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Motor neuron nuclei in Ts65Dn mice, a model of Down syndrome, are chronically stimulated by physical training
By Chiara Rita Ingosio Chiara Rita Ingosio Sillett Preprints.org Google Scholar Maria Assunta Laccavalla Maria Assunta Laccavalla Sillett Preprints.org Google Scholar Publications Barbara Cisterna Barbara Cisterna Sillett Preprints.org View Google Scholar Publications Carlo Zancanaro Zancanaro Sillett Preprints.org * Google Scholar View publication Manuela Malatesta Manuela Malatesta Sylhet Preprints.org Google Scholar Publications
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Department of Biology, Biomedical Sciences and Exercise, Department of Anatomy and Histology, University of Verona, Strada Le Grazie 8, I-37134 Verona, Italy
Original application received: April 20, 2023 / Date of revision: May 20, 2023 / Date of acceptance: May 24, 2023 / Date of publication: May 27, 2023
Down syndrome (DS) is a genetic disease based on trisomy 21 (Hsa21). DS is characterized by intellectual disability associated with several pathological features, including premature aging and changes in motor coordination. Physical training and passive exercise have been found to be beneficial in combating motor dysfunction in DS subjects. In this study, we used Ts65Dn mice, a widely accepted animal model of DS, to study the ultrastructure of the myelinated motor neuron nucleus, which is considered an indicator of cell functional status. We investigated in detail possible trisomy-related changes in nuclear components using transmission electron microscopy, ultrastructure, and immunohistochemistry. The size and distribution of nuclear components are known to change with nuclear activity and its effects. . Give them proper physical training. The results showed that trisomy itself affects nuclear components to a limited extent. However, conditioned physical training can chronically stimulate transcriptional activity and pre-mRNA processing in motor neuron nuclei of triploid mice, although to a lesser extent than in diploid littermates. These findings are a step toward understanding the mechanisms underlying the positive effects of physical activity in DS.
Down syndrome (DS) or trisomy 21 (Hsa21) is a genetic disease that affects approximately 1 in 700 newborns [1]. Normal gene expression is altered in DS, leading to intellectual disability and several pathological features, including craniofacial changes, congenital heart disease, early Alzheimer’s disease, and gastrointestinal disorders. In addition, DS patients have altered motor coordination (despite large individual differences) [2, 3], the most characteristic feature is the slowing of voluntary movements. Therefore, people with DS take longer to respond to stimuli and even longer to perform motor tasks. In addition, DS patients experience muscle hypotonia, muscle weakness, and ligament weakness [4, 5], all of which contribute to poor movement patterns [6]. In addition, DS patients show premature aging at several organ levels [7, 8]. This, similar to the muscle atrophy that occurs physiologically in healthy older adults, inevitably affects muscle mass, strength, and function [9] and also contributes to motor impairment in the DS.
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Physical training and passive exercise have been shown to be beneficial in counteracting muscle hypotonia in children with DS [10, 11] and reducing motor dysfunction and increasing muscle strength in adolescents with DS [12, 13], has shown that it improves some outcomes in humans. I have DS [10, 11] and I suffer from DS [14]. Physical exercise has been shown to be beneficial for muscle mass and function in age-related muscular dystrophies (recently reviewed in [15]), and coordination and motor learning [16]. Due to obvious ethical issues, studies of this type have provided very little morphological evidence of the effects of physical exercise on skeletal muscle or central nervous system (CNS) regions involved in motor function. Instead, animal models are suitable tools to achieve this goal.
Ts65Dn mice (which have trisomy on a portion of chromosome 16 containing a gene orthologous to Hsa21) [17] have a phenotype somewhat similar to human DS [18], including locomotor and myopathic features. , which is widely accepted as a model for DS. total [19, 20], lack of grip strength, motor coordination, running and swimming speed [21]. Ts65Dn mice also show significant delays in the development of many sensory and motor functions [19, 22]. Ts65Dn mice were found to have structural and compositional changes in skeletal muscle [23] due to age-related muscular dystrophy [24]. Several studies have shown neurological benefits after exercise in Ts65Dn mice [25, 26, 27]. Modified physical training with high tolerance in Ts65Dn mice also showed some beneficial effects on spinal muscles analyzed using nuclear magnetic resonance [28] or transmission electron microscopy [29].
To deepen our knowledge of the structural basis of neuromuscular deficits in DS and the potential benefits of physical training, we shifted our attention from muscles to spinal motor neurons. In this study, we focused on the ultrastructure of motor motor nuclei, which can be seen as an indicator of the functional state of the cell. Interestingly, a relationship between changes in nuclear structure and function in hippocampal neurons and cognitive impairment was recently suggested in triploid Ts65Dn mice [30]. By analyzing heterochromatin aggregates, nuclear RNP-containing structures (perichromatin fibers, perichromatin granules, and interchromatin granules), and nuclei using transmission electron microscopy, ultrastructure, and immunohistochemistry. . Structural components (medium fiber, dense fiber components, granular components; see [31] for these nomenclature), and their volume and distribution vary according to nuclear activity (e.g. [32, 33, 34 , 35 ]). The effect of modified physical training on the above nuclear components was also evaluated.
Ts65Dn [strain: B6EiC3Sn. BLia-Ts(17 16)65Dn/DnJ] was obtained from Jackson Laboratory, Maine, USA. This colony was maintained by mating triploid female mice to male euploid B6EiC3Sn.BLiAF1/J mice. Pups were weaned on the 21st day of life. Genotypic analysis Materials were obtained from tail sections of P11 mice. Genotyping was performed with Mmu17 translocation points isolated by PCR [36]. Eight (4 triploid, 4 euploid) Ts65Dn males, aged 8 ± 3.10 months, were used for this study. They were genotypically maintained under standard conditions (ambient temperature 24 ± 1°C., relative humidity 60 ± 15%, 12 hour light/dark cycle) and fed a standard commercially available diet ad libitum. According to a previous study [19], trisomy mice showed deficits in balance and motor coordination at 4 months of age. Four animals (two haploids and two triploids, hereafter referred to as stable haploids and stable trisomies, respectively) showed spontaneous locomotor activity only in the cage, but not elsewhere. ) showed spontaneous locomotor activity only inside the cage. Trained triploids, each) ran for 45 min daily at a speed of 8 m × min on a Harvard Instruments treadmill (Crisel Instruments, Rome, Italy).
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Belt speed (0% incline), 5 days a week for 1 month [28]. The current treadmill protocol for adult mice involves exercise for 1 h per day at belt speeds greater than 10 m × min.
. In this study, physical training was modified to improve training compliance in three-quarter length rats. In order to avoid adverse effects of running with the persistent effects of physical training, all animals were examined 3 days after the last treadmill training session.
Ts65Dn mice were deeply anesthetized with tribromoethanol and perfused transcardially with 0.1 M phosphate buffered saline (PBS) followed by 4% paraformaldehyde in PBS. After perfusion, the spinal cord was quickly and carefully removed, and the distal half was divided transversely into slices approximately 2 mm in length. The samples were then treated with a solution of 2.5% glutaraldehyde and 2% paraformaldehyde (for samples intended for ultrastructural morphology and morphometry) or a solution of 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1M PBS (were prepared for immunohistochemistry) ultrastructural. left at 4 °C for 2 hours. infrastructure). . After fixation, ultrastructural morphology samples were rinsed in PBS, postfixed in 1% OsO4 for 2 h at 4 °C, dehydrated in graded acetone, and embedded in Epon 812 resin. For immunohistochemistry, Wash samples with PBS and treat with 0.5 M NH.
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