaviors and vocalization outcomes, and (2) exercise increases optical density of serotonin (5-HT) in the hypoglossal nucleus, and tyrosine hydroxylase immunoreactive (Th-ir) cell counts in the LC. At two months of age Pink1-/- rats were randomized to exercise or non-exercise treatment. Age-matched wildtype (WT) control rats were assigned to non-exercise treatment. Tongue force and timing behaviors and ultrasonic vocalizations were measured at baseline (two months) and final (four months) timepoints. Optical density of 5-HT in the hypoglossal nucleus and TH-ir cell counts in the LC were obtained. Pink1-/- rats produced greater tongue forces, faster tongue contraction, and higher-intensity vocalization following exercise. There were no differences in LC TH-ir. The non-exercised Pink1-/- group had reduced density of 5-HT in the hypoglossal nucleus compared to the WT control group. The changes to tongue function and vocalization after targeted exercise suggests exercise intervention may be beneficial in early PD.

Ultrasonic vocalization phenotypes in the Ts65Dn and Dp(16)1Yey mouse models of Down syndrome.

Down syndrome (DS) is a developmental disorder associated with a high incidence of challenges in vocal communication. DS can involve medical co-morbidities and structural social factors that may impact communication outcomes, which can present difficulties for the study of vocal communication challenges. Mouse models of DS may be used to study vocal communication differences associated with this syndrome and allow for greater control and consistency of environmental factors. Prior work has demonstrated differences in ultrasonic vocalization (USV) of the Ts65Dn mouse model of DS at a young adult age, however it is not known how USV characteristics are manifested at mature ages. Given that the aging process and age-related co-morbidities may also impact communication in DS, addressing this gap in knowledge may be of value for efforts to understand communication difficulties in DS across the lifespan. The current study hypothesized that the Ts65Dn and Dp(16)1Yey mouse models of DS would demonstrate differences in multiple measures of USV communication at a mature adult age of 5 months. METHODS: Ts65Dn mice (n = 16) and euploid controls (n = 19), as well as Dp(16)1Yey mice (n = 20) and wild-type controls (n = 22), were evaluated at 5 months of age for USV production using a mating paradigm. Video footage of USV sessions were analyzed to quantify social behaviors of male mice during USV testing sessions. USV recordings were analyzed using Deepsqueak software to identify 10 vocalization types, which were quantified for 11 acoustic measures. RESULTS: Ts65Dn, but not Dp(16)1Yey, showed significantly lower proportions of USVs classified as Step Up, Short, and Frequency Steps, and significantly higher proportions of USVs classified as Inverted U, than euploid controls. Both Ts65Dn and Dp(16)1Yey groups had significantly greater values for power and tonality for USVs than respective control groups. While Ts65Dn showed lower frequencies than controls, Dp(16)1Yey showed higher frequencies than controls. Finally, Ts65Dn showed reductions in a measure of complexity for some call types. No significant differences between genotype groups were identified in analysis of behaviors during testing sessions. CONCLUSION: While both Ts65Dn and Dp(16)1Yey show significant differences in USV measures at 5 months of age, of the two models, Ts65Dn shows a relatively greater numbers of differences. Characterization of communication phenotypes in mouse models of DS may be helpful in laying the foundation for future translational advances in the area of communication difficulties associated with DS.

Bioenergetic Evaluation of Muscle Fatigue in Murine Tongue.

Muscle fatigue is the diminution of force required for a particular action over time. Fatigue may be particularly pronounced in aging muscles, including those used for swallowing actions. Because risk for swallowing impairment (dysphagia) increases with aging, the contribution of muscle fatigue to age-related dysphagia is an emerging area of interest. The use of animal models, such as mice and rats (murine models) allows experimental paradigms for studying the relationship between muscle fatigue and swallowing function with a high degree of biological precision that is not possible in human studies. The goal of this article is to review basic experimental approaches to the study of murine tongue muscle fatigue related to dysphagia. Traditionally, murine muscle fatigue has been studied in limb muscles through direct muscle stimulation and behavioral exercise paradigms. As such, physiological and bioenergetic markers of muscle fatigue that have been validated in limb muscles may be applicable in studies of cranial muscle fatigue with appropriate modifications to account for differences in muscle architecture, innervation ratio, and skeletal support. Murine exercise paradigms may be used to elicit acute fatigue in tongue muscles, thereby enabling study of putative muscular adaptations. Using these approaches, hypotheses can be developed and tested in mice and rats to allow for future focused studies in human subjects geared toward developing and optimizing treatments for age-related dysphagia.

Progressive Protrusive Tongue Exercise Does Not Alter Aging Effects in Retrusive Tongue Muscles.

Purpose: Exercise-based treatment approaches for dysphagia may improve swallow function in part by inducing adaptive changes to muscles involved in swallowing and deglutition. We have previously shown that both aging and progressive resistance tongue exercise, in a rat model, can induce biological changes in the genioglossus (GG); a muscle that elevates and protrudes the tongue. However, the impacts of progressive resistance tongue exercise on the retrusive muscles (styloglossus, SG; hyoglossus, HG) of the tongue are unknown. The purpose of this study was to examine the impact of a progressive resistance tongue exercise regimen on the retrusive tongue musculature in the context of aging. Given that aging alters retrusive tongue muscles to more slowly contracting fiber types, we hypothesized that these biological changes may be mitigated by tongue exercise. Methods: Hyoglossus (HG) and styloglossus (SG) muscles of male Fischer 344/Brown Norway rats were assayed in age groups of young (9 months old, n = 24), middle-aged (24 months old, n = 23), and old (32 months old, n = 26), after receiving an 8-week period of either progressive resistance protrusive tongue exercise, or sham exercise conditions. Following exercise, HG and SG tongue muscle contractile properties were assessed in vivo. HG and SG muscles were then isolated and assayed to determine myosin heavy chain isoform (MyHC) composition. Results: Both retrusive tongue muscle contractile properties and MyHC profiles of the HG and SG muscles were significantly impacted by age, but were not significantly impacted by tongue exercise. Old rats had significantly longer retrusive tongue contraction times and longer decay times than young rats. Additionally, HG and SG muscles showed significant MyHC profile changes with age, in that old groups had slower MyHC profiles as compared to young groups. However, the exercise condition did not induce significant effects in any of the biological outcome measures. Conclusion: In a rat model of protrusive tongue exercise, aging induced significant changes in retrusive tongue muscles, and these age-induced changes were unaffected by the tongue exercise regimen. Collectively, results are compatible with the interpretation that protrusive tongue exercise does not induce changes to retrusive tongue muscle function.

Functional characterization of extrinsic tongue muscles in the Pink1-/- rat model of Parkinson disease.

Parkinson disease (PD) is associated with speech and swallowing difficulties likely due to pathology in widespread brain and nervous system regions. In post-mortem studies of PD, pathology has been reported in pharyngeal and laryngeal nerves and muscles. However, it is unknown whether PD is associated with neuromuscular changes in the tongue. Prior work in a rat model of PD (Pink1-/-) showed oromotor and swallowing deficits in the premanifest stage which suggested sensorimotor impairments of these functions. The present study tested the hypothesis that Pink1-/- rats show altered tongue function coinciding with neuromuscular differences within tongue muscles compared to wildtype (WT). Male Pink1-/- and WT rats underwent behavioral tongue function assays at 4 and 6 months of age (n = 7-8 rats per group), which are time points early in the disease. At 6 months, genioglossus (GG) and styloglossus (SG) muscles were analyzed for myosin heavy chain isoforms (MyHC), α-synuclein levels, myofiber size, centrally nucleated myofibers, and neuromuscular junction (NMJ) innervation. Pink1-/- showed greater tongue press force variability, and greater tongue press forces and rates as compared to WT. Additionally, Pink1-/- showed relative increases of MyHC 2a in SG, but typical MyHC profiles in GG. Western blots revealed Pink1-/- had more α-synuclein protein than WT in GG, but not in SG. There were no differences between Pink1-/- and WT in myofiber size, centrally-nucleated myofibers, or NMJ innervation. α-synuclein protein was observed in nerves, NMJ, and vessels in both genotypes. Findings at these early disease stages suggest small changes or no changes in several peripheral biological measures, and intact motor innervation of tongue muscles. Future work should evaluate these measures at later disease stages to determine when robust pathological peripheral change contributes to functional change, and what CNS deficits cause behavioral changes. Understanding how PD affects central and peripheral mechanisms will help determine therapy targets for speech and swallowing disorders.

The Adult Ts65Dn Mouse Model of Down Syndrome Shows Altered Swallow Function.

There are increased risks for deglutition disorders in people with Down syndrome (DS). Although mouse models have been used to study the biological underpinnings of DS in other areas, relatively little is known about swallowing phenotypes in these models. We hypothesized that swallowing performance would be affected in adult mouse models of DS, relative to typical control mice. Videofluoroscopic swallow studies (VFSS) were conducted on adults of two mouse models of DS: Ts65Dn and Dp(16)1Yey, and evaluated in comparison with age-matched controls. Relative to other groups, adult Ts65Dn showed significantly slower swallow rates, longer inter-swallow intervals (ISI), and greater numbers of jaw excursion cycles preceding each swallow. In contrast, adult Dp(16)1Yey mice showed swallowing performance similar to control mice. Exploratory quantitative analyses of the intrinsic tongue (transverse muscle), and extrinsic tongue muscles [genioglossus (GG), styloglossus (SG), and hyoglossus (HG)] showed no significant differences between genotype groups in myosin heavy chain isoform profiles. Collectively, these findings suggest that while swallowing is typical in adult Dp(16)1Yey, swallowing in adult Ts65Dn is atypical due to unknown causes. The finding that adult Ts65Dn may have utility as a model of dysphagia provides new opportunities to elucidate biological underpinnings of dysphagia associated with DS.

Laryngeal muscle biology in the Pink1-/- rat model of Parkinson disease.

Neuromuscular pathology is found in the larynx and pharynx in humans with Parkinson disease (PD); however, it is unknown when this pathology emerges. We hypothesized that pathology occurs in early (premanifest) stages. To address this, we used the Pink1-/- rat model of PD, which shows age-dependent dopaminergic neuron loss, locomotor deficits, and deficits related to laryngeal function. We report findings in the thyroarytenoid muscle (TA) in Pink1-/- rats compared with wild-type (WT) control rats at 4 and 6 mo of age. TAs were analyzed for force production, myosin heavy chain isoform (MyHC), centrally nucleated myofibers, neural cell adhesion molecule, myofiber size, and muscle section size. Compared with WT, Pink1-/- TA had reductions in force levels at 1-Hz stimulation and 20-Hz stimulation, increases in relative levels of MyHC 2L, increases in incidence of centrally nucleated myofibers in the external division of the TA, and reductions in myofiber size of the vocalis division of the TA at 6 mo of age. Alterations of laryngeal muscle biology occur in a rat model of premanifest PD. Although these alterations are statistically significant, their functional significance remains to be determined. NEW & NOTEWORTHY Pathology of peripheral nerves and muscle has been reported in the larynx and pharynx of humans diagnosed with Parkinson disease (PD); however, it is unknown whether differences of laryngeal muscle occur at premanifest stages. This study examined the thyroarytenoid muscles of the Pink1-/- rat model of PD for differences of muscle biology compared with control rats. Thyroarytenoid muscles of Pink1-/- rats at premanifest stages show differences in multiple measures of muscle biology.

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

Down syndrome (DS) in humans is associated with differences of the central nervous system and 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.

Digastric Muscle Phenotypes of the Ts65Dn Mouse Model of Down Syndrome.

Down syndrome is frequently associated with complex difficulties in oromotor development, feeding, and swallowing. However, the muscle phenotypes underlying these deficits are unclear. We tested the hypotheses that the Ts65Dn mouse model of DS has significantly altered myosin heavy chain (MyHC) isoform profiles of the muscles involved in feeding and swallowing, as well as reductions in the speed of these movements during behavioral assays. SDS-PAGE, immunofluorescence, and qRT-PCR were used to assess MyHC isoform expression in pertinent muscles, and functional feeding and swallowing performance were quantified through videofluoroscopy and mastication assays. We found that both the anterior digastric (ADG) and posterior digastric (PDG) muscles in 11-day old and 5-6 week old Ts65Dn groups showed significantly lower MyHC 2b protein levels than in age-matched euploid control groups. In videofluoroscopic and videotape assays used to quantify swallowing and mastication performance, 5-6 week old Ts65Dn and euploid controls showed similar swallow rates, inter-swallow intervals, and mastication rates. In analysis of adults, 10-11 week old Ts65Dn mice revealed significantly less MyHC 2b mRNA expression in the posterior digastric, but not the anterior digastric muscle as compared with euploid controls. Analysis of MyHC 2b protein levels across an adult age range (10-53 weeks of age) revealed lower levels of MyHC 2b protein in the PDG of Ts65Dn than in euploids, but similar levels of MyHC 2b in the ADG. Cumulatively, these results indicate biochemical differences in some, but not all, muscles involved in swallowing and jaw movement in Ts65Dn mice that manifest early in post-natal development, and persist into adulthood. These findings suggest potential utility of this model for future investigations of the mechanisms of oromotor difficulties associated with Down syndrome.

Effect of radiation dose-rate on hematopoietic cell engraftment in adult zebrafish.

Although exceptionally high radiation dose-rates are currently attaining clinical feasibility, there have been relatively few studies reporting the biological consequences of these dose-rates in hematopoietic cell transplant (HCT). In zebrafish models of HCT, preconditioning before transplant is typically achieved through radiation alone. We report the comparison of outcomes in adult zebrafish irradiated with 20 Gy at either 25 or 800 cGy/min in the context of experimental HCT. In non-transplanted irradiated fish we observed no substantial differences between dose-rate groups as assessed by fish mortality, cell death in the kidney, endogenous hematopoietic reconstitution, or gene expression levels of p53 and ddb2 (damage-specific DNA binding protein 2) in the kidney. However, following HCT, recipients conditioned with the higher dose rate showed significantly improved donor-derived engraftment at 9 days post transplant (p ≤ 0.0001), and improved engraftment persisted at 31 days post transplant. Analysis for sdf-1a expression, as well as transplant of hematopoietic cells from cxcr4b -/- zebrafish, (odysseus), cumulatively suggest that the sdf-1a/cxcr4b axis is not required of donor-derived cells for the observed dose-rate effect on engraftment. Overall, the adult zebrafish model of HCT indicates that exceptionally high radiation dose-rates can impact HCT outcome, and offers a new system for radiobiological and mechanistic interrogation of this phenomenon. Key words: Radiation dose rate, Total Marrow Irradiation (TMI), Total body irradiation (TBI), SDF-1, Zebrafish, hematopoietic cell transplant.

Migration of cardiomyocytes is essential for heart regeneration in zebrafish.

Adult zebrafish possess a significant ability to regenerate injured heart tissue through proliferation of pre-existing cardiomyocytes, which contrasts with the inability of mammals to do so after the immediate postnatal period. Zebrafish therefore provide a model system in which to study how an injured heart can be repaired. However, it remains unknown what important processes cardiomyocytes are involved in other than partial de-differentiation and proliferation. Here we show that migration of cardiomyocytes to the injury site is essential for heart regeneration. Ventricular amputation induced expression of cxcl12a and cxcr4b, genes encoding a chemokine ligand and its receptor. We found that cxcl12a was expressed in the epicardial tissue and that Cxcr4 was expressed in cardiomyocytes. We show that pharmacological blocking of Cxcr4 function as well as genetic loss of cxcr4b function causes failure to regenerate the heart after ventricular resection. Cardiomyocyte proliferation was not affected but a large portion of proliferating cardiomyocytes remained localized outside the injury site. A photoconvertible fluorescent reporter-based cardiomyocyte-tracing assay demonstrates that cardiomyocytes migrated into the injury site in control hearts but that migration was inhibited in the Cxcr4-blocked hearts. By contrast, the epicardial cells and vascular endothelial cells were not affected by blocking Cxcr4 function. Our data show that the migration of cardiomyocytes into the injury site is regulated independently of proliferation, and that coordination of both processes is necessary for heart regeneration.

Zebrafish stromal cells have endothelial properties and support hematopoietic cells.

The goal of this study was to determine if we could establish a mesenchymal stromal line from zebrafish that would support hematopoietic cells. Such a coculture system would be a great benefit to study of the hematopoietic cell-stromal cell interaction in both in vitro and in vivo environments. Zebrafish stromal cells (ZStrC) were isolated from the "mesenchymal" tissue of the caudal tail and expanded in a specialized growth media. ZStrC were evaluated for phenotype, gene expression, and ability to maintain zebrafish marrow cells in coculture experiments. ZStrC showed mesenchymal and endothelial gene expression. Although ZStrC lacked the ability to differentiate into classic mesenchymal stromal cell lineages (i.e., osteocytes, adipocytes, chondrocytes), they did have the capacity for endotube formation on Matrigel and low-density lipoprotein uptake. ZStrC supported marrow cells for >2 weeks in vitro. Importantly, marrow cells were shown to retain homing ability in adoptive transfer experiments. ZStrC were also shown to improve hematopoietic recovery after sublethal irradiation after adoptive transfer. As the zebrafish model grows in popularity and importance in the study of hematopoiesis, new tools to aid in our understanding of the hematopoietic cell-stromal cell interaction are required. ZStrC represent an additional tool in the study of hematopoiesis and will be useful in understanding the factors that mediate the stromal cell-hematopoietic cell interactions that are important in hematopoietic cell maintenance.

Stromal cell-derived factor-1 and hematopoietic cell homing in an adult zebrafish model of hematopoietic cell transplantation.

In mammals, stromal cell-derived factor-1 (SDF-1) promotes hematopoietic cell mobilization and migration. Although the zebrafish, Danio rerio, is an emerging model for studying hematopoietic cell transplantation (HCT), the role of SDF-1 in the adult zebrafish has yet to be determined. We sought to characterize sdf-1 expression and function in the adult zebrafish in the context of HCT. In situ hybridization of adult zebrafish organs shows sdf-1 expression in kidney tubules, gills, and skin. Radiation up-regulates sdf-1 expression in kidney to nearly 4-fold after 40 Gy. Assays indicate that zebrafish hematopoietic cells migrate toward sdf-1, with a migration ratio approaching 1.5 in vitro. A sdf-1a:DsRed2 transgenic zebrafish allows in vivo detection of sdf-1a expression in the adult zebrafish. Matings with transgenic reporters localized sdf-1a expression to the putative hematopoietic cell niche in proximal and distal renal tubules and collecting ducts. Importantly, transplant of hematopoietic cells into myelosuppressed recipients indicated migration of hematopoietic cells to sdf-1a-expressing sites in the kidney and skin. We conclude that sdf-1 expression and function in the adult zebrafish have important similarities to mammals, and this sdf-1 transgenic vertebrate will be useful in characterizing the hematopoietic cell niche and its interactions with hematopoietic cells.

Expression of telomerase and telomere length are unaffected by either age or limb regeneration in Danio rerio.

BACKGROUND: The zebrafish is an increasingly popular model for studying many aspects of biology. Recently, ztert, the zebrafish homolog of the mammalian telomerase gene has been cloned and sequenced. In contrast to humans, it has been shown that the zebrafish maintains telomerase activity for much of its adult life and has remarkable regenerative capacity. To date, there has been no longitudinal study to assess whether this retention of telomerase activity equates to the retention of chromosome telomere length through adulthood. METHODOLOGY/PRINCIPAL FINDINGS: We have systematically analyzed individual organs of zebrafish with regard to both telomere length and telomerase activity at various time points in its adult life. Heart, gills, kidney, spleen, liver, and intestine were evaluated at 3 months, 6 months, 9 months, and 2 years of age by Southern blot analysis. We found that telomeres do not appreciably shorten throughout the lifespan of the zebrafish in any organ. In addition, there was little difference in telomere lengths between organs. Even when cells were under the highest pressure to divide after fin-clipping experiments, telomere length was unaffected. All aged (2 year old) tissues examined also expressed active amounts of telomerase activity as assessed by TRAP assay. CONCLUSIONS/SIGNIFICANCE: In contrast to several other species including humans, the retention of lifelong telomerase and telomeres, as we have reported here, would be necessary in the zebrafish to maintain its tremendous regenerative capacity. The ongoing study of the zebrafish's ability to maintain telomerase activity may be helpful in unraveling the complexity involved in the maintenance (or lack thereof) of telomeres in other species such the mouse or human.

Thin-sheet laser imaging microscopy for optical sectioning of thick tissues.

We report the development of a modular and optimized thin-sheet laser imaging microscope (TSLIM) for nondestructive optical sectioning of organisms and thick tissues such as the mouse cochlea, zebrafish brain/inner ear, and rat brain at a resolution that is comparable to wide-field fluorescence microscopy. TSLIM optically sections tissue using a thin sheet of light by inducing a plane of fluorescence in transparent or fixed and cleared tissues. Moving the specimen through the thinnest portion of the light sheet and stitching these image columns together results in optimal resolution and focus across the width of a large specimen. Dual light sheets and aberration-corrected objectives provide uniform section illumination and reduce absorption artifacts that are common in light-sheet microscopy. Construction details are provided for duplication of a TSLIM device by other investigators in order to encourage further use and development of this important technology.